Overexpression of the groESL Operon Enhances the Heat and Salinity Stress Tolerance of the Nitrogen-Fixing Cyanobacterium Anabaena sp. Strain PCC7120

The bicistronic groESL operon, encoding the Hsp60 and Hsp10 chaperonins, was cloned into an integrative expression vector, pFPN, and incorporated at an innocuous site in the Anabaena sp. strain PCC7120 genome. In the recombinant Anabaena strain, the additional groESL operon was expressed from a strong cyanobacterial P_psbA1 promoter without hampering the stress-responsive expression of the native groESL operon. The net expression of the two groESL operons promoted better growth, supported the vital activities of nitrogen fixation and photosynthesis at ambient conditions, and enhanced the tolerance of the recombinant Anabaena strain to heat and salinity stresses.Nitrogen-fixing cyanobacteria, especially strains of Nostoc and Anabaena, are native to tropical agroclimatic conditions, such as those of Indian paddy fields, and contribute to the carbon (C) and nitrogen (N) economy of these soils (22, 30). However, their biofertilizer potential decreases during exposure to high temperature, salinity, and other such stressful environments (1). A common target for these stresses is cellular proteins, which are denatured and inactivated during stress, resulting in metabolic arrest, cessation of growth, and eventually loss of viability. Molecular chaperones play a major role in the conformational homeostasis of cellular proteins (13, 16, 24, 26) by (i) proper folding of nascent polypeptide chains; (ii) facilitating protein translocation and maturation to functional conformation, including multiprotein complex assembly; (iii) refolding of misfolded proteins; (iv) sequestering damaged proteins to aggregates; and (v) solubilizing protein aggregates for refolding or degradation. Present at basal levels under optimum growth conditions in bacteria, the expression of chaperonins is significantly enhanced during heat shock and other stresses (2, 25, 32).The most common and abundant cyanobacterial chaperones are Hsp60 proteins, and nitrogen-fixing cyanobacteria possess two or more copies of the hsp60 or groEL gene (http://genome.kazusa.or.jp/cyanobase). One occurs as a solitary gene, cpn60 (17, 21), while the other is juxtaposed to its cochaperonin encoding genes groES and constitutes a bicistronic operon groESL (7, 19, 31). The two hsp60 genes encode a 59-kDa GroEL and a 61-kDa Cpn60 protein in Anabaena (2, 20). Both the Hsp60 chaperonins are strongly expressed during heat stress, resulting in the superior thermotolerance of Anabaena, compared to the transient expression of the Hsp60 chaperonins in Escherichia coli (20). GroEL and Cpn60 stably associate with thylakoid membranes in Anabaena strain PCC7120 (14) and in Synechocystis sp. strain PCC6803 (15). In Synechocystis sp. strain PCC6803, photosynthetic inhibitors downregulate, while light and redox perturbation induce cpn60 expression (10, 25, 31), and a cpn60 mutant exhibits a light-sensitive phenotype (http://genome.kazusa.or.jp/cyanobase), indicating a possible role for Cpn60 in photosynthesis. GroEL, a lipochaperonin (12, 28), requires a cochaperonin, GroES, for its folding activity and has wider substrate selectivity. In heterotrophic nitrogen-fixing bacteria, such as Klebsiella pneumoniae and Bradyrhizobium japonicum, the GroEL protein has been implicated in nif gene expression and the assembly, stability, and activity of the nitrogenase proteins (8, 9, 11).Earlier work from our laboratory demonstrated that the Hsp60 family chaperonins are commonly induced general-stress proteins in response to heat, salinity, and osmotic stresses in Anabaena strains (2, 4). Our recent work elucidated a major role of the cpn60 gene in the protection from photosynthesis and the nitrate reductase activity of N-supplemented Anabaena cultures (21). In this study, we integrated and constitutively overexpressed an extra copy of the groESL operon in Anabaena to evaluate the importance and contribution of GroEL chaperonin to the physiology of Anabaena during optimal and stressful conditions.Anabaena sp. strain PCC7120 was photoautotrophically grown in combined nitrogen-free (BG11⁻) or 17 mM NaNO₃-supplemented (BG11⁺) BG11 medium (5) at pH 7.2 under continuous illumination (30 μE m⁻² s⁻¹) and aeration (2 liters min⁻¹) at 25°C ± 2°C. Escherichia coli DH5α cultures were grown in Luria-Bertani medium at 37°C at 150 rpm. For E. coli DH5α, kanamycin and carbenicillin were used at final concentrations of 50 μg ml⁻¹ and 100 μg ml⁻¹, respectively. Recombinant Anabaena clones were selected on BG11⁺ agar plates supplemented with 25 μg ml⁻¹ neomycin or in BG11⁻ liquid medium containing 12.5 μg ml⁻¹ neomycin. The growth of cyanobacterial cultures was estimated either by measuring the chlorophyll a content as described previously (18) or the turbidity (optical density at 750 nm). Photosynthesis was measured as light-dependent oxygen evolution at 25 ± 2°C by a Clark electrode (Oxy-lab 2/2; Hansatech Instruments, England) as described previously (21). Nitrogenase activity was estimated by acetylene reduction assays, as described previously (3). Protein denaturation and aggregation were measured in clarified cell extracts containing ∼500 μg cytosolic proteins treated with 100 μM 8-anilino-1-naphthalene sulfonate (ANS). The pellet (protein aggregate) was solubilized in 20 mM Tris-6 M urea-2% sodium dodecyl sulfate (SDS)-40 mM dithiothreitol for 10 min at 50°C. The noncovalently trapped ANS was estimated using a fluorescence spectrometer (model FP-6500; Jasco, Japan) at a λ_excitation of 380 nm and a λ_emission of 485 nm, as described previously (29).The complete bicistronic groESL operon (2.040 kb) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ608815","term_id":"222354886","term_text":"FJ608815"}}FJ608815) was PCR amplified from PCC7120 genomic DNA using specific primers (Table ​(Table1)1) and the amplicon cloned into the NdeI-BamHI restriction sites of plasmid vector pFPN, which allows integration at a defined innocuous site in the PCC7120 genome and expression from a strong cyanobacterial P_psbA1 promoter (6). The resulting construct, designated pFPNgro (Table ​(Table1),1), was electroporated into PCC7120 using an exponential-decay wave form electroporator (200 J capacitive energy at a full charging voltage of 2 kV; Pune Polytronics, Pune, India), as described previously (6). The electroporation was carried out at 6 kV cm⁻¹ for 5 ms, employing an external autoclavable electrode with a 2-mm gap. The electroporation buffer contained high concentrations of salt (10 mM HEPES, 100 mM LiCl, 50 mM CaCl₂), as have been recommended for plant cells (23) and other cell types (27). The electrotransformants, selected on BG11⁺ agar plates supplemented with 25 μg ml⁻¹ neomycin by repeated subculturing for at least 25 weeks to achieve complete segregation, were designated AnFPNgro.

TABLE 1.

Plasmids, strains, and primers used in this study

Efficient Production of 2-Oxobutyrate from 2-Hydroxybutyrate by Using Whole Cells of Pseudomonas stutzeri Strain SDM

2-Oxobutyrate is an important intermediate in the chemical, drug, and food industries. Whole cells of Pseudomonas stutzeri SDM, containing NAD-independent lactate dehydrogenases, effectively converted 2-hydroxybutyrate into 2-oxobutyrate. Under optimal conditions, the biocatalytic process produced 2-oxobutyrate at a high concentration (44.4 g liter⁻¹) and a high yield (91.5%).2-Oxobutyrate (2-OBA) is used as a raw material in the synthesis of chiral 2-aminobutyric acid, isoleucine, and some kinds of medicines (1, 8). There is no suitable starting material for 2-OBA production by chemical synthesis; therefore, the development of innovative biotechnology-based techniques for 2-OBA production is desirable (12).2-Hydroxybutyrate (2-HBA) is cheaper than 2-OBA and can be substituted for 2-OBA in the production of isoleucine, as reported previously (9, 10). The results of those studies also indicated that it might be possible to produce 2-OBA from 2-HBA by a suitable biocatalytic process. In the presence of NAD, NAD-dependent 2-hydroxybutyrate dehydrogenase can catalyze the oxidation of 2-HBA to 2-OBA (4). However, due to the high cost of pyridine cofactors (11), it is preferable to use a biocatalyst that directly catalyzes the formation of 2-OBA from 2-HBA without any requirement for NAD as a cofactor.In our previous report, we confirmed that NAD-independent lactate dehydrogenases (iLDHs) in the pyruvate-producing strain Pseudomonas stutzeri SDM (China Center for Type Culture Collection no. M206010) could oxidize lactate and 2-HBA (6). Therefore, in addition to pyruvate production from lactate, P. stutzeri SDM might also have a potential application in 2-OBA production.To determine the 2-OBA production capability of P. stutzeri SDM, the strain was first cultured at 30°C in a minimal salt medium (MSM) supplemented with 5.0 g liter⁻¹ dl-lactate as the sole carbon source (5). The whole-cell catalyst was prepared by centrifuging the medium and resuspending the cell pellet, and biotransformation was then carried out under the following conditions using 2-HBA as the substrate and whole cells of P. stutzeri SDM as the biocatalyst: 2-HBA, 10 g liter⁻¹; dry cell concentration, 6 g liter⁻¹; buffer, 100 mM potassium phosphate (pH 7.0); temperature, 30°C; shaking speed, 300 rpm. After 4 h of reaction, the mixture was analyzed by high-performance liquid chromatography (HPLC; Agilent 1100 series; Hewlett-Packard) using a refractive index detector (3). The HPLC system was fitted with a Bio-Rad Aminex HPX-87 H column. The mobile phase consisted of 10 mM H₂SO₄ pumped at 0.4 ml min⁻¹ (55°C). Biotransformation resulted in the production of a compound that had a retention time of 19.57 min, which corresponded to the peak of authentic 2-OBA (see Fig. S1 in the supplemental material).After acidification and vacuum distillation, the new compound was analyzed by negative-ion mass spectroscopy. The molecular ion ([M − H]⁻, m/z 101.1) signal of the compound was consistent with the molecular weight of 2-OBA, i.e., 102.1 (see Fig. S2 in the supplemental material). These results confirmed that 2-HBA was oxidized to 2-OBA by whole cells of P. stutzeri SDM.To investigate whether iLDHs are responsible for 2-OBA production in the above-described biocatalytic process, 2-HBA oxidation activity in P. stutzeri SDM was probed by native polyacrylamide gel electrophoresis. After electrophoresis, the gels were soaked in a substrate solution [50 mM Tris-HCl buffer (pH 8.0) containing 0.1 mM phenazine methosulfate, 0.1 mM 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, and 1 mM l-lactate, dl-lactate, or dl-2-HBA] and gently shaken. As shown in Fig. ​Fig.1,1, d- and l-iLDH migrated as two bands with distinct mobilities. The activities responsible for d- and l-2-HBA oxidation were located at the same positions as the d- and l-iLDH activities, respectively. No other bands responsible for d- and l-2-HBA oxidation were detected. Moreover, the dialysis of the crude cell extract did not lead to loss of 2-HBA oxidation activity and the addition of 10 mM NAD⁺ could not stimulate the reaction (see Table S1 in the supplemental material). These results implied that in the biocatalytic system, 2-HBA was oxidized to 2-OBA by iLDHs present in P. stutzeri SDM.Open in a separate windowFIG. 1.Activity staining of iLDHs after native polyacrylamide gel electrophoresis with lactate or 2-HBA as the substrate.Although the SDM strain could not use 2-HBA or 2-OBA for growth (see Fig. S3 in the supplemental material), 2-HBA might induce some of the enzymes responsible for 2-OBA production in the biocatalytic process. To exclude this possibility, the SDM strain was cultured in MSM containing dl-lactate or pyruvate as the sole carbon source. As shown in Fig. ​Fig.2,2, the enzyme activities that catalyzed lactate and 2-HBA oxidation were simultaneously present in the cells cultured on lactate and were absent in those cultured on pyruvate. After the lactate or pyruvate was exhausted, 5.05 g liter⁻¹ dl-2-HBA was added to the medium. It was observed that dl-2-HBA was efficiently converted to 2-OBA in the medium containing dl-lactate (Fig. ​(Fig.2a).2a). No 2-OBA production was detected in the medium containing pyruvate. Because 2-HBA addition did not induce the enzymes involved in 2-HBA oxidation (Fig. 2a and b), we concluded that the iLDHs induced by dl-lactate catalyzed 2-HBA oxidation in this biocatalytic process.Open in a separate windowFIG. 2.Time course of P. stutzeri SDM growth on media containing dl-lactate (a) and pyruvate (b). 2-HBA was added to the medium after the exhaustion of lactate or pyruvate. Symbols: ▴, lactate; ▵, pyruvate; •, 2-HBA; ○, 2-OBA; ▪, cell density; ▧, iLDHs activity with dl-lactate as the substrate; ▒, iLDHs activity with dl-2-HBA as the substrate.iLDHs could catalyze the oxidation of the substrate in a flavin-dependent manner and might use membrane quinone as the electron acceptor. Unlike the oxidases, which directly use the oxygen as the electron acceptor, this substrate oxidation mechanism could prevent the formation of H₂O₂ (see Fig. S4 in the supplemental material). The P. stutzeri SDM strain efficiently converted dl-2-HBA to 2-OBA with high yields (4.97 g liter⁻¹ 2-OBA was produced from 5.05 g liter⁻¹ dl-2-HBA); therefore, 2-OBA production by this strain can be a valuable and technically feasible process. To increase the efficiency of P. stutzeri SDM in the biotechnological production of 2-OBA, the conditions for biotransformation using whole cells of P. stutzeri SDM were first optimized. The influence of the reaction pH and 2-HBA concentration on 2-OBA production was determined in 100 mM phosphate buffer containing whole cells harvested from the medium containing dl-lactate as the sole carbon source. The reaction was initiated by adding the whole cells and 2-HBA at 37°C, followed by incubation for 10 min. After stopping the reaction by adding 1 M HCl, the 2-OBA concentration was determined by HPLC.As shown in Fig. ​Fig.3a,3a, ​,2-OBA2-OBA production was highest at pH 7.0. Under acidic or alkaline conditions, the transformation of 2-HBA to 2-OBA decreased. The optimal 2-HBA concentration was found to be 0.4 M, as shown in Fig. ​Fig.3b.3b. 2-OBA production increased as the 2-HBA concentration increased up to about 0.4 M and decreased thereafter. The concentration of the whole-cell catalyst was then optimized using 0.4 M 2-HBA as the substrate at pH 7.0. As shown in Fig. ​Fig.3c,3c, the highest 2-OBA concentration was obtained with 20 g (dry cell weight [DCW]) liter⁻¹ of P. stutzeri SDM. The 2-OBA concentration decreased with any increase beyond this cell concentration.Open in a separate windowFIG. 3.Optimization of the biocatalysis conditions. (a) Effect of pH on 2-OBA production activity. (b) Effect of 2-HBA concentrations on 2-OBA production activity. (c) Effect of the concentration of P. stutzeri SDM on biotransformation. OD, optical density.After optimizing the biocatalytic conditions, we studied the biotechnological production of 2-OBA from 2-HBA by using the whole-cell catalyst P. stutzeri SDM. As shown in Fig. ​Fig.4,4, when 20 g (DCW) liter⁻¹ P. stutzeri SDM was used as the biocatalyst, 48.5 g liter⁻¹ 2-HBA was biotransformed into 44.4 g liter⁻¹ 2-OBA in 24 h.Open in a separate windowFIG. 4.Time course of production of 2-OBA from 2-HBA under the optimum conditions. Symbols: ▪, 2-OBA; •, 2-HBA.Biocatalytic production of 2-OBA was carried out using crotonic acid, propionaldehyde, 1,2-butanediol, or threonine as the substrate (2, 7, 8, 12). Resting cells of the strain Rhodococcus erpi IF0 3730 produced 15.7 g liter⁻¹ 2-OBA from 20 g liter⁻¹ 1,2-butanediol, which is the highest reported yield of 2-OBA to date (8). By using the whole-cell catalyst P. stutzeri SDM, it was possible to produce 2-OBA at a high concentration (44.4 g liter⁻¹) and a high yield (91.5%). Due to the simple composition of the biocatalytic system (see Fig. S5 in the supplemental material), 2-HBA and 2-OBA could be easily separated on a column using a suitable resin. Separation of 2-OBA from the biocatalytic system was relatively inexpensive. The biocatalytic process presented in this report could be a promising alternative for the biotechnological production of 2-OBA.      

Assessing the Potential of an Induced-Mutation Strategy for Avermectin Overproducers

Mutant libraries of avermectin-producing Streptomyces avermitilis strains were constructed by different mutagenesis strategies. A metric was applied to assess the mutation spectrum by calculating the distribution of average phenotypic distance of each population. The results showed for the first time that a microgravity environment could introduce larger phenotype distribution and diversity than UV and N-methyl-N-nitro-N-nitrosoguanidine (NTG) could.Induced mutagenesis is a classical and successful method for improving strains to increase the productivity of commercially significant microbial metabolites. To evaluate different induced-mutagenesis approaches, Klein-Marcuschamer and Stephanopoulos presented a metric based on the quantification of phenotypic diversity to evaluate strain improvement approaches (14).New approaches of inducing mutagenesis emerged with the development of biotechnology, and of these new approaches, spaceflight-induced mutagenesis has led to great progress in strain improvement (6, 15, 26). In outer space, cosmic rays, high vacuum, intense magnetic field, and microgravity induced chromosomal aberrations, which lead to genetic mutations in microorganisms (13). However, it is difficult to carry out spaceflight-induced mutagenesis extensively owing to the limitations of high cost and few chances to board spaceships. Therefore, ground-based simulated experiments have greater practical significance, and high-magnetogravity experiments are a good choice to simulate the space environment (16).Avermectins and its analogues, produced by Streptomyces avermitilis, are major commercial antiparasitic agents for animal health, agriculture, and human infections (7). A variety of mutagenesis methods have been developed to increase the productivity of S. avermitilis (18, 19, 21-23, 25). Though most of them can produce higher mutation rates, the potential of their success in strain improvement is different.In this study, mutant libraries of S. avermitilis strains were constructed by three mutagenesis-inducing strategies: UV, N-methyl-N-nitro-N-nitrosoguanidine (NTG), and high-magnetogravitational environment (HMGE). For each population, the distribution of average phenotypic distance was calculated on the basis of the modified version (15) of the metric of Klein-Marcuschamer and Stephanopoulos (14). The mutation rate was also calculated. A good correlation between the distribution of average phenotypic distance and the percent improvement was found and analyzed. In this way, the potential to produce mutations among different induced-mutagenesis approaches was evaluated to find the most effective one for S. avermitilis.The industrial avermectin-producing S. avermitilis 3-115 strain and the mutants derived from strain 3-115 were grown on YMG agar medium (10). For diversity quantification and preliminary screening, fermentation was carried out in high-throughput format at 28°C. For confirmation of results and secondary screening, mutants that exhibited a higher yield than the wild-type strain were inoculated into shake flasks (10, 11). A high-magnetogravitational experimental platform using the large gradient superconducting magnet was described in detail by Qian et al. (20).The mutant libraries were prepared from S. avermitilis 3-115 by three different mutagenesis strategies. Spore suspensions were prepared in sterile water (10⁶ spores/ml). For UV-induced mutagenesis, 4-ml aliquots of the spore suspension were transferred into sterile petri dishes with a diameter of 80 mm. The petri dishes were then exposed to UV light in a UV-dispensing cabinet fitted with 15-W lamps with about 90% of its radiation at 265 nm. The dishes were placed at a distance of 30.0 cm away from the center of the UV light source and exposed to UV light for 15, 30, 45, 60, 75, and 90 s. The UV-exposed aliquots were then stored in the dark overnight to avoid photoreactivation. For NTG-induced mutagenesis, 9 ml of the spore suspension was added to 1 ml of a sterile solution of NTG (3 mg/ml NTG in phosphate buffer; solution freshly prepared 1 h before use). The samples were shaken at 28°C for 30 min and immediately centrifuged for 10 min at 5,000 rpm, and the supernatant was decanted. The cells were washed three times with sterile water and resuspended in 10 ml of sterile phosphate buffer (pH 7.2). All of the experimental samples were serially diluted with sterile water and plated on YMG plates. For HMGE-induced mutagenesis, the superconducting magnet generated three different magnetic force fields in different places, which corresponded to three apparent levels of gravity (0, 1, and 2 g) and two magnetic induction intensities (12 and 16 T). Specifically, there were three treatment groups in this study: 0-g group (0 g, 12 T), 2-g group (2 g, 12 T), and 1-g group (1 g, 16 T). The YMG plates (plated with 4 ml of spore suspension) were placed at the corresponding places in the platform for 7 days at 28°C to simulate strains grown in space. For all of above induced-mutagenesis experiments, when single colonies were visible on YMG agar medium, they were transferred to 96-well plates for cultivation.To quantify avermectin production, UV absorbance of culture was measured on a multiplate reader (11), and all experiments were repeated twice except where specifically noted. More than 165 clones from each library were screened. The phenotypic distributions of five different populations (including the control) were quantified. We used the optical density at 245 nm (OD₂₄₅) of the culture as the phenotype for diversity quantification of each mutant library. All data were analyzed with MATLAB (MathWorks, USA). Average phenotypic distance (d) was calculated as follows: (1) where the brackets indicate an average over all pairs of members of the population (colonies i and j) and P_i is the phenotype of colony i and P_j is the phenotype of colony j. In this case, the logarithm of the OD₂₄₅ was used as the phenotypic measure (O_i) because it was found to be lognormally distributed: (2) The strains from improved wells with respect to the control OD₂₄₅ were cultured in shake flasks to verify the results. The production of avermectins was determined by high-performance liquid chromatography (HPLC) (Agilent 1200) (8, 11). The positive mutants were defined as the strains that showed increased avermectin B1a production (increased by more than 10%) compared to the original strains. The negative mutants were defined as the strains that showed decreased avermectin B1a production (decreased by more than 10%) compared with the original strains. The mutation rate was calculated as the number of either positive or negative mutants divided by the total number of screened mutants, and the calculation was based on the results of preliminary screening.The distributions of the average phenotypic distances of five different populations were calculated. Bootstrapping was used to derive these distributions, and the results were displayed in a histogram (Fig. ​(Fig.11 a). To see the statistical significance of the difference between the different groups, the mean and standard deviation of each histogram in Fig. ​Fig.1a1a were calculated (Fig. ​(Fig.1b).1b). Homogeneous populations had small average phenotypic distances, whereas diverse populations had larger ones. Larger distance implied larger phenotypic dissimilarity among members of a population. Figure 1a and b showed that the phenotypic diversities in decreasing order were HMGE, NTG, and UV. Among HMGE-induced-mutagenesis libraries, phenotypic diversities in decreasing order were 0-g group (0 g, 12 T), 2-g group (2 g, 12 T), and 1-g group (1 g, 16 T) (Fig. 1a and b). For comparison, the traditional evaluation index, the mutation rate, was also calculated (Fig. ​(Fig.1c).1c). By using the mutation rate, the positive mutation rate of NTG was the highest, while that of UV was the lowest. Among HMGE-induced-mutagenesis libraries, the mutation rates in decreasing order were 1-g group (1 g, 16 T), 2-g group (2 g, 12 T), and 0-g group (0 g, 12 T) (Fig. ​(Fig.1c1c).Open in a separate windowFIG. 1.Avermectin production spectrum evaluation of five different mutagenized S. avermitilis populations and the untreated control. Mutations were induced by UV, NTG (N-methyl-N-nitro-N-nitrosoguanidine), or high-magnetogravitational environment (HMGE) treatment. There were three HMGE groups: 0-g group (0 g, 12 T), 2-g group (2 g, 12 T), and 1-g group (1 g, 16 T). (a) Bars in a histogram that reflect the probabilistic distributions of the estimated average phenotypic distance (d in equation 1). (b) To assess the statistical significance of this metric more directly, the mean and standard deviation of each bar in the histogram in panel a were calculated. (c) Mutation rates of the five populations.Figure ​Figure22 shows the percentages of mutants that exhibited higher yield in both preliminary and secondary screening (percent improved), considering the instability of mutants in induced mutagenesis. The results parallel the findings of the diversity metric (Fig. ​(Fig.1b),1b), not the mutation rate (Fig. ​(Fig.1c).1c). To investigate the predictability of the divergence for improved phenotypes, the correlation between the divergence (average phenotype distance) and the occurrence of a mutant with improved production was investigated. Figure ​Figure33 shows the correlation between divergence and the percent improved. A sigmoid fit and a correlation of R² = 1 were obtained. The results indicated that for medium divergence (0.6 < divergence < 0.8), improved diversity increased rapidly with the probability of isolating mutants with improved phenotype, while for divergence which was less than 0.6 or more than 0.8, the correlation of divergence with the probability of finding improved mutants was relatively low.Open in a separate windowFIG. 2.Percentage of improved mutants that produce 10% more avermectin B1a than the parent strain. The percentage represents the fraction of “successful” screening events (produce more avermectin B1a on secondary screening) and is a measure of the probability of finding improved mutants in a population.Open in a separate windowFIG. 3.Correlation of divergence and percentage of improved mutants. A sigmoid fit was used (equations shown in the figure).Traditionally, the positive mutation rate is used to evaluate mutation spectrum. However, the positive mutation rate describes only the mutations that occur and cannot describe the extent of mutation or how broad the mutation spectrum is. Therefore, in this study, divergence of mutant libraries was calculated to assess the mutation effect, and divergence was used to evaluate the effect of different induced-mutation strategies, including HMGE, a new spaceflight-simulated mutation strategy. The results of this study indicated the following. (i) HMGE-induced mutagenesis enhanced average phenotype distance and diversity better than UV and NTG mutagenesis did for S. avermitilis. (ii) Microgravity introduced the largest diversity in the genome of S. avermitilis under HMGE conditions. (iii) For medium divergence (0.6 < divergence < 0.8), improved diversity increased the probability of isolating mutants with improved phenotype.NTG was far more efficient than UV irradiation, a conclusion that many other researchers have reached; however, in a study on the efficiency of mutagenesis on spectinomycin resistance in Streptomyces fradiae, it was reported that NTG was more efficient than UV (1-5, 17). The platform of HMGE was developed to simulate the space environment, which has been reported to improve production of certain antibiotics in microorganisms (20). The results indicated that the simulated weightless environment significantly affected cell population and suggested that microgravity may cause the main mutagenic effects on the strains. Results reported here add empirical support to the hypothesis that microgravity is the most important mutagenic factor in spaceflight (12). Microgravity may increase the growth rate of microorganisms, because under microgravity conditions, oxygen in air can be supplied to microorganisms on all surfaces equally, an advantage in the production of biological matter in space (13). It has been hypothesized that microgravity may disturb the system of DNA repair, which blocks or delays the repair of DNA strand breakage. However, some researchers (24) have demonstrated that is not true, and the mutation mechanism is still not clear.Diversity has been reported to be correlated with the probability of finding improved mutants, and improved diversity would increase the probability of isolating mutants with improved phenotype (14). Results from our study partly supported this view. For medium divergence (0.6 < divergence < 0.8), there was a significant correlation between diversity and the probability of isolating mutants with improved phenotype. An optimal mutation rate, which functioned as a balance between uniqueness and retention of function, was proved to exist (9). In addition, those findings demonstrated how optimal error-prone PCR mutation rates may be calculated and indicated that “optimal” rates depended on both the protein and the mutagenesis protocol. Our results concurred with the above findings and showed that no significant correlation was detected for divergence which is less than 0.6 or more than 0.8 but that an optimal mutation rate for medium divergence (0.6 < divergence < 0.8) exists. The findings indicated the existence of a balance between mutation rate and improved phenotype, which implies that too high a mutation rate would cause dysfunction in some gene sequences, while certain mutation rates would produce a few mutated gene sequences helpful for improving productivity.     

Important Role for the Murid Herpesvirus 4 Ribonucleotide Reductase Large Subunit in Host Colonization via the Respiratory Tract

Viral enzymes that process small molecules provide potential chemotherapeutic targets. A key constraint—the replicative potential of spontaneous enzyme mutants—has been hard to define with human gammaherpesviruses because of their narrow species tropisms. Here, we disrupted the murid herpesvirus 4 (MuHV-4) ORF61, which encodes its ribonucleotide reductase (RNR) large subunit. Mutant viruses showed delayed in vitro lytic replication, failed to establish infection via the upper respiratory tract, and replicated to only a very limited extent in the lower respiratory tract without reaching lymphoid tissue. RNR could therefore provide a good target for gammaherpesvirus chemotherapy.Cellular deoxyribonucleotide synthesis is strongly cell cycle dependent. DNA viruses replicating in noncycling cells must therefore either induce cellular enzymes or supply their own. Most herpesviruses encode multiple homologs of nucleotide metabolism enzymes, including both subunits of the cellular ribonucleotide reductase (RNR) (4). While most in vivo cells are resting, most in vitro cell lines divide continuously (29). The importance of viral RNRs may therefore only be apparent in vivo (14). In contrast to alpha- and betaherpesviruses, gammaherpesviruses cause disease mainly through latency-associated cell proliferation. However, gamma-2 herpesviruses show lytic gene expression in sites of latency (9, 17), and lytic reactivation could potentially alleviate some gammaherpesvirus-infected cancers (7, 8). Therefore, it is important also to understand the pathogenetic roles of gammaherpesvirus lytic cycle enzymes, such as RNR.The known human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV) have narrow species tropisms that preclude most pathogenesis studies. In contrast, murid herpesvirus 4 (MuHV-4) (21, 26) allows gammaherpesvirus host colonization to be studied in vivo. After intranasal (i.n.) inoculation, MuHV-4 replicates lytically in lung epithelial cells before seeding to lymphoid tissue (27). Long-term virus loads are independent of extensive primary lytic spread (25). However, whether persistence requires some lytic gene expression remains unclear. Replication-deficient viral DNA reached the spleen after intraperitoneal (i.p.) but not i.n. virus inoculation (15, 20, 28), suggesting that virus dissemination from the lung to lymphoid tissue requires lytic replication. In addition, less invasive inoculations may increase further the viral functions required to establish a persistent infection. Thymidine kinase (TK)-deficient MuHV-4 given i.n. without general anesthesia, in which method the wild-type virus infects the upper respiratory tract and reaches lymphoid tissue without infecting the lungs (18), fails to colonize in mice at all (12). The implication is that virions using a likely physiological route of host entry must replicate in terminally differentiated cells to establish a significant infection. However, some unusual features of gammaherpesvirus TKs (11) suggest that they have functions besides thymidine phosphorylation. We therefore targeted here another enzyme linked to viral DNA replication, the MuHV-4 RNR. We aimed to define the in vivo importance of a potential therapeutic target and to advance generally our understanding of gammaherpesvirus pathogenesis.Transposon insertions in the MuHV-4 RNR small (ORF60) and large (ORF61) RNR subunit genes have been described as either attenuating or not for lytic replication in vitro (19, 23). We disrupted ORF61 (RNR⁻) by inserting stop codons close to its 5′ end (Fig. ​(Fig.11 a). An EcoRI-L genomic clone (coordinates 80644 to 84996) in pUC19 (6) was digested with AleI to remove nucleotides 82320 to 82534 of ORF61 (82865 to 80514). An oligonucleotide encoding multiple stop codons and an EcoRI restriction site (5′-CTAGCATGCTAGAATTCTAGCATGCATG-3′) was ligated in place. Nucleotides 81365 to 83883 were then PCR amplified, including a BamHI site in the 81365 primer, cloned as a BglII/BamHI fragment into the BamHI site of pST76K-SR, and recombined into a MuHV-4 bacterial artificial chromosome (BAC) (1). A revertant virus was made by reconstituting the corresponding, unmutated genomic fragment. Southern blots (5) of viral DNA (Fig. ​(Fig.1b)1b) confirmed the expected genomic structures, and immunoblots (5) of infected cell lysates (Fig. ​(Fig.1c)1c) established that mutant viruses no longer expressed the RNR large subunit.Open in a separate windowFIG. 1.Disruption of the MuHV-4 ORF61. (a) Schematic diagram of the ORF61 (RNR large) locus, showing the mutation introduced and relevant restriction sites. (b) Viral DNA was digested with EcoRI and probed for ORF61. Oligonucleotide insertion into ORF61 changes a 4,352-bp wild-type band to 2,462 bp plus 1,676 bp. The 2,462-bp fragment is not visible because it overlaps the probe by only 331 nucleotides (nt) and comigrates with a background band of unknown origin. WT, wild type; REV, revertant; RNR⁻, mutant; RNR⁻ ind, independent mutant. WT luc⁺ is MuHV-4 expressing luciferase from an ORF57/ORF58 intergenic cassette. RNR⁻ luc⁺ and RNR⁻ luc⁺ind have ORF61 disrupted on this background. (c) Infected cell lysates were immunoblotted for gp150 (virion envelope glycoprotein, monoclonal antibody [MAb] T1A1), ORF17 (capsid component, MAb 150-7D1), TK (tegument component, MAb CS-4A5), and ORF61 (MAb PS-8A7). (d) BHK-21 cells were infected with RNR⁺ or RNR⁻ viruses (0.01 eGFP units/cell, 2 h, 37°C), washed two times with phosphate-buffered saline (PBS) to remove unbound virions, and cultured at 37°C to allow virus spread. Infectivity (in eGFP units) at each time point was determined on fresh BHK-21 cells in the presence of phosphonoacetic acid to prevent further viral spread, with the number of eGFP-postive cells counted 18 h later by flow cytometry. (e) BHK-21 cells were infected with RNR⁺ or RNR⁻ viruses (2 eGFP units/cell, 2 h, 37°C), washed in medium (pH 3) to inactivate nonendocytosed virions, and cultured at 37°C to allow virus replication. The infectivity of replicate cultures was then assayed as described in the legend of panel d. (f) BHK-21 cells were incubated with RNR⁺ or RNR⁻ viruses (0.3 eGFP units/cell, 37°C) for the times indicated, and the numbers of eGFP-positive cells in the cultures were then determined by flow cytometry.RNR⁻ viruses were noticeably slower than RNR⁺ viruses when spreading through BHK-21 cell monolayers after BAC DNA transfection. Normalizing by immunoblot signal, RNR⁻ virus stocks had titers similar to that of the wild type by viral enhanced green fluorescent protein (eGFP) expression but 10- to 100-fold lower plaque titers. Using eGFP expression as a readout, RNR⁻ virion production after a low multiplicity of infection lagged 1 day behind that of the wild type (Fig. ​(Fig.1d).1d). Maximum infectivity yields were also reduced, but once BHK-21 cells become confluent, they support MuHV-4 lytic infection poorly, so this was probably a consequence of the slower lytic spread. After a high multiplicity of infection (Fig. ​(Fig.1e),1e), RNR⁻ mutants showed a 10-h lag in virion production and no difference in the final yield. They showed no defect in single-cycle eGFP expression (Fig. ​(Fig.1f),1f), implying normal virion entry. Therefore, the main RNR⁻ defect lay in infectious virion production.For in vivo experiments, the loxP-flanked viral BAC-eGFP cassette must be removed (1). Therefore, to monitor infection in vivo without having to rely on new virion production as a readout, we transferred the RNR mutation onto a luciferase-positive (luc⁺) background (18). Viral luciferase expression (from an early lytic promoter) by in vitro luminometry (18) was independent of either viral DNA replication or RNR expression (Fig. ​(Fig.22 a). After i.n. inoculation of anesthetized mice, RNR⁻ luciferase signals measured in vivo by i.p. luciferin injection and IVIS Lumina charge-coupled-device (CCD) camera scanning (18) were visible in lungs (Fig. ​(Fig.2b)2b) but were 100-fold lower than those of the RNR⁺ controls (Fig. ​(Fig.2c).2c). A severe impairment of RNR⁻ lytic replication was confirmed by plaque assay (18) (Fig. ​(Fig.2d);2d); the difference between RNR⁻ and RNR⁺ plaque titers greatly exceeded any difference in plaquing efficiency.Open in a separate windowFIG. 2.Host colonization by RNR⁻ MuHV-4 mutants. (a) BHK-21 cells were left uninfected or infected overnight with RNR⁺ or RNR⁻ luc⁺ MuHV-4 and then assayed for luciferase expression by luminometry. Phosphonoacetic acid (PAA; 100 μg/ml) was either added or not to cultures to block viral late gene expression. Each point shows the mean ± standard deviation from triplicate cultures. (b) BALB/c mice were infected i.n. under general anesthesia with RNR⁻ or RNR⁺ luc⁺ MuHV-4 (5 × 10³ PFU) and then assayed for luciferase expression by luciferin injection and CCD camera scanning. The images are from 5 days postinfection. Note that the RNR⁺ and RNR⁻ images have different sensitivity scales. (c) For quantitation, dorsal and ventral luciferase signals were summed. Each point shows 1 mouse. The dashed lines show detection thresholds. The RNR⁺ signal was significantly greater than the RNR⁻ signal for all sites and time points (P < 0.001 by Student''s t test). (d) C57BL/6 mice were infected i.n. under anesthesia with RNR⁻ or RNR⁺ MuHV-4 (5 × 10³ PFU). Five days later, infectious virus loads in noses and lungs were measured by plaque assay. Each point shows 1 mouse. RNR⁻ infections yielded no plaques and therefore are shown at the sensitivity limits of each assay. (e) BALB/c mice were infected i.n. with RNR⁻ or RNR⁺ MuHV-4 without anesthesia and then monitored by luciferin injection and CCD camera scanning. Each point shows the summed ventral and dorsal signals of the relevant region for 1 mouse. Neck signals correspond to the superficial cervical lymph nodes (SCLN). The dashed lines show detection thresholds. RNR⁻ luciferase signals were undetectable at all time points.No RNR⁻ luciferase signals were visible in noses, nor did RNR⁻ MuHV-4 give signals in the superficial cervical lymph nodes (SCLN), which drain the nose (Fig. ​(Fig.2c).2c). This lack of live imaging signals from the upper respiratory tract was confirmed by ex vivo imaging of SCLN at day 14 postinfection. We examined upper respiratory tract infection further with an independently derived luc⁺ RNR⁻ mutant, inoculating i.n. without anesthesia so as to avoid virus aspiration into the lungs. No RNR⁻ luciferase signals were detected, while wild-type signals were readily observed in the nose and superficial cervical lymph nodes (Fig. ​(Fig.2e2e).Like RNR⁻ MuHV-4, TK⁻ mutants are severely attenuated for lytic replication in the lower respiratory tract. However, they eventually establish a reactivatable latent infection and induce virus-specific antibody (3). Latent virus titers in spleens peak at 1 month postinoculation. Infectious center assays showed no RNR⁻ infection of spleens at that time (Fig. ​(Fig.33 a). We also looked for viral DNA in spleens by quantitative PCR (Fig. ​(Fig.3b).3b). Genomic coordinates 4166 to 4252 were amplified and hybridized to a probe with coordinates 4218 to 4189. Viral genome copies, relative to the cellular adenosine phosphoribosyl transferase copy number, were calculated from standard curves of cloned plasmid DNA (10). No RNR⁻ viral DNA was detected. ELISA for MuHV-4-specific serum IgG (24) detected an antibody response after lung infection but not upper respiratory tract infection of BALB/c mice with RNR⁻ MuHV-4 (Fig. ​(Fig.3c).3c). There was a similar lack of antibody 1 month after upper respiratory tract infection of C57BL/6 mice with independently derived RNR⁻ mutants (Fig. ​(Fig.3d)3d) and 3 months after exposure of 6 BALB/c mice to RNR⁻ luc⁺ MuHV-4. In contrast, i.p. RNR⁻ luc⁺ MuHV-4 gave lower luciferase signals than RNR⁺ luc⁺ MuHV-4 (Fig. ​(Fig.44 a), but RNR⁻ infectious centers (Fig. ​(Fig.4b)4b) and viral genomes (Fig. ​(Fig.4c)4c) were detected in spleens, and enzyme-linked immunosorbent assays (ELISAs) (Fig. ​(Fig.4d)4d) showed MuHV-4-specific serum IgG.Open in a separate windowFIG. 3.Spleen colonization by RNR⁻ MuHV-4. (a) BALB/c or C57BL/6 mice were infected i.n. either with general anesthesia (lung infection) or without (nose infection). One month later, spleens were assayed for recoverable latent virus by infectious center assay. Lower detection limit, 10 infectious centers per spleen. (b) The spleens described in the legend of panel a were further analyzed for viral DNA by quantitative PCR. Copy numbers are expressed relative to the cellular adenosine phosphoribosyl transferase copy number in each sample. The dashed lines show lower detection limits (1 viral copy/10,000 cellular copies). (c) Sera from BALB/c mice after i.n. infection either with (lung infection) or without (nose infection) general anesthesia were assayed for MuHV-4-specific IgG by ELISA. Each line shows the absorbance curve for 1 mouse. The dashed lines show naive serum. (d) Sera from C57BL/6 mice 1 month after infection with independent RNR mutants were analyzed for MuHV-4-specific IgG, as described in the legend to panel c.Open in a separate windowFIG. 4.Intraperitoneal infection with RNR⁺ and RNR⁻ MuHV-4. (a) Mice were infected i.p. with RNR⁻ luc⁺ or RNR⁺ luc⁺ MuHV-4 and then monitored for luciferase expression. Each point shows the total abdominal signal of 1 mouse. The x axis is at the lower limit of signal detection above the background. (b) Spleens were assayed for recoverable virus by infectious center assay 10 days after i.p. infection with RNR⁻ luc⁺ or RNR⁺ luc⁺ MuHV-4. Each point shows the titer of 1 mouse. One log₁₀ infectious center per mouse corresponds to the lower limit of detection. (c) Spleen DNA was analyzed for viral genome content by quantitative PCR. Each point shows viral copy/cellular copy for the mean of triplicate reactions for 1 mouse. (d) Sera taken 10 days after i.p. infection with RNR⁻ luc⁺ or RNR⁺ luc⁺ MuHV-4 were assayed for MuHV-4-specific IgG by ELISA. Each line shows the absorbance values for the serum of 1 mouse. “Naive” represents age-matched, uninfected controls.The failure of both the RNR large subunit (ORF61) and TK⁻ MuHV-4 mutants to infect via the upper respiratory tract argues that this requires viral replication in a nucleotide-poor cell. The additional lack of lymphoid RNR⁻ infection after inoculation into the lungs seemed likely to reflect a defect in virus transport, as RNR⁻ MuHV-4 did colonize the spleen after i.p. inoculation. It is also possible that the first cells infected simply produced no infectious virions, although this seemed a more likely explanation for upper respiratory tract infection being undetectable; lung infection progressed sufficiently to give detectable luciferase expression and to induce an antiviral antibody response. How transport from lung to lymphoid tissue occurs is unknown, but likely scenarios include latently infected dendritic cells (22) carrying MuHV-4 along afferent lymphatics to germinal centers and cell-free virions being captured in lymph nodes by subcapsular sinus macrophages (13). Therefore, RNR may be important for MuHV-4 to spread from myeloid cells to B cells.The difference between RNR⁻ and TK⁻ mutants in host colonization via the lung—TK⁻ mutants reached lymphoid tissue whereas RNR⁻ mutants did not—could reflect additional ORF61 functions, as precedent exists for functional drift (2, 16). Alternatively, RNR may be needed more than TK for MuHV-4 replication in some cell types. Formidable hurdles to RNR-based therapies remain: human gammaherpesvirus infections rarely present until latency is well established, so blocking virus spread to lymphoid tissue may have a limited impact, and no drugs sufficiently selective to target viral RNRs in a clinical setting have yet emerged. Nevertheless, the severe in vivo attenuation of RNR⁻ MuHV-4 suggested that RNR may be a viable target for limiting gammaherpesvirus lytic spread.     

Streptomyces Development in Colonies and Soils

Streptomyces development was analyzed under conditions resembling those in soil. The mycelial growth rate was much lower than that in standard laboratory cultures, and the life span of the previously named first compartmentalized mycelium was remarkably increased.Streptomycetes are gram-positive, mycelium-forming, soil bacteria that play an important role in mineralization processes in nature and are abundant producers of secondary metabolites. Since the discovery of the ability of these microorganisms to produce clinically useful antibiotics (2, 15), they have received tremendous scientific attention (12). Furthermore, its remarkably complex developmental features make Streptomyces an interesting subject to study. Our research group has extended our knowledge about the developmental cycle of streptomycetes, describing new aspects, such as the existence of young, fully compartmentalized mycelia (5-7). Laboratory culture conditions (dense inocula, rich culture media, and relatively elevated temperatures [28 to 30°C]) result in high growth rates and an orderly-death process affecting these mycelia (first death round), which is observed at early time points (5, 7).In this work, we analyzed Streptomyces development under conditions resembling those found in nature. Single colonies and soil cultures of Streptomyces antibioticus ATCC 11891 and Streptomyces coelicolor M145 were used for this analysis. For single-colony studies, suitable dilutions of spores of these species were prepared before inoculation of plates containing GYM medium (glucose, yeast extract, malt extract) (11) or GAE medium (glucose, asparagine, yeast extract) (10). Approximately 20 colonies per plate were obtained. Soil cultures were grown in petri dishes with autoclaved oak forest soil (11.5 g per plate). Plates were inoculated directly with 5 ml of a spore suspension (1.5 × 10⁷ viable spores ml⁻¹; two independent cultures for each species). Coverslips were inserted into the soil at an angle, and the plates were incubated at 30°C. To maintain a humid environment and facilitate spore germination, the cultures were irrigated with 3 ml of sterile liquid GAE medium each week.The development of S. coelicolor M145 single colonies growing on GYM medium is shown in Fig. ​Fig.1.1. Samples were collected and examined by confocal microscopy after different incubation times, as previously described (5, 6). After spore germination, a viable mycelium develops, forming clumps which progressively extend along the horizontal (Fig. 1a and b) and vertical (Fig. 1c and d) axes of a plate. This mycelium is fully compartmentalized and corresponds to the first compartmentalized hyphae previously described for confluent surface cultures (Fig. 1e, f, and j) (see below) (5); 36 h later, death occurs, affecting the compartmentalized hyphae (Fig. 1e and f) in the center of the colony (Fig. ​(Fig.1g)1g) and in the mycelial layers below the mycelial surface (Fig. 1d and k). This death causes the characteristic appearance of the variegated first mycelium, in which alternating live and dead segments are observed (Fig. 1f and j) (5). The live segments show a decrease in fluorescence, like the decrease in fluorescence that occurs in solid confluent cultures (Fig. ​(Fig.11 h and i) (5, 9). As the cycle proceeds, the intensity of the fluorescence in these segments returns, and the segments begin to enlarge asynchronously to form a new, multinucleated mycelium, consisting of islands or sectors on the colony surfaces (Fig. 1m to o). Finally, death of the deeper layers of the colony (Fig. ​(Fig.1q)1q) and sporulation (Fig. ​(Fig.1r)1r) take place. Interestingly, some of the spores formed germinate (Fig. ​(Fig.1s),1s), giving rise to a new round of mycelial growth, cell death, and sporulation. This process is repeated several times, and typical, morphologically heterogeneous Streptomyces colonies grow (not shown). The same process was observed for S. antibioticus ATCC 11891, with minor differences mainly in the developmental time (not shown).Open in a separate windowFIG. 1.Confocal laser scanning fluorescence microscopy analysis of the development-related cell death of S. coelicolor M145 in surface cultures containing single colonies. Developmental culture times (in hours) are indicated. The images in panels l and n were obtained in differential interference contrast mode and correspond to the same fields as in panels k and m, respectively. The others are culture sections stained with SYTO 9 and propidium iodide. Panels c, d, k, l, p, and q are cross sections; the other images are longitudinal sections (see the methods). Panels h and i are images of the same field taken with different laser intensities, showing low-fluorescence viable hyphae in the center of the colonies that develop into a multinucleated mycelium. The arrows in panels e and s indicate septa (e) and germinated spores (s). See the text for details.Figure ​Figure22 shows the different types of mycelia present in S. coelicolor cultures under the conditions described above, depending on the compartmentalization status. Hyphae were treated with different fluorescent stains (SYTO 9 plus propidium iodide for nucleic acids, CellMask plus FM4-64 for cell membranes, and wheat germ agglutinin [WGA] for cell walls). Samples were processed as previously described (5). The young initial mycelia are fully compartmentalized and have membranous septa (Fig. 2b to c) with little associated cell wall material that is barely visible with WGA (Fig. ​(Fig.2d).2d). In contrast, the second mycelium is a multinucleated structure with fewer membrane-cell wall septa (Fig. 2e to h). At the end of the developmental cycle, multinucleated hyphae begin to undergo the segmentation which precedes the formation of spore chains (Fig. 2i to m). Similar results were obtained for S. antibioticus (not shown), but there were some differences in the numbers of spores formed. Samples of young and late mycelia were freeze-substituted using the methodology described by Porta and Lopez-Iglesias (13) and were examined with a transmission electron microscope (Fig. 2n and o). The septal structure of the first mycelium (Fig. ​(Fig.2n)2n) lacks the complexity of the septal structure in the second mycelium, in which a membrane with a thick cell wall is clearly visible (Fig. ​(Fig.2o).2o). These data coincide with those previously described for solid confluent cultures (4).Open in a separate windowFIG. 2.Analysis of S. coelicolor hyphal compartmentalization with several fluorescent indicators (single colonies). Developmental culture times (in hours) are indicated. (a, e, and i) Mycelium stained with SYTO 9 and propidium iodide (viability). (b, f, and j) Hyphae stained with Cell Mask (a membrane stain). (c, g, and l) Hyphae stained with FM 4-64 (a membrane stain). (d, h, and m) Hyphae stained with WGA (cell wall stain). Septa in all the images in panels a to j, l, and m are indicated by arrows. (k) Image of the same field as panel j obtained in differential interference contrast mode. (n and o) Transmission electron micrographs of S. coelicolor hyphae at different developmental phases. The first-mycelium septa (n) are comprised of two membranes separated by a thin cell wall; in contrast, second-mycelium septa have thick cell walls (o). See the text for details. IP, propidium iodide.The main features of S. coelicolor growing in soils are shown in Fig. ​Fig.3.3. Under these conditions, spore germination is a very slow, nonsynchronous process that commences at about 7 days (Fig. 3c and d) and lasts for at least 21 days (Fig. 3i to l), peaking at around 14 days (Fig. 3e to h). Mycelium does not clump to form dense pellets, as it does in colonies; instead, it remains in the first-compartmentalized-mycelium phase during the time analyzed. Like the membrane septa in single colonies, the membrane septa of the hyphae are stained with FM4-64 (Fig. 3j and k), although only some of them are associated with thick cell walls (WGA staining) (Fig. ​(Fig.3l).3l). Similar results were obtained for S. antibioticus cultures (not shown).Open in a separate windowFIG. 3.Confocal laser scanning fluorescence microscopy analysis of the development-related cell death and hyphal compartmentalization of S. coelicolor M145 growing in soil. Developmental culture times (in days) are indicated. The images in panels b, f, and h were obtained in differential interference contrast mode and correspond to the same fields as the images in panels a, e, and g, respectively. The dark zone in panel h corresponds to a particle of soil containing hyphae. (a, c, d, e, g, i, j, and k) Hyphae stained with SYTO 9, propidium iodide (viability stain), and FM4-64 (membrane stain) simultaneously. (i) SYTO 9 and propidium iodide staining. (j) FM4-64 staining. The image in panel k is an overlay of the images in panels i and j and illustrates that first-mycelium membranous septa are not always apparent when they are stained with nucleic acid stains (SYTO 9 and propidium iodide). (l) Hyphae stained with WGA (cell wall stain), showing the few septa with thick cell walls present in the cells. Septa are indicated by arrows. IP, propidium iodide.In previous work (8), we have shown that the mycelium currently called the substrate mycelium corresponds to the early second multinucleated mycelium, according to our nomenclature, which still lacks the hydrophobic layers characteristic of the aerial mycelium. The aerial mycelium therefore corresponds to the late second mycelium which has acquired hydrophobic covers. This multinucleated mycelium as a whole should be considered the reproductive structure, since it is destined to sporulate (Fig. ​(Fig.4)4) (8). The time course of lysine 6-aminotransferase activity during cephamycin C biosynthesis has been analyzed by other workers using isolated colonies of Streptomyces clavuligerus and confocal microscopy with green fluorescent protein as a reporter (4). A complex medium and a temperature of 29°C were used, conditions which can be considered similar to the conditions used in our work. Interestingly, expression did not occur during the development of the early mycelium and was observed in the mycelium only after 80 h of growth. This suggests that the second mycelium is the antibiotic-producing mycelium, a hypothesis previously confirmed using submerged-growth cultures of S. coelicolor (9).Open in a separate windowFIG. 4.Cell cycle features of Streptomyces growing under natural conditions. Mycelial structures (MI, first mycelium; MII, second mycelium) and cell death are indicated. The postulated vegetative and reproductive phases are also indicated (see text).The significance of the first compartmentalized mycelium has been obscured by its short life span under typical laboratory culture conditions (5, 6, 8). In previous work (3, 7), we postulated that this structure is the vegetative phase of the bacterium, an hypothesis that has been recently corroborated by proteomic analysis (data not shown). Death in confluent cultures begins shortly after germination (4 h) and continues asynchronously for 15 h. The second multinucleated mycelium emerges after this early programmed cell death and is the predominant structure under these conditions. In contrast, as our results here show, the first mycelium lives for a long time in isolated colonies and soil cultures. As suggested in our previous work (5, 6, 8), if we assume that the compartmentalized mycelium is the Streptomyces vegetative growth phase, then this phase is the predominant phase in individual colonies (where it remains for at least 36 h), soils (21 days), and submerged cultures (around 20 h) (9). The differences in the life span of the vegetative phase could be attributable to the extremely high cell densities attained under ordinary laboratory culture conditions, which provoke massive differentiation and sporulation (5-7, 8).But just exactly what are “natural conditions”? Some authors have developed soil cultures of Streptomyces to study survival (16, 17), genetic transfer (14, 17-19), phage-bacterium interactions (3), and antibiotic production (1). Most of these studies were carried out using amended soils (supplemented with chitin and starch), conditions under which growth and sporulation were observed during the first few days (1, 17). These conditions, in fact, might resemble environments that are particularly rich in organic matter where Streptomyces could conceivably develop. However, natural growth conditions imply discontinuous growth and limited colony development (20, 21). To mimic such conditions, we chose relatively poor but more balanced carbon-nitrogen soil cultures (GAE medium-amended soil) and less dense spore inocula, conditions that allow longer mycelium growth times. Other conditions assayed, such as those obtained by irrigating the soil with water alone, did not result in spore germination and mycelial growth (not shown). We were unable to detect death, the second multinucleated mycelium described above, or sporulation, even after 1 month of incubation at 30°C. It is clear that in nature, cell death and sporulation must take place at the end of the long vegetative phase (1, 17) when the imbalance of nutrients results in bacterial differentiation.In summary, the developmental kinetics of Streptomyces under conditions resembling conditions in nature differs substantially from the developmental kinetics observed in ordinary laboratory cultures, a fact that should be born in mind when the significance of development-associated phenomena is analyzed.     

ARG1 and ARL2 form an actin-based gravity-signaling chaperone complex in root statocytes?

Plants are acutely sensitive to the directional information provided by gravity. They have evolved statocytes, which are specialized cells that sense gravity and, upon integration of the corresponding information with that of other environmental stimuli, control the growth behavior of their organs. The cellular mechanisms that allow statocytes to sense and transduce gravitational information likely involve detecting the sedimentation of, or the tension/pressure exerted by, starch-filled amyloplasts—the presumptive statoliths—within their cytoplasm. Gravity signaling in root statocytes controls the direction of transport of signaling compounds, especially auxin, across the root cap, establishing a lateral gradient that is transmitted to cells in the elongation zone and results in gravitropic curvature. The Arabidopsis J-domain proteins ARG1 and ARL2 function as gravity-signal transducers in root statocytes. In the January issue of The Plant Journal, we reported that ARG1 and ARL2 function non-redundantly in a common gravity signaling pathway required for accumulation of the auxin efflux facilitator PIN3 on the new bottom side of statocytes following gravity stimulation, and lateral redistribution of auxin toward the new lower flank of stimulated roots. Here we present data suggesting that ARG1 physically associates with ARL2, the J-domain co-chaperone HSC70, and actin in vivo. We briefly discuss potential mechanisms by which ARG1 and ARL2 might function in gravity signaling in light of this information.Key words: gravitropism, statocytes, actin, HSC70/HSP70, auxinGravitropic growth in plants requires the ability to sense an organ''s orientation within the gravity field, transduce that information into a biochemical signal, and mount a differential cellular-elongation response that corrects growth to follow a defined gravity set point angle. Gravity sensation in roots occurs mainly in statocytes of the root cap columella, either via detection of the position or movement of, or the pressure exerted by, amyloplasts within these cells, or by perceiving the pressure exerted by the statocyte''s protoplast within its cell wall.^1,2 Several models address the mechanism by which statocytes use amyloplasts as gravity susceptors. One model postulates that gravity signal transduction initiates when sedimenting amyloplasts promote the opening of mechano-sensitive ion channels, either directly or through interaction with the actin cytoskeleton. Alternatively, signal transduction may initiate upon sedimentation, when amyloplast-borne ligands interact with receptors located on sensitive structures within the statocytes.¹ Nevertheless, the activated pathway promotes a fast and transient cytoplasmic alkalinization and redistribution of the auxin efflux carrier PIN3 to the lower membrane of the statocytes. These changes contribute to lateral, downward, redistribution of auxin across the cap, resulting in the formation of a lateral auxin gradient that, upon transmission to the elongation zone, promotes tip curvature (reviewed in ref. ¹).Along with experiments demonstrating the importance of amyloplasts in gravity susception, genetic analysis in Arabidopsis has identified ARG1 and ARL2 as gravity signal transducers in root and hypocotyl statocytes.^3–6 ARG1 and ARL2 function in a gravity-signaling pathway that links gravistimulation to the accumulation of PIN3 within the PM at the lower side of the statocytes and a redistribution of auxin across the cap.^4–6 ARG1 and ARL2 are membrane-associated proteins. While ARG1 localizes throughout the endosomal/secretory pathway, ARL2 associates primarily with the PM, and both proteins are found at the cell plate during cytokinesis.^4,6 ARG1 and ARL2 contain J-domains near their N-termini. J-domain proteins typically function as molecular co-chaperones by interacting with HSC70, coupling substrate binding to HSC70 ATPase activity.¹⁰ A direct role for HSC70 in gravity signaling has not been reported, though proteomics approaches have identified cytosolic HSC70 isoforms as gravity-regulated proteins within Arabidopsis root tips.^7,8The C-termini of ARG1 and ARL2 contain putative coiled-coil domains with similarity to predicted coiled-coils found in proteins that interact with the cytoskeleton, including Rho-associated kinases, myosin, heavy chain kinesins and tropomyosins.³ Interestingly, a biochemical fractionation experiment detected possible interaction between ARG1 and polymerized actin in plant extracts.⁴ Furthermore, proteomic analyses of root gravitropism also identified actin as a gravity-regulated protein^7,8 whereas other studies strongly suggest a role for the actin cytoskeleton in the modulation of gravity signaling in roots.⁹To investigate possible in vivo interactions between ARG1, ARL2, HSC70 and/or actin, we generated transgenic plants expressing GFP fusions to ARG1 or ARL2, in the arg1-2 or arl2-3 mutant backgrounds (Ws alleles), and used them in co-immunoprecipitation experiments. All constructs were tested for functional rescue prior to use. Functional GFP fusions to ARG1 or ARL2 were isolated from plant extracts using immobilized anti-GFP antibodies. Initial experiments indicated that both N and C-terminal GFP fusions to ARG1 and C-terminal fusions to ARL2 could be purified by this method (Fig. 1A). However, we consistently noticed lower relative abundance of ARL2-GFP purified from plants compared to ARG1-GFP, even when both were expressed under the same promoter (Fig. 1A). This is consistent with lower fluorescence intensity and fusion protein abundance of ARL2-GFP compared to ARG1-GFP in the many transgenic lines analyzed (data not shown).Open in a separate windowFigure 1HSC70, actin and ARL2 co-purify with ARG1 from plant extracts. Total protein extracts from four-week-old liquid grown plants of the indicated genotypes (top) were affinity-purified using anti-GFP antibodies (gift of Richard Vierstra, UW-Madison) cross-linked to Protein A-Sepharose beads and detected with the indicated antibodies. In (A) the affinity-purified eluate was probed with anti-GFP antibody to indicate the presence of both full-length GFP fusions and breakdown products. In (B) western blots of total protein extracts (Input, In) and affinity-purified eluates (E) were probed with anti-HSC70 (diluted 1:10,000; Stressgen, San Diego, CA), monoclonal anti-actin (diluted 1:1000; clone C4 from Chemicon Int., Temecula, CA), or anti-a-tubulin antibodies (Sigma, St Louis, MO). In (C) western blots of affinity-purified eluates (top) were probed with two anti-ARG1 antibodies raised in rabbits against a bacterially expressed His6-ARG1 protein fusion (anti-ARG1 I) and against a peptide corresponding to the 15 C-terminal residues of ARG1 (anti-ARG1 II), both used at 1:1000 dilutions, as well as with a polyclonal anti-ARL2 antibody raised in rabbits against a synthetic ARL2 peptide, used at 1:3000 dilution. Note that anti-ARG1 II did not recognize the ARG1-GFP protein probably because the corresponding C-terminal epitope was masked in this C-terminal fusion. A western blot of total protein extracts used for affinity purification (In, bottom) was probed with anti-HSP70 antibody (gift of Elizabeth Craig, UW-Madison; 1:20,000 dilution) to show relative amounts of protein prior to extraction. Experimental procedures. Plants expressing ARG1::GFP-ARG1 and 35S::ARL2-GFP are described elsewhere (reviewed in refs. ⁴ and ⁶, respectively). Plants expressing 35S::ARG1-GFP were generated by transformation with a T-DNA derived from the binary vector pK7FWG2²⁵ containing the ARG1 cDNA, whereas plants expressing AtMDR1::GFP were a gift from Edgar Spalding (UW-Madison). For immunoprecipitation, anti-GFP serum was cross-linked to Protein A Sepharose (Amersham, Piscataway, NJ) using the cross-linking reagent disuccinimidyl suberate according to the manufacturer''s recommendations (Pierce, Rockford, Ill.). Extracts from liquid-grown plants were generated by grinding in 1:1 (vol:wt) ice cold grinding buffer [20 mM Tris, 1 mM EDTA, 100 mM NaCl, 0.1% Triton X-100 pH7.5, containing 1:100 dilution of protease inhibitor cocktail (Sigma, or Calbiochem, San Diego, CA) and 1 mM phenylmethylsulphonyl fluoride] in a mortar, then adding more buffer to a ratio of 4:1 (vol:wt). Homogenates were cleared by filtering through Miracloth (Calbiochem) and spinning twice at 13,000 g for 15 min each to pellet cellular debris. Protein concentration in the resulting supernatant was then quantified by a modified Lowry assay, using BSA as a standard. Extracts were gently rocked with anti-GFP beads for one hour at room temperature. The beads were washed extensively (five times with ≥5× bead vol.) in extraction buffer, and the bound protein was eluted with low pH (0.1 M glycine pH 2.5) into a small amount of neutralizing 1 M tris buffer (pH 8.0), precipitated with TCA, separated by SDS-PAGE, blotted to PVDF membrane (Millipore, Billerica, MA), and probed with the appropriate antibodies. Positive signals were detected using HRP-conjugated secondary antibodies at 1:20,000 (Sigma).Proteins that interact with ARG1 or ARL2 are expected to bind to the anti-GFP-coated beads in this assay, along with their GFP-ARG1 or GFP-ARL2 partner, and to co-elute with them upon washing the beads with a low-pH solution. Their identity can be revealed by western blot analysis of bead eluates, using specific antibodies. Figure 1B shows that HSC70 co-purifies with ARG1-GFP and GFP-ARG1 when extracts from plants expressing these fusions are tested in this assay. However, HSC70 was not present in the eluate when plants expressing GFP alone under the control of the CaMV35S or AtMDR1 ectopic promoters were tested (Fig. 1B). Interestingly, we did not detect HSC70 in bead eluates when ARL2-GFP-expressing plants were subjected to this co-immunoprecipitation assay, suggesting that ARG1 and ARL2 differ in their affinity for HSC70. It should however be noted that the low amount of ARL2-GFP precipitated in these experiments does not allow for direct comparison (Fig. 1B). These results indicate an in vivo interaction between ARG1 and HSC70, suggesting that ARG1 is a bona fide co-chaperone.Figure 1B also shows that actin is present specifically in immunocomplexes containing ARG1. In a parallel experiment, ARL2-GFP precipitates did not contain detectable actin (Fig. 1B). However, here again, the low amount of ARL2 in these precipitates does not allow direct comparison to ARG1. Together these data suggest that ARG1 physically associates with actin in vivo, even though they do not address whether this association involves monomeric or polymerized actin. Interestingly, the biochemical fractionation experiments reported by Boonsirichai et al. (2003) suggested that some ARG1 might associate with polymerized actin in vivo.Plants with mutations in ARG1, ARL2, or both, result in very similar gravity signaling defects, demonstrating that ARG1 and ARL2 function as non-redundant members of the same signaling pathway.⁵ One interpretation of this data is that ARG1 and ARL2 function in the same signaling complex, the presence of both being required for complex activity in gravity signaling. In support of this model, we have detected an in vivo interaction between ARG1 and ARL2 by immunoprecipitation. Figure 1C shows that endogenous ARG1 (∼45 kDa) co-immunoprecipitates with ARL2-GFP from extracts derived from plants expressing 35S::ARL2-GFP, but not from extracts derived from negative control plants. We were not surprised by the failure to detect ARG1 in precipitates from plants expressing ARL2-GFP under the control of its endogenous promoter (ARL2::ARL2-GFP). Indeed, its expression is restricted to a few cells—therefore, the protein is present at low abundance in whole-plant extracts.⁶ Similarly, native ARL2 was not detected in ARG1 immunoprecipitates likely due to the low level of endogenous ARL2 expression. Together, these results suggest that ARG1 and ARL2 associate in a complex in vivo, consistent with the function of these proteins in a common signaling pathway.⁵The sub-cellular localization of a putative ARG1- and ARL2-containing complex is unknown, but is likely associated with the PM of root statocytes, where ARL2-GFP localizes in cells expressing ARL2::ARL2-GFP.⁶ Such a complex may serve as a membrane-associated signaling module that recruits cytosolic HSC70 to a membrane site in a gravity-regulated manner. In agreement with this model, we found that HSP70 increases in abundance in a biochemical fraction that includes membrane-associated proteins extracted from Arabidopsis root tips within 10 min of a gravistimulus.⁸ A similar recruitment model has been proposed for J-protein function in several HSC70-mediated processes, including the regulation of vesicle dynamics and signaling by HSC70.¹⁰ In animal systems, HSC70 activity is required for several steps in clatherin-dependent vesicle recycling,¹¹ a process that appears to mediate PIN protein trafficking¹². Recruitment of HSC70 activity to clatherin-coated vesicles is mediated mainly by the J-domain protein auxillin, which has paralogs other than ARG1 and ARL2 in plants.¹⁰ HSC70 function is also required for Ca²⁺-induced exocytosis in Drosophila, and depends on the J-domain-containing cysteine-string protein, apparent homologs of which are absent from plants.¹³ GRV2, a locus required for normal shoot tropisms, encodes a membrane-associated J-domain protein homologous to animal RME-8.¹⁴ RME-8 is a membrane-associated J-domain protein that interacts with cytosolic HSC70 and is required for endocytosis and endosomal trafficking in D. melanogaster and C. elegans.^15,16 GRV2 appears to regulate dynamics of late vacuolar endosomes,¹⁷ which is important for gravity signaling in shoots but not roots.¹⁸ GRV2/RME-8, auxillin, and cysteine-string proteins are each specific for endosomal trafficking involving particular molecules or vesicles, thus illustrating the specificity that J-domain proteins can confer to HSC70-mediated processes.One possibility is that the putative ARG1/ARL2 complex recruits HSC70 to alter actin dynamics in response to gravistimulation. The actin network in root statocytes appears to be intimately associated with the mechanism of gravity sensation, as treatments with low concentrations of the actin-depolymerizing drug Latrunculin B enhances gravity signaling events including amyloplast sedimentation, alkalinization of the statocyte cytoplasm, with subsequent enhancement of both auxin redistribution and organ curvature.⁹ arg1-2 mutants are deficient in gravity-induced statocyte alkalinization and auxin redistribution, suggesting that ARG1 and microfilaments regulate this gravity signaling response.⁴The lateral polarization of the statocytes upon gravistimulation likely involves dramatic changes in vesicle trafficking. Mechanisms that lead to accumulation of PIN3 upon the new bottom side of root statocytes are unknown, but require ARG1 and ARL2 either directly or as upstream signaling components. The prominent localization of ARG1 and ARL2 to the cell plate during cytokinesis suggests that they are present in areas of intense vesicle dynamics. The recent discovery that members of an Arabidopsis retromer complex are required for cell polarization, PIN trafficking, and gravitropic growth suggests they may function in redirection of auxin following gravistimulation.^19,20 Animal homologs of these retromer components have been identified in association with clathrin-coated vesicles,²¹ and disruption of Drosophila Vps35 or human SNX9 affect endocytosis and actin organization.^22,23 The mechanisms of clathrin-dependent endocytosis are unresolved, but appear to depend on dynamic cortical microfilaments in animals and yeast suggesting that similar mechanisms may function in plants.²⁴ ARG1 and ARL2 may mediate statocyte polarization via regulating actin and/or vesicle dynamics. Of course, we have not yet excluded the possibility that ARG1 and ARL2 might, in fact, transduce the gravity signal in an HSC70 and actin-independent manner through interactions with yet unidentified molecules. In this case, the interactions identified in this study would be relevant to other, yet uncharacterized, functions potentially associated with ARG1 and ARL2 in plants.^4,5 Work aimed at identifying additional ARG1 and ARL2-interacting proteins is underway.     

Self-Assembly of the Bacterial Cytoskeleton-Associated RNA Helicase B Protein into Polymeric Filamentous Structures

The Escherichia coli RNA degradosome proteins are organized into a helical cytoskeletal-like structure within the cell. Here we describe the ATP-dependent assembly of the RhlB component of the degradosome into polymeric filamentous structures in vitro, which suggests that extended polymers of RhlB are likely to comprise a basic core element of the degradosome cytoskeletal structures.The RNA degradosome plays an essential role in normal RNA processing and degradation. Within the cell, the degradosome proteins (RNA helicase B [RhlB], RNase E, polynucleotide phosphorylase [PNPase], and enolase) (4, 13, 15, 16) are organized into coiled structures that resemble the pole-to-pole helical structures of the MreB and MinCDE bacterial cytoskeletal systems (4, 12, 13). However, the degradosomal structures are also present in cells that lack the MreB and MinCDE cytoskeletal elements, suggesting that the degradosomal structures may be part of an independent class of prokaryotic cytoskeletal elements (19-21).One of the degradosomal proteins, RhlB, is organized into similar helical cellular structures in cells that lack the other degradosome proteins (Fig. ​(Fig.11 A). In addition, RhlB recruits PNPase to the helical framework in the absence of other degradosome proteins, suggesting that the RhlB structures are core elements of the degradosomal cytoskeletal-like elements of the cell (Fig. ​(Fig.1B)1B) (20). The cellular RhlB structures could be generated in two ways: (i) individual RhlB molecules may bind to an as-yet-undefined underlying track, or (ii) RhlB may polymerize to form the filamentous helical structures independent of any underlying template.Open in a separate windowFIG. 1.The RhlB filamentous cytoskeletal-like structures. (A) Cellular organization of RhlB based on immunofluorescence microscopy using purified anti-RhlB antibody in the absence of RNase E filamentous elements in AT8 cells (rne^1-417), which fail to generate RNase E coiled structures because of the absence of the RNase E cytoskeletal localization domain (20). (B) Proposed model for the cytoskeletal-like organization of the RNA degradosome (modified from reference 20). Arcs depict the RNase E (blue) and RhlB (red) helical strands. It is not known whether the RNase E helical strand is formed by RNase E polymerization or by the association of RNase E with an unknown underlying cytoskeletal structure. Enolase (Eno) and PNPase are shown in gray. Molecular dimensions and stoichiometry of the proteins were arbitrarily chosen to simplify the figure. (C to H) Electron micrographs of uranyl acetate-stained RhlB filaments (C, E, and H) and RhlB sheets (D). Unless otherwise indicated, the sample contained 9 μM RhlB, 2 mM ATP, 5 mM MgCl₂, and 5 mM CaCl₂. (E) Calcium was omitted. (F) ATP was omitted. (G and H) ATP was replaced by ATPγS (G) or AMP-PNP (H). Samples were loaded on glow-discharged 300-mesh carbon-coated copper grids and then stained. Images were taken with a JEOL 100CX transmission electron microscope. Magnification, ×10,000 to 50,000.Here we report that RhlB can self-assemble into extended polymeric structures in vitro in a process that requires ATP binding but not ATP hydrolysis. It is likely that extended RhlB polymers such as those described here are the basic components of the RhlB filamentous helical elements that comprise the core of the degradosomal cytoskeletal structures of the Escherichia coli cell.Evidence that RhlB can self-assemble into filamentous polymeric structures came from electron microscopic studies of purified His-tagged RhlB negatively stained with 2% uranyl acetate. This staining showed large numbers of long uniform filamentous structures when the purified protein was incubated in the presence of ATP and Ca²⁺ (Fig. ​(Fig.1C).1C). The filaments were 25 ± 1.8 nm wide (n = 91; mean ± standard deviation) and were generally more than 10 μm long. Some wider sheets were also observed (Fig. ​(Fig.1D).1D). Optimal assembly of the RhlB filamentous structures required ATP and Ca²⁺, as shown by the observation that only occasional single structures were present when the polymerization reaction was carried out in the absence of Ca²⁺ (Fig. ​(Fig.1E)1E) or ATP (Fig. ​(Fig.1F).1F). The polymeric RhlB-His structures were observed with approximately similar frequencies when ATP was replaced by the nonhydrolyzable ATP analog adenosine 5′-(γ-thiotriphosphate) (ATPγS) or AMP-PNP (Fig. 1G and H).Cellular localization studies showed that the presence of the His tag did not interfere with the ability of RhlB to form the helical cellular structures. Thus, RhlB-His was present in extended helical filamentous structures that were indistinguishable from those formed by untagged RhlB (20, 21). Similarly, the RhlB-His structures recruited PNPase to the helical framework in a manner similar to untagged RhlB (20) (see Fig. S1 in the supplemental material).Immunogold staining showed that the filaments and sheets were decorated with gold particles when stained with mouse anti-His tag antibody and gold-labeled secondary antibody (Fig. ​(Fig.22 A to C), confirming that the structures were composed of RhlB. In contrast, the structures were not decorated with gold particles in the absence of the primary antibody or when mouse anti-His tag antibody was replaced by nonimmune mouse IgG (Fig. ​(Fig.2D2D and E). The polymeric structures were observed with C-terminally His-tagged RhlB, which is functional in terms of helicase activity (8), but not when the tag was present at the amino terminus of the protein, where the His tag may interfere with RhlB self-assembly.Open in a separate windowFIG. 2.Immunogold electron microscopy of RhlB structures. Samples were prepared as describe for Fig. ​Fig.1C,1C, except that the grids were stained with 2% uranyl acetate after exposure to primary and/or secondary antibodies as indicated. (A to C) RhlB structures decorated with 10-nm gold particles in samples stained with mouse anti-His tag monoclonal antibody and gold-labeled secondary antibody. (A) single filaments; (B) clustered filaments; (C) a single RhlB filament and RhlB sheet. Arrows indicate gold particles. (D) The primary mouse anti-His tag antibody was replaced by mouse IgG. (E) The primary antibody was omitted.Changes in light scattering were used to follow the course of polymerization and to compare polymerization conditions in a more quantitative way than is possible by electron microscopy. The initial rate of increase in scattering was used to estimate polymerization rate (see Table S1 in the supplemental material). Significant rates of polymerization were observed in the presence of ATP and Ca²⁺, whereas there was very little increase in light scattering in the absence of nucleotide and/or Ca²⁺ (Fig. ​(Fig.33 A). ADP was less effective than ATP, whereas AMP and cyclic AMP (cAMP) were inactive (Fig. ​(Fig.3B).3B). In the presence of Ca²⁺, the extent and rate of RhlB polymerization varied as a function of ATP concentration (Fig. ​(Fig.3C).3C). Millimolar concentrations of Ca²⁺ were required to produce a measurable rate of polymerization in the light scattering assay (Fig. ​(Fig.3A).3A). It is not known how these relatively high concentrations of Ca²⁺ promote the in vitro polymerization of RhlB and other cytoskeletal proteins, such as MreB and FtsZ (1, 11, 12, 14, 23).Open in a separate windowFIG. 3.RhlB polymerization. (A and B) RhlB polymerization as shown by 90° light scattering is indicated in arbitrary units (a.u.). RhlB polymerization was followed at room temperature in a 1-cm light path quartz cuvette using a Hitachi fluorometer (FL-2500) set to 400 V with excitation and emission wavelengths set at 455 nm and a slit width of 10 nm. The reaction (100 μl volume) was performed in a polymerization buffer (50 mM Tris, 50 mM KCl, 5 mM MgCl₂; pH 8) as indicated. (A) The sample contained 9 μM RhlB-His, 1 mM ATP, and either 10 mM CaCl₂, 7.5 mM CaCl₂, or no CaCl₂ (squares). In the lower three curves the samples lacked ATP or CaCl₂, as indicated. (B) The sample contained 9 μM RhlB-His, 7.5 mM CaCl₂, and 1 mM adenosine nucleotides: ATP, ADP, AMP, cAMP, AMP-PNP, and ATPγS. (C) The sample contained 9 μM RhlB-His, 7.5 mM CaCl₂, and ATP as indicated (1 mM, 0.75 mM, 0.5 mM, 0.25 mM, or 0.1 mM ATP). (D) Effects of Ca²⁺ concentration on RhlB sedimentation in the presence of 2 mM ATP. RhlB in the pellet, expressed as a percentage of total RhlB present in the polymerization reaction mixture, was plotted against calcium concentration. The insert shows an example of a Coomassie blue-stained gel of supernatant (S) and pellet (P) fractions from the sedimentation assay in the presence of ATP and Ca²⁺ (see results for ATP in Table ​Table11).The nonhydrolyzable ATP analogs ATPγS and AMP-PNP were approximately equivalent to ATP in promoting polymerization as monitored by the light scattering assay (Fig. ​(Fig.3B)3B) as well as in the electron microscopic studies. This suggests that nucleotide binding, but not hydrolysis, is required to promote RhlB polymerization. In this regard, RhlB resembles a number of other proteins, including F-actin, MreB, and MinD, where polymerization is induced by nucleotide binding (2, 5, 6, 18, 22). In these systems, subsequent ATP hydrolysis induces depolymerization, providing the basis for the dynamic behavior of the polymers within the cell. RhlB is an RNA-dependent ATPase (7), but it is not yet known whether ATP hydrolysis is associated with depolymerization in the RhlB system.Similar results were obtained when the extent of polymerization was monitored by a sedimentation assay, measuring the proportion of RhlB in the pellet fraction after centrifugation at 278,000 × g for 10 min (Table ​(Table1;1; Fig. ​Fig.3D).3D). Essentially all of the protein was sedimentable at pH 8 in the presence of Ca²⁺ and ATP. RhlB sedimentation returned to background levels when EGTA or EDTA was added to the reaction mixture (Table ​(Table1),1), confirming the Ca²⁺ requirement for RhlB polymerization in the electron microscopic and light scattering analyses. ATPγS was equivalent to ATP in the sedimentation assay, confirming the results described above. The relatively high background of RhlB sedimentation was not affected by prespinning the samples prior to addition of nucleotides and/or Ca²⁺.

TABLE 1.

Sedimentation assay for RhlB polymerization

l-Ribose Production from l-Arabinose by Using Purified l-Arabinose Isomerase and Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

Two enzymes, l-arabinose isomerase and mannose-6-phosphate isomerase, from Geobacillus thermodenitrificans produced 118 g/liter l-ribose from 500 g/liter l-arabinose at pH 7.0, 70°C, and 1 mM Co²⁺ for 3 h, with a conversion yield of 23.6% and a volumetric productivity of 39.3 g liter⁻¹ h⁻¹.l-Ribose, a potential starting material for the synthesis of many l-nucleoside-based pharmaceutical compounds, is not abundant in nature (4, 15, 20). l-Ribose has been synthesized primarily from l-arabinose, l-xylose, d-glucose, d-galactose, d-ribose, and d-mannono-1,4-lactone (1, 13, 20). Recombinant cells containing a NAD-dependent mannitol-1-dehydrogenase produced 52 g/liter l-ribose from 100 g/liter ribitol after fermentation for 72 h (14). However, the volumetric productivity of l-ribose was 26-fold lower than that of the chemical synthetic method starting from l-arabinose (6). l-Ribose isomerase from an Acinetobacter sp., which is most active with l-ribose, showed poor efficiency in the conversion of l-ribulose to l-ribose (9). Recently, l-ribulose was produced with a conversion yield of 19% from the inexpensive sugar l-arabinose using l-arabinose isomerase (AI) from Geobacillus thermodenitrificans (18). l-Ribose has been produced from l-ribulose using mannose-6-phosphate isomerase (MPI) from Bacillus subtilis with a conversion yield of 70% (17). In this study, the production of l-ribose from l-arabinose was demonstrated via a two-enzyme system from G. thermodenitrificans, in which l-ribulose was first produced from l-arabinose by AI and subsequently converted to l-ribose by MPI.The analysis of monosaccharides and the purification and thermostability of AI and MPI from G. thermodenitrificans (2) isolated from compost were performed as described previously (7, 18, 19). The cross-linked enzymes were obtained from the treatment of 0.5% glutaraldehyde (10, 16). The reaction was performed by replacing the reaction solution with 100 g/liter l-arabinose and 1 mM Co²⁺ every 6 h at 70°C and pH 7.0. The reaction volume of 10 ml contained 5 g of the cross-linked enzymes with 8 U/ml AI and 20 U/ml MPI. One unit of AI or MPI activity, which corresponded to 0.0625 or 2.5 mg protein, respectively, was defined as the amount of enzyme required to produce 1 μmol of l-ribulose or l-ribose, respectively, per min at 70°C, pH 7.0, and 1 mM Co²⁺. Unless otherwise stated, the reaction was carried out in 50 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer (pH 7.0) in the presence of 1 mM Co²⁺ at 70°C for 4 h. All experiments were performed in triplicate.The recombinant Escherichia coli ER2566 (New England Biolabs, Ipswich, MA) containing pTrc99A plasmid (Pharmacia Biotech, Piscataway, NJ) and the AI or MPI gene was cultivated in a 7-liter fermentor containing 3 liters of chemically defined medium (11). When the cell mass reached 2 g/liter, 10 g/liter lactose was added for enzyme induction. After 14 h, 40 g/liter cells with 13,400 U/liter of AI or 34 g/liter cells with 630 U/liter of MPI was obtained. The enzyme was purified by heat treatment and Hi-Trap anion-exchange chromatography. The purification yields of AI and MPI were 21 and 78%, respectively, and the levels of purity for the concentrated AI and MPI by gene scanning were 48 and 92%, respectively. Maximum l-ribose production from l-arabinose by AI and by MPI in 10 ml of total volume was observed at pH 7.0, 70°C, and 1 mM Co²⁺ (data not shown). Half-lives for the two-enzyme system containing 10 mM l-arabinose, 0.2 U/ml AI, and 0.5 U/ml MPI at 60, 65, 70, 75, and 80°C were 1,216, 235, 48, 26, and 12 h, respectively. The use of Co²⁺ may be disadvantageous, as it is fairly toxic. This problem can be solved by using Mn²⁺ instead of Co²⁺. When Mn²⁺ was used in the reaction with the same amounts of enzymes, the conversion yield was the same as that obtained with Co²⁺, even though the volumetric productivity was lower than that with Co²⁺ (data not shown).The effect of the ratio of AI to MPI in the two-step enzymatic production of l-ribose from l-arabinose was investigated by mixing the enzyme solutions (8 U/ml AI and 20 U/ml MPI) to obtain AI/MPI ratios ranging from 10:90 to 90:10 (vol/vol) (Fig. ​(Fig.1).1). The reactions were run with 300 g/liter l-arabinose. Maximum l-ribose production was observed at a volume ratio of 50:50 of the enzyme solutions. The effects of enzyme concentration on l-ribose production were investigated at the optimal unit ratio (AI/MPI ratio, 1:2.5) with 500 g/liter l-arabinose and AI and MPI concentrations from 0.4 and 1.0 U/ml, respectively, to 9.2 and 23.0 U/ml, respectively (Fig. ​(Fig.2A).2A). l-Ribose production increased with increasing amounts of enzymes until reaching a plateau at 8 U/ml AI and 20 U/ml MPI. The effect of substrate concentration on l-ribose production was evaluated at l-arabinose concentrations ranging from 15 to 500 g/liter with 8 U/ml AI and 20 U/ml MPI (Fig. ​(Fig.2B).2B). The production of both l-ribose and l-ribulose, an intermediate, increased with increasing substrate level. The results suggest that concentrations of substrate above 500 g/liter l-arabinose might cause the increased production. The conversion yields of l-ribose and l-ribulose from l-arabinose were constant at 32% and 14%, respectively, within an initial concentration of 100 g/liter l-arabinose, indicating that the reactions reached equilibrium at an l-arabinose/l-ribulose/l-ribose ratio of 54:14:32, which was in agreement with the calculated equilibrium (17). However, at l-arabinose concentrations above 100 g/liter, the conversion yields of l-ribose and l-ribulose from l-arabinose decreased with increasing l-arabinose concentration. The l-arabinose/l-ribulose/l-ribose ratio, with an initial l-arabinose concentration of 300 g/liter, was 71:6:23 after 4 h of reaction. To obtain near-equilibrium (54:14:32) at this high concentration of l-arabinose, more effective enzymes are required.Open in a separate windowFIG. 1.Effect of the ratio of AI to MPI on l-ribose production from l-arabinose by the purified AI and MPI from G. thermodenitrificans. Data are the means for three separate experiments, and error bars represent standard deviations. Symbols: •, l-ribose; ▪, l-ribulose.Open in a separate windowFIG. 2.(A) Effect of enzyme concentration on l-ribose production from l-arabinose at the optimal unit ratio (AI/MPI ratio, 1:2.5). Symbols: •, l-ribose; ▪, l-ribulose; ○, l-arabinose. (B) Effect of l-arabinose concentration on l-ribose production. Symbols: •, l-ribose; ▪, l-ribulose. Data are the means for three separate experiments, and error bars represent standard deviations.A time course reaction of l-ribose production from l-arabinose was monitored for 3 h with 8 U/ml AI and 20 U/ml MPI (Fig. ​(Fig.3).3). As a result, 118 g/liter l-ribose was obtained from an initial l-arabinose concentration of 500 g/liter after 3 h, with a conversion yield of 23.6% and a productivity of 39.3 g liter⁻¹ h⁻¹. Recombinant E. coli containing MDH yielded 52 g/liter l-ribose from an initial ribitol concentration of 100 g/liter after 72 h, with a productivity of 0.72 g liter⁻¹ h⁻¹ (14). The production and productivity obtained in the current study using AI and MPI from G. thermodenitrificans were 2.3- and 55-fold higher, respectively, than those obtained from ribitol and 17- and 21-fold higher than those obtained with the production of l-ribose from l-arabinose using resting cells of recombinant Lactobacillus plantarum (5). The chemical synthetic method is capable of producing 56.5 g/liter l-ribose from 250 g/liter l-arabinose after 3 h, corresponding to a productivity of 18.8 g liter⁻¹ h⁻¹ (6). Still, both the production and productivity of l-ribose using the method described herein were 2.1-fold higher. Thus, the method of production of l-ribose in the present study exhibited the highest productivity and production, compared to other fermentation methods and chemical syntheses.Open in a separate windowFIG. 3.Time course of l-ribose production from l-arabinose by purified AI and MPI from G. thermodenitrificans. Data are the means for three separate experiments, and error bars represent standard deviations. Symbols: •, l-ribose; ▪, l-ribulose; ○, l-arabinose.Several rounds of conversion reusing the cross-linked enzymes were performed (Fig. ​(Fig.4).4). The immobilized enzymes showed more than 20% conversion of l-ribose from l-arabinose for the 9th batch, and the concentration of l-ribose was reduced to 43% after the 20th batch. These results suggest that the immobilization of enzyme facilitates separation of product and enzyme, and it enables the enzyme to function continuously, as reported previously (3, 8, 12). Thus, the reuse of enzyme by immobilization improves the economic viability of this enzymatic process.Open in a separate windowFIG. 4.Reuse of immobilized AI and MPI from G. thermodenitrificans for l-ribose production from 100 g/liter l-arabinose. Data are the means for three separate experiments, and error bars represent standard deviations.     

Metabolic Engineering of the Tricarboxylic Acid Cycle for Improved Lysine Production by Corynebacterium glutamicum

In the present work, lysine production by Corynebacterium glutamicum was improved by metabolic engineering of the tricarboxylic acid (TCA) cycle. The 70% decreased activity of isocitrate dehydrogenase, achieved by start codon exchange, resulted in a >40% improved lysine production. By flux analysis, this could be correlated to a flux shift from the TCA cycle toward anaplerotic carboxylation.With an annual market volume of more than 1,000,000 tons, lysine is one of the dominating products in biotechnology. In recent years, rational metabolic engineering has emerged as a powerful tool for lysine production (16, 18, 22). Hereby, different target enzymes and pathways in the central metabolism could be identified and successfully modified to create superior production strains (1, 2, 5, 8, 10, 17-20). The tricarboxylic acid (TCA) cycle has not been rationally engineered so far, despite its major role in Corynebacterium glutamicum (6). From metabolic flux studies, however, it seems that the TCA cycle might offer a great potential for optimization (Fig. ​(Fig.1),1), which is also predicted from in silico pathway analysis (13, 22). Experimental evidence comes from studies with Brevibacterium flavum exhibiting increased lysine production due to an induced bottleneck toward the TCA cycle (21). In the present work, we performed TCA cycle engineering by downregulation of isocitrate dehydrogenase (ICD). ICD is the highest expressed TCA cycle enzyme in C. glutamicum (7). Downregulation was achieved by start codon exchange, controlling ICD expression on the level of translation.Open in a separate windowFIG. 1.Stoichiometric correlation of lysine yield (%), biomass yield (g/mol) and TCA cycle flux (%; entry flux through citrate synthase) determined by ¹³C metabolic flux analysis achieved by paraboloid fitting of the data set (parameters were determined with Y0 = 105.1, a = −1.27, b = 0.35, c = −9.35 × 10⁻³, d = −11.16 × 10⁻³). The data displayed represent values from 18 independent experiments with different C. glutamicum strains taken from previous studies (1-3, 11, 12, 15, 23).     

Isoprenoids Are Essential for Fruiting Body Formation in Myxococcus xanthus

It was recently shown that Myxococcus xanthus harbors an alternative and reversible biosynthetic pathway to isovaleryl coenzyme A (CoA) branching from 3-hydroxy-3-methylglutaryl-CoA. Analyses of various mutants in these pathways for fatty acid profiles and fruiting body formation revealed for the first time the importance of isoprenoids for myxobacterial development.Myxobacteria are unique among the prokaryotes as (i) they can form highly complex fruiting bodies under starvation conditions, even up to microscopic tree-like structures (28); (ii) they can move on solid surfaces using different motility mechanisms (16); (iii) they produce some of the most cytotoxic secondary metabolites, with epothilone already in clinical use against cancer (2, 3); and (iv) they harbor the largest prokaryotic genomes found so far (15, 27). The large genome might be directly related to their complex life-style and the diverse secondary (3) and primary (9) metabolisms. Already in 2002 we found that myxobacteria are able to produce isovaleryl coenzyme A (IV-CoA) and compounds derived thereof via a new pathway that branches from 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which is the central intermediate of the well-known mevalonate-dependent isoprenoid biosynthesis (Fig. ​(Fig.1)1) (22, 23). Usually IV-CoA is derived from leucine degradation via the branched-chain keto acid dehydrogenase (BKD) complex (24), which is also the preferred pathway to IV-CoA in the myxobacteria Myxococcus xanthus and Stigmatella aurantiaca (Fig. ​(Fig.2A).2A). However, in bkd mutants, where no or only residual leucine degradation is possible (30), the alternative pathway is induced (Fig. ​(Fig.2B),2B), presumably to ensure the production of iso-fatty acids (iso-FAs) (5). A possible reason for this alternative pathway is the importance of IV-CoA-derived compounds in the complex myxobacterial life cycle, which is the starvation-induced formation of fruiting bodies in which the cells differentiate into myxospores. We showed that this pathway is induced during fruiting body formation in M. xanthus when leucine is limited. Under these conditions, this pathway might be more important for protein synthesis than for lipid remodeling, as lipids are present in excess during development due to the surface reduction from vegetative rods to round myxospores as described previously (29). Examples of IV-CoA-derived compounds are the unusual iso-branched ether lipids, which are almost exclusively produced in the developing myxospores. They might serve as structural lipids and signaling compounds during fruiting body formation (26).Open in a separate windowFIG. 1.Biosynthesis of IV-CoA and compounds derived thereof and biosynthesis of isoprenoids in M. xanthus. Broken arrows indicate multistep reactions; supplementation (double-lined arrows) with MVL and IVA can be used to complement selected mutants.Open in a separate windowFIG. 2.Short representations of proposed metabolic fluxes through the IV-CoA/isoprenoid network. Broken arrows indicate no metabolic flux. (A) DK1622 (wild type); (B) DK5643 (Δbkd); (C) DK5624 (Δbkd mvaS::kan); (D) HB002 (Δbkd liuC::kan); (E) HB002 with 1 mM IVA; (F) HB002 with 1 mM MVL. Ac-CoA, acetyl-CoA; MVA, mevalonic acid.In M. xanthus, we could recently identify candidate genes involved in the alternative pathway from HMG-CoA to IV-CoA. We also described the genes required for the degradation pathway of leucine and subsequently also those involved in the transformation of IV-CoA to HMG-CoA (4). In myxobacteria leucine is an important precursor for isoprenoid biosynthesis, as was already shown elsewhere for the biosynthesis of steroids (7) and prenylated secondary metabolites like aurachin (22) or leupyrrins (6), as well as volatiles like geosmin or germacradienol in M. xanthus and S. aurantiaca (11, 13). The interconnection of iso-FAs and isoprenoid biosynthesis made it difficult to assign functions to these compound classes during fruiting body formation in M. xanthus because it cannot be excluded that reduced leucine degradation also impairs isoprenoid biosynthesis. A mutant strain of M. xanthus that was blocked in the degradation of leucine and the alternative pathway had a deletion in the bkd locus as well as a plasmid insertion in the mvaS gene encoding the HMG-CoA synthase (strain DK5624). This double mutation severely affected isoprenoid biosynthesis (5), and cultures of DK5624 must be supplemented with mevalonolactone (MVL; the cyclized form of mevalonic acid) in order to enable growth (Fig. ​(Fig.2C).2C). Since we have identified the genes involved in IV-CoA biosynthesis and the mevalonate pathway (4), we can now start to identify differences between strains that show deficiencies in iso-FAs and strains that show deficiencies in isoprenoids via simple analysis of the FA profile and analysis of the myxobacterial development of selected mutants.All mutants used in this study (HB002 [Δbkd liuC::kan], HB015 [Δbkd MXAN_4265::kan], DK5624 [Δbkd mvaS::kan], HB019 [Δbkd mvaS::kan mvaS⁺], and HB020 [Δbkd MXAN_4265::kan mvaS⁺]) have been published previously (4), and FA analysis as well as myxobacterial fruiting body formation has also been described previously (26).M. xanthus HB002 (Δbkd liuC) shows only residual amounts of iso-FAs, as both leucine degradation and the alternative pathway to IV-CoA are blocked (Fig. ​(Fig.2D)2D) and its capability to form fruiting bodies is strongly reduced (Fig. ​(Fig.3).3). The residual amount of iso-FAs results from a second BKD activity in M. xanthus that has been identified by residual leucine incorporation as well as by residual enzymatic activity in bkd mutants (23, 30). This second BKD activity might be a side activity of the pyruvate dehydrogenase or a related chemical oxidative decarboxylation, as no second bkd locus could be identified in the genome (unpublished results). Moreover, growth of HB002 is not MVL dependent because the block in the alternative pathway does not affect isoprenoid biosynthesis, as liuC encodes a dehydratase/hydratase that is involved in the conversion of HMG-CoA to 3-methylglutaconyl-CoA and vice versa (4). As expected, the FA profile (4) as well as the developmental phenotype (data not shown) can be complemented (Fig. ​(Fig.2E)2E) by the addition of isovaleric acid (IVA), the free acid of IV-CoA, indicating the importance of iso-branched compounds for development in M. xanthus. Unexpectedly, addition of MVL (Fig. ​(Fig.2F)2F) also partially restored fruiting body formation without restoring the FA profile (Fig. ​(Fig.3).3). Similarly, M. xanthus HB015 (Δbkd MXAN_4265::kan) can produce only traces of iso-FAs, as both pathways to IV-CoA are blocked. MXAN_4265 encodes a protein with similarity to a glutaconyl-CoA transferase subunit, but from our previous results, we postulated it to be involved in the alternative pathway to IV-CoA (Fig. ​(Fig.1)1) (4). The respective mutant shows a severely impaired developmental phenotype, which can be complemented not only by the addition of IVA (not shown) but also by the addition of MVL (Fig. ​(Fig.3).3). Again, no change in the FA profile was observed after the addition of MVL. However, a plasmid insertion into MXAN_4265 has a polar effect on mvaS, which is the last gene in this five-gene operon and which is crucial for HMG-CoA formation from acetoacetyl-CoA and acetyl-CoA. Therefore, we assume that both pathways to HMG-CoA are blocked in HB015: no HMG-CoA can be made from acetyl-CoA and hardly any can be made via leucine degradation. In order to prove this hypothesis, we complemented HB015 with an additional copy of mvaS under the constitutive T7A1 promoter as described previously, using the plasmid pCK4267exp (4). The resulting strain, HB020 (Δbkd MXAN_4265::kan mvaS⁺), showed a restored developmental phenotype but still produced only trace amounts of iso-FAs.Open in a separate windowFIG. 3.Fruiting body formation on TPM agar in selected mutants at 24, 48, and 72 h after starvation. Numbers refer to the relative amounts (in percentages) of the most abundant iso-FA, iso-15:0, which is indicative of iso-FAs in general. Strains were DK1622 (wild type), HB002 (Δbkd liuC::kan), HB015 (Δbkd MXAN_4265::kan), DK5624 (Δbkd mvaS::kan), HB019 (Δbkd mvaS::kan mvaS⁺), and HB020 (Δbkd MXAN_4265::kan mvaS⁺). DK5624 was grown with 0.3 mM MVL prior to starvation, and the cells were washed and plated on TPM with or without 1 mM of MVL.The data from HB002, HB015, and HB020 indicate an important function of the mevalonate-dependent isoprenoid pathway for fruiting body formation in M. xanthus. Therefore, MVL addition can at least partially complement the developmental phenotype of DK5624, which cannot form fruiting bodies without MVL (Fig. ​(Fig.3).3). However, genetic complementation with mvaS in HB019 resulted in the expected complementation of the fruiting body formation and the FA profile (Fig. ​(Fig.3,3, bottom row).Leucine is one of the most abundant proteinogenic amino acids. It is also an essential amino acid for M. xanthus (8), which has a predatory life-style (1), as it lives on other bacteria and fungi that contain a lot of leucine. Moreover, leucine is very efficiently incorporated into isoprenoids like geosmin and aurachin (10, 22). Thus, one can conclude that in fact leucine degradation is the major pathway for HMG-CoA biosynthesis instead of the usual formation via acetoacetyl-CoA and acetyl-CoA by the HMG-CoA synthase MvaS as indicated in Fig. ​Fig.2A.2A. No difference in growth was observed between culture with and culture without MVL for HB002 (Δbkd liuC::kan) and HB015 (Δbkd MXAN_4265::kan) in rich medium (data not shown), probably due to the complete MvaS activity (in HB002) or residual BKD activity (in HB002 and HB015), resulting in all precursors for the mevalonate-dependent isoprenoid biosynthesis still being present in excess under these conditions. However, under starvation conditions a small reduction in HMG-CoA biosynthesis caused by completely blocked leucine degradation (as in HB002 due to the mutation in liuC [Fig. ​[Fig.2D])2D]) or reduced leucine degradation and a mutation in mvaS (as in HB015) might each result in a reduced isoprenoid level, which can be complemented at least partially by the addition of MVL. This would also explain the difference in the developmental phenotypes of HB002 and HB015, with the phenotype being more severe in HB002 (Fig. ​(Fig.3).3). The fact that complementation with IVA is in all cases more efficient than that with MVL can be explained by the role of the already-mentioned isolipids. They can be produced only after IVA addition, which also complements the (developmental) phenotype of some of these mutants (26).As isoprenoids represent probably the most diverse class of natural products (14), it is very hard to predict which particular isoprenoids might be responsible for the observed effects. Several isoprenoids (7, 11-13), prenylated secondary metabolites (6, 22), and carotenoids (18-21) are known from myxobacteria in general, and a major volatile compound from M. xanthus is the terpenoid geosmin (13). In order to test whether geosmin might be required for fruiting body formation, we constructed a plasmid insertion mutant in MXAN_6247, which is involved in the cyclization of farnesyl diphosphate to geosmin, following published procedures (4, 5). The resulting strain, HB022, showed the expected loss in geosmin production but no developmental phenotype (data not shown).Additionally, it cannot be excluded that prenylated proteins, sugars, or quinones from the respiratory chain are important for fruiting body formation. Moreover, stigmolone has been described as a pheromone involved in fruiting body formation in S. aurantiaca (25). Although its biosynthesis has not been elucidated yet, stigmolone could be an isoprenoid as well, which is deducible from the two iso-branched residues within its chemical structure (17). Nevertheless, the importance of isoprenoids for M. xanthus is evident from the data presented, and clearly more work is needed to identify the compound(s) involved.     

Metabolic Engineering of Saccharomyces cerevisiae for Production of Novel Cyanophycins with an Extended Range of Constituent Amino Acids

Cyanophycin (multi-l-arginyl-poly-l-aspartic acid; also known as cyanophycin grana peptide [CGP]) is a putative precursor for numerous biodegradable technically used chemicals. Therefore, the biosynthesis and production of the polymer in recombinant organisms is of special interest. The synthesis of cyanophycin derivatives consisting of a wider range of constituents would broaden the applications of this polymer. We applied recombinant Saccharomyces cerevisiae strains defective in arginine metabolism and expressing the cyanophycin synthetase of Synechocystis sp. strain PCC 6308 in order to synthesize CGP with citrulline and ornithine as constituents. Strains defective in arginine degradation (Car1 and Car2) accumulated up to 4% (wt/wt) CGP, whereas strains defective in arginine synthesis (Arg1, Arg3, and Arg4) accumulated up to 15.3% (wt/wt) of CGP, which is more than twofold higher than the previously content reported in yeast and the highest content ever reported in eukaryotes. Characterization of the isolated polymers by different analytical methods indicated that CGP synthesized by strain Arg1 (with argininosuccinate synthetase deleted) consisted of up to 20 mol% of citrulline, whereas CGP from strain Arg3 (with ornithine carbamoyltransferase deleted) consisted of up to 8 mol% of ornithine, and CGP isolated from strain Arg4 (with argininosuccinate lyase deleted) consisted of up to 16 mol% lysine. Cultivation experiments indicated that the incorporation of citrulline or ornithine is enhanced by the addition of low amounts of arginine (2 mM) and also by the addition of ornithine or citrulline (10 to 40 mM), respectively, to the medium.Cyanophycin (multi-l-arginyl-poly-[l-aspartic acid]), also referred to as cyanophycin grana peptide (CGP), represents a polydisperse nonribosomally synthesized polypeptide consisting of poly(aspartic acid) as backbone and arginine residues bound to each aspartate (49) (Fig. ​(Fig.1).1). One enzyme only, referred to as cyanophycin synthetase (CphA), catalyzes the synthesis of the polymer from amino acids (55). Several CphAs originating from different bacteria exhibit specific features (2, 7, 5, 32, 50, 51). CphAs from the cyanobacteria Synechocystis sp. strain PCC 6308 and Anabaena variabilis ATCC 29413, respectively, exhibit a wide substrate range in vitro (2, 7), whereas CphA from Acinetobacter baylyi or Nostoc ellipsosporum incorporates only aspartate and arginine (23, 24, 32). CphA from Thermosynechococcus elongatus catalyzes the synthesis of CGP primer independently (5); CphA from Synechococcus sp. strain MA19 exhibits high thermostability (22). Furthermore, two types of CGP were observed concerning its solubility behavior: (i) a water-insoluble type that becomes soluble at high or low pH (34, 48) and (ii) a water-soluble type that was only recently observed in recombinant organisms (19, 26, 42, 50, 56). In the past, bacteria were mainly applied for the synthesis of CGP (3, 14, 18, 53), whereas recently there has been greater interest in synthesis in eukaryotes (26, 42, 50). CGP was accumulated to almost 7% (wt/wt) of dry matter in recombinant Nicotiana tabacum and Saccharomyces cerevisiae (26, 50).Open in a separate windowFIG. 1.Chemical structures of dipeptide building blocks of CGP variants detected in vivo. Structure: 1, aspartate-arginine; 2, aspartate-lysine; 3, aspartate-citrulline; 4, aspartate-ornithine. Aspartic acid is presented in black; the second amino acid of the dipeptide building blocks is shown in gray. The nomenclature of the carbon atoms is given.In S. cerevisiae the arginine metabolism is well understood and has been investigated (30) (see Fig. ​Fig.2).2). Arginine is synthesized from glutamate via ornithine and citrulline in eight successive steps. The enzymes acetylglutamate synthase, acetylglutamate kinase, N-acetyl-γ-glutamylphosphate reductase, and acetylornithine aminotransferase are involved in the formation of N-α-acetylornithine. The latter is converted to ornithine by acetylornithine acetyltransferase. In the next step, ornithine carbamoyltransferase (ARG3) condenses ornithine with carbamoylphosphate, yielding citrulline. Citrulline is then converted to l-argininosuccinate by argininosuccinate synthetase. The latter is in the final step cleaved into fumarate and arginine by argininosuccinate lyase (ARG4). The first five steps occur in the mitochondria, whereas the last three reactions occur in the cytosol (28, 54). Arginine degradation is initiated by arginase (CAR1) and ornithine aminotransferase (CAR2) (10, 11, 38, 39).Open in a separate windowFIG. 2.Schematic overview of the arginine metabolism in S. cerevisiae. Reactions shown in the shaded area occur in the mitochondria, while the other reactions are catalyzed in the cytosol. Abbreviations: ARG2, acetylglutamate synthase; ARG6, acetylglutamate kinase; ARG5, N-acetyl-γ-glutamyl-phosphate reductase; ARG8, acetylornithine aminotransferase; ECM40, acetylornithine acetyltransferase; ARG1, argininosuccinate synthetase; ARG3, ornithine carbamoyltransferase; ARG4, argininosuccinate lyase; CAR1, arginase; CAR2, ornithine aminotransferase.A multitude of putative applications for CGP derivatives are available (29, 41, 45, 47), thus indicating a need for efficient biotechnological production and for further investigations concerning the synthesis of CGP with alternative properties and different constituents. It is not only the putative application of the polymer as a precursor for poly(aspartic acid), which is used as biodegradable alternative for poly(acrylic acid) or for bulk chemicals, that makes CGP interesting (29, 45-47). In addition, a recently developed process for the production of dipeptides from CGP as a precursor makes the synthesis of CGP variants worthwhile (43). Dipeptides play an important role in medicine and pharmacy, e.g., as additives for malnourished patients, as treatments against liver diseases, or as aids for muscle proliferation (43). Because dipeptides are synthesized chemically (40) or enzymatically (6), novel biotechnological production processes are welcome.