Use of the valine biosynthetic pathway to convert glucose into isobutanol

Microbiological synthesis of higher alcohols (1-butanol, isobutanol, 2-methyl-1-butanol, etc.) from plant biomass is critically important due to their advantages over ethanol as a motor fuel. In recent years, the use of branched-chain amino acid (BCAA) biosynthesis pathways together with heterologous Ehrlich pathway enzyme system (Hazelwood et al. in Appl Environ Microbiol 74:2259–2266, 2008) has been proposed by the Liao group as an alternative approach to aerobic production of higher alcohols as new-generation biofuels (Atsumi et al. in Nature 451:86–90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651–657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89–98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769–5775, 2008; Shen and Liao in Metab Eng 10:312–320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471–479, 2009). On the basis of these remarkable investigations, we re-engineered Escherichia coli valine-producing strain H-81, which possess overexpressed ilvGMED operon, for the aerobic conversion of sugar into isobutanol. To redirect valine biosynthesis to the production of alcohol, we also—as has been demonstrated previously (Atsumi et al. in Nature 451:86–90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651–657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89–98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769–5775, 2008; Shen and Liao in Metab Eng 10:312–320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471–479, 2009)—used enzymes of Ehrlich pathway. In particular, in our study, the following heterologous proteins were exploited: branched-chain 2-keto acid decarboxylase (BCKAD) encoded by the kdcA gene from Lactococcus lactis with rare codons substituted, and alcohol dehydrogenase (ADH) encoded by the ADH2 gene from Saccharomyces cerevisiae. We show that expression of both of these genes in the valine-producing strain H-81 results in accumulation of isobutanol instead of valine. Expression of BCKAD alone also resulted in isobutanol accumulation in the culture broth, supporting earlier obtained data (Atsumi et al. in Appl Microbiol Biotechnol 85:651–657, 2010) that native ADHs of E. coli are also capable of isobutanol production. Thus, in this work, isobutanol synthesis by E. coli was achieved using enzymes similar to but somewhat different from those previously used.     

Identification of "Candidatus Thioturbo danicus," a microaerophilic bacterium that builds conspicuous veils on sulfidic sediments

Molecular analysis of bacteria enriched under in situ-like conditions and mechanically isolated by micromanipulation showed that a hitherto-uncultivated microaerophilic bacterium thriving in oxygen-sulfide counter-gradients (R. Thar and M. Kühl, Appl. Environ. Microbiol. 68:6310-6320, 2000) is affiliated with the epsilon-subdivision of the Proteobacteria. The affiliation was confirmed by the use of whole-cell hybridization with newly designed specific oligonucleotide probes. The bacterium belongs to a new genus and received the provisional name "Candidatus Thioturbo danicus."     

Evaluation of a Fluorescent Lectin-Based Staining Technique for Some Acidophilic Mining Bacteria

A fluorescence-labeled wheat germ agglutinin staining technique (R. K. Sizemore et al., Appl. Environ. Microbiol. 56:2245–2247, 1990) was modified and found to be effective for staining gram-positive, acidophilic mining bacteria. Bacteria identified by others as being gram positive through 16S rRNA sequence analyses, yet clustering near the divergence of that group, stained weakly. Gram-negative bacteria did not stain. Background staining of environmental samples was negligible, and pyrite and soil particles in the samples did not interfere with the staining procedure.     

Bactericidal Activity of Glycinecin A, a Bacteriocin Derived from Xanthomonas campestris pv. glycines, on Phytopathogenic Xanthomonas campestris pv. vesicatoria Cells

The ability of glycinecin A, a bacteriocin derived from Xanthomonas campestris pv. glycines 8ra, to kill closely related bacteria has been demonstrated previously by our group (S. G. Heu et al., Appl. Environ. Microbiol. 67:4105-4110, 2001). In the present study, we aimed at determining the glycinecin A-induced cause of death. Treatment with glycinecin A caused slow dissipation of membrane potential and rapid depletion of the pH gradient. Glycinecin A treatment also induced leakage of potassium ions from X. campestris pv. vesicatoria YK93-4 cells and killed sensitive bacterial cells in a dose-dependent manner. Sensitive cells were killed within 2 h of incubation, most likely due to the potassium ion efflux caused by glycinecin A. These results suggest that the bactericidal mechanism of action of glycinecin A is correlated with the permeability of membranes to hydroxyl and potassium ions, leading to the lethal activity of the bacteriocin on the target bacteria.     

Biotransformation of 1,2,3-tri- and 1,2,3,4,7,8-hexachlorodibenzo-p- dioxin by Sphingomonas wittichii strain RW1

Sphingomonas wittichii RW1 is able to catabolize 1,2,3,4-tetrachlorodibenzo-p-dioxin (H. B. Hong, Y. S. Chang, I. H. Nam, P. Fortnagel, and S. Schmidt, Appl. Environ. Microbiol. 68:2584-2588, 2002). Here we demonstrate the aerobic bacterial catabolism of the ubiquitous toxic diaryl ether pollutant 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin by this strain. The products of this biotransformation were identified as tetrachlorocatechol and 2-methoxy-3,4,5,6-tetrachlorophenol by comparing mass spectra recorded before and after n-butylboronate and N,O-bis(trimethylsilyl)-trifluoroacetamide derivatization with those of authentic compounds. Additional experiments showed that the less-chlorinated 1,2,3,7,8-pentachlorodibenzo-p-dioxin was not transformed by the strain RW1. The importance of substitution patterns for the degradability of individual congeners was illustrated by the fact that the 1,2,3-trichlorodibenzo-p-dioxin was catabolized to yield 3,4,5-trichlorocatechol, whereas the 2,3,7-trichlorodibenzo-p-dioxin was not attacked.     

Heterologous gene expression in <Emphasis Type="Italic">Thermus thermophilus</Emphasis>: β-galactosidase,dibenzothiophene monooxygenase,PNB carboxy esterase, 2-aminobiphenyl-2,3-diol dioxygenase,and chloramphenicol acetyl transferase

Enzymes from thermophiles are preferred for industrial applications because they generally show improved tolerance to temperature, pressure, solvents, and pH as compared with enzymes from mesophiles. However, nearly all thermostable enzymes used in industrial applications or available commercially are produced as recombinant enzymes in mesophiles, typically Escherichia coli. The development of high-temperature bioprocesses, particularly those involving cofactor-requiring enzymes and/or multi-step enzymatic pathways, requires a thermophilic host. The extreme thermophile most amenable to genetic manipulation is Thermus thermophilus, but the study of expression of heterologous genes in T. thermophilus is in its infancy. While several heterologous genes have previously been expressed in T. thermophilus (Fridjonsson et al. in J Bacteriol 184:3385–3391, 2002, Koyama et al. in Appl Environ Microbiol 56:2251–225, 1990, Lasa et al. in J Bacteriol 174:6424–6431, 1992, Mathew et al. in Appl Environ Microbiol 58:421–425, 1992, Takagi et al. in J Ind Microbiol Biotechnol 23:214–217, 1999, Tamakoshi et al. in Extremophiles 5:17–22 2001), the data reported here include the first examples of the functional expression of a gene from an archaeal hyperthermophile (bglA from Pyrococcus woesei), a cofactor-requiring enzyme (dszC from Rhodococcus erythropolis IGTS8), and a two-component enzyme (carBa and carBb from Sphingomonas sp. GTIN11). A thermostable derivative of pnbA from Bacillus subtilis was also expressed, further expanding the list of genes from heterologous hosts that have been expressed in T. thermophilus.     

Identification of “Candidatus Thioturbo danicus,” a Microaerophilic Bacterium That Builds Conspicuous Veils on Sulfidic Sediments

Molecular analysis of bacteria enriched under in situ-like conditions and mechanically isolated by micromanipulation showed that a hitherto-uncultivated microaerophilic bacterium thriving in oxygen-sulfide counter-gradients (R. Thar and M. Kühl, Appl. Environ. Microbiol. 68:6310-6320, 2000) is affiliated with the -subdivision of the Proteobacteria. The affiliation was confirmed by the use of whole-cell hybridization with newly designed specific oligonucleotide probes. The bacterium belongs to a new genus and received the provisional name “Candidatus Thioturbo danicus.”     

Mechanism of pyrite dissolution in the presence of Thiobacillus ferrooxidans.

In spite of the environmental and commercial interests in the bacterial leaching of pyrite, two central questions have not been answered after more than 35 years of research: does Thiobacillus ferrooxidans enhance the rate of leaching above that achieved by ferric sulfate solutions under the same conditions, and if so, how do the bacteria affect such an enhancement? Experimental conditions of previous studies were such that the concentrations of ferric and ferrous ions changed substantially throughout the course of the experiments. This has made it difficult to interpret the data obtained from these previous works. The aim of this work was to answer these two questions by employing an experimental apparatus designed to maintain the concentrations in solution at a constant value. This was achieved by using the constant redox potential apparatus described previously (P. I. Harvey, and F. K. Crundwell, Appl. Environ. Microbiol. 63:2586-2592, 1997; T. A. Fowler, and F. K. Crundwell, Appl. Environ. Microbiol. 64:3570-3575, 1998). Experiments were conducted in both the presence and absence of T. ferrooxidans, maintaining the same conditions in solution. The rate of dissolution of pyrite with bacteria was higher than that without bacteria at the same concentrations of ferrous and ferric ions in solution. Analysis of the dependence of the rate of leaching on the concentration of ferric ions and on the pH, together with results obtained from electrochemical measurements, provided clear evidence that the higher rate of leaching with bacteria is due to the bacteria increasing the pH at the surface of the pyrite.     

Viability of heterotrophic bacteria in the Bay of Marseilles

Marine microorganism activities are commonly assessed by bulk methods and assigned to the total cell count. The presence in significant amounts of ghost, dead, and damaged cells makes such as assignment a non-correct one. A Nucleic Acid Double Staining protocol (NADS) of fresh water bacteria (Barbesti et al., Cytometry 40 (2000) 214-218) has been adapted to resolve viable, damaged and dead cells in marine environments (Grégori et al., Appl. Environ. Microbiol. 67 (2001) 4662-4670). The present reports the first in situ application of this approach, conducted in the Bay of Marseilles in winter and spring periods at two sites with contrasted features.     

Comparison of different PCR tests for detecting Shiga toxin-producing Escherichia coli O157 and development of an ELISA-PCR assay for specific identification of the bacteria

In an attempt to develop a standard for ELISA-PCR detection of Shiga toxin producing Escherichia coli (STEC) O157, six published PCR tests were tested in a comparative study on a panel of 277 bacterial strains isolated from foods, animals and humans. These tests were based on the detection of the genes rfbE [J. Clin. Microbiol. 36 (1998) 1801] and rfbB [Appl. Environ. Microbiol. 65 (1999) 2954], the 3' end of the eae gene [Epidemiol. Infect. 112 (1994) 449], the region immediately flanking the 5' end of the eae gene [Int. J. Food. Microbiol. 32 (1996) 103], the flicH7 gene [J. Clin. Microbiol. 35 (1997) 656], or a part of the recently described 2634-bp Small Inserted Locus (SILO(157) locus) of STEC O157 [J. Appl. Microbiol. 93 (2002) 250]. Unlike the other PCR assays, those amplifying the rfb sequences were unable to distinguish toxigenic from nontoxigenic O157. These assays were relatively specific to STEC O157, giving essentially a cross reaction with clonally related E. coli O55 and to a lesser extent with E. coli O145, O125, O126. They also detected the Shiga toxin (stx)-negative derivatives of STEC O157. Based on these results, an ELISA-PCR assay consisting of the solution hybridization of amplicons with two probes that ensured the specificity of the amplification was developed. The ELISA-PCR assay, which used an internal control (IC) of inhibition, was able to detect 1 to 10 copies of STEC O157 in the PCR tube. Adaptation of PCR into ELISA-PCR assay format facilitates specific and sensitive detection of PCR amplification products and constitutes a method of choice for screening STEC O157.     

Comparison of methods for isolating Campylobacter jejuni from raw milk.

The method of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) was compared with that of Lovett et al. (Appl. Environ. Microbiol. 46:459-462, 1983) for the ability to recover Campylobacter jejuni strains inoculated into raw milk at a concentration of less than 1 cell per g. The method of Lovett et al. gave significantly greater recovery proportions.     

Comparison of methods for isolating Campylobacter jejuni from raw milk

The method of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) was compared with that of Lovett et al. (Appl. Environ. Microbiol. 46:459-462, 1983) for the ability to recover Campylobacter jejuni strains inoculated into raw milk at a concentration of less than 1 cell per g. The method of Lovett et al. gave significantly greater recovery proportions.     

Extracellular enzyme activity in anaerobic bacterial cultures: evidence of pullulanase activity among mesophilic marine bacteria.

The extracellular enzymatic activity of a mixed culture of anaerobic marine bacteria enriched on pullulan [alpha(1,6)-linked maltotriose units] was directly assessed with a combination of gel permeation chromatography (GPC) and nuclear magnetic resonance spectroscopy (NMR). Hydrolysis products of pullulan were separated by GPC into three fractions with molecular weights of > or = 10,000, approximately 5,000, and < or = 1,200. NMR spectra of these fractions demonstrated that pullulan was rapidly and specifically hydrolyzed at alpha(1,6) linkages by pullulanase enzymes, most likely type II pullulanase. Although isolated pullulanase enzymes have been shown to hydrolyze pullulan completely to maltotriose (S. H. Brown, H. R. Costantino, and R. M. Kelly, Appl. Environ. Microbiol. 56:1985-1991, 1990; M. Klingeberg, H. Hippe, and G. Antranikian, FEMS Microbiol. Lett. 69:145-152, 1990; R. Koch, P. Zablowski, A. Spreinat, and G. Antranikian, FEMS Microbiol. Lett. 71:21-26, 1990), the smallest carbohydrate detected in the bacterial cultures consisted of two maltotriose units linked through one alpha(1,6) linkage. Either the final hydrolysis step was closely linked to substrate uptake, or specialized porins similar to maltoporin might permit direct transport of large oligosaccharides into the bacterial cell. This is the first report of pullulanase activity among mesophilic marine bacteria. The combination of GPC and NMR could easily be used to assess other types of extracellular enzyme activity in bacterial cultures.     

Mechanism of Pyrite Dissolution in the Presence of Thiobacillus ferrooxidans

In spite of the environmental and commercial interests in the bacterial leaching of pyrite, two central questions have not been answered after more than 35 years of research: does Thiobacillus ferrooxidans enhance the rate of leaching above that achieved by ferric sulfate solutions under the same conditions, and if so, how do the bacteria affect such an enhancement? Experimental conditions of previous studies were such that the concentrations of ferric and ferrous ions changed substantially throughout the course of the experiments. This has made it difficult to interpret the data obtained from these previous works. The aim of this work was to answer these two questions by employing an experimental apparatus designed to maintain the concentrations in solution at a constant value. This was achieved by using the constant redox potential apparatus described previously (P. I. Harvey, and F. K. Crundwell, Appl. Environ. Microbiol. 63:2586–2592, 1997; T. A. Fowler, and F. K. Crundwell, Appl. Environ. Microbiol. 64:3570–3575, 1998). Experiments were conducted in both the presence and absence of T. ferrooxidans, maintaining the same conditions in solution. The rate of dissolution of pyrite with bacteria was higher than that without bacteria at the same concentrations of ferrous and ferric ions in solution. Analysis of the dependence of the rate of leaching on the concentration of ferric ions and on the pH, together with results obtained from electrochemical measurements, provided clear evidence that the higher rate of leaching with bacteria is due to the bacteria increasing the pH at the surface of the pyrite.     

Early Interactions of Rhizobium leguminosarum bv. phaseoli and Bean Roots: Specificity in the Process of Adsorption and Its Requirement of Ca(sup2+) and Mg(sup2+) Ions

Roots of Phaseolus vulgaris L. were incubated with dilute suspensions (1 x 10(sup3) to 3 x 10(sup3) bacteria ml(sup-1)) of an antibiotic-resistant indicator strain of Rhizobium leguminosarum bv. phaseoli in mineral medium and washed four times by a standardized procedure prior to quantitation of adsorption (G. Caetano-Anolles and G. Favelukes, Appl. Environ. Microbiol. 52:371-376, 1986). The population of rhizobia remaining adsorbed on roots after washing was homogeneous, as indicated by the first-order course of its desorption by hydrodynamic shear. Rhizobia were maximally active for adsorption in the early stationary phase of growth. The process leading to adsorption was rapid, without an initial lag, and slowed down after 1 h. Adsorption of the indicator strain at 10(sup3) bacteria ml(sup-1) was inhibited to different extents in the presence of 10(sup3) to 10(sup8) antibiotic-sensitive competitor rhizobia ml(sup-1). After a steep rise above 10(sup4) bacteria ml(sup-1), inhibition by heterologous competitors in the concentration range of 10(sup5) to 10(sup7) bacteria ml(sup-1) was markedly less than by homologous strains, while at 10(sup8) bacteria ml(sup-1) it approached the high level of inhibition by the latter. At 10(sup7) bacteria ml(sup-1), all of the heterologous strains tested were consistently less inhibitory than homologous competitors (P < 0.001). These differences in competitive behavior indicate that in the process of adsorption of R. leguminosarum bv. phaseoli to its host bean roots, different modes of adsorption occur and that some of these modes are specific for the microsymbiont (as previously reported for the alfalfa system [G. Caetano-Anolles and G. Favelukes, Appl. Environ. Microbiol. 52:377-381, 1986]). Moreover, whereas the nonspecific process occurred either in the absence or in the presence of Ca(sup2+) and Mg(sup2+) ions, expression of specificity was totally dependent on the presence of those cations. R. leguminosarum bv. phaseoli bacteria adsorbed in the presence of Ca(sup2+) and Mg(sup2+) were more resistant to desorption by shear forces than were rhizobia adsorbed in their absence. These results indicate that (i) symbiotic specificity in the P. vulgaris-R. leguminosarum bv. phaseoli system is expressed already during the early process of rhizobial adsorption to roots, (ii) Ca(sup2+) and Mg(sup2+) ions are required by R. leguminosarum bv. phaseoli for that specificity, and (iii) those cations cause tighter binding of rhizobia to roots.     

Microbial responses to microgravity and other low-shear environments.

Microbial adaptation to environmental stimuli is essential for survival. While several of these stimuli have been studied in detail, recent studies have demonstrated an important role for a novel environmental parameter in which microgravity and the low fluid shear dynamics associated with microgravity globally regulate microbial gene expression, physiology, and pathogenesis. In addition to analyzing fundamental questions about microbial responses to spaceflight, these studies have demonstrated important applications for microbial responses to a ground-based, low-shear stress environment similar to that encountered during spaceflight. Moreover, the low-shear growth environment sensed by microbes during microgravity of spaceflight and during ground-based microgravity analogue culture is relevant to those encountered during their natural life cycles on Earth. While no mechanism has been clearly defined to explain how the mechanical force of fluid shear transmits intracellular signals to microbial cells at the molecular level, the fact that cross talk exists between microbial signal transduction systems holds intriguing possibilities that future studies might reveal common mechanotransduction themes between these systems and those used to sense and respond to low-shear stress and changes in gravitation forces. The study of microbial mechanotransduction may identify common conserved mechanisms used by cells to perceive changes in mechanical and/or physical forces, and it has the potential to provide valuable insight for understanding mechanosensing mechanisms in higher organisms. This review summarizes recent and future research trends aimed at understanding the dynamic effects of changes in the mechanical forces that occur in microgravity and other low-shear environments on a wide variety of important microbial parameters.     

Biotransformation of 1,2,3-Tri- and 1,2,3,4,7,8-Hexachlorodibenzo-p- Dioxin by Sphingomonas wittichii Strain RW1

Sphingomonas wittichii RW1 is able to catabolize 1,2,3,4-tetrachlorodibenzo-p-dioxin (H. B. Hong, Y. S. Chang, I. H. Nam, P. Fortnagel, and S. Schmidt, Appl. Environ. Microbiol. 68:2584-2588, 2002). Here we demonstrate the aerobic bacterial catabolism of the ubiquitous toxic diaryl ether pollutant 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin by this strain. The products of this biotransformation were identified as tetrachlorocatechol and 2-methoxy-3,4,5,6-tetrachlorophenol by comparing mass spectra recorded before and after n-butylboronate and N,O-bis(trimethylsilyl)-trifluoroacetamide derivatization with those of authentic compounds. Additional experiments showed that the less-chlorinated 1,2,3,7,8-pentachlorodibenzo-p-dioxin was not transformed by the strain RW1. The importance of substitution patterns for the degradability of individual congeners was illustrated by the fact that the 1,2,3-trichlorodibenzo-p-dioxin was catabolized to yield 3,4,5-trichlorocatechol, whereas the 2,3,7-trichlorodibenzo-p-dioxin was not attacked.     

Microbial Responses to Microgravity and Other Low-Shear Environments

Microbial adaptation to environmental stimuli is essential for survival. While several of these stimuli have been studied in detail, recent studies have demonstrated an important role for a novel environmental parameter in which microgravity and the low fluid shear dynamics associated with microgravity globally regulate microbial gene expression, physiology, and pathogenesis. In addition to analyzing fundamental questions about microbial responses to spaceflight, these studies have demonstrated important applications for microbial responses to a ground-based, low-shear stress environment similar to that encountered during spaceflight. Moreover, the low-shear growth environment sensed by microbes during microgravity of spaceflight and during ground-based microgravity analogue culture is relevant to those encountered during their natural life cycles on Earth. While no mechanism has been clearly defined to explain how the mechanical force of fluid shear transmits intracellular signals to microbial cells at the molecular level, the fact that cross talk exists between microbial signal transduction systems holds intriguing possibilities that future studies might reveal common mechanotransduction themes between these systems and those used to sense and respond to low-shear stress and changes in gravitation forces. The study of microbial mechanotransduction may identify common conserved mechanisms used by cells to perceive changes in mechanical and/or physical forces, and it has the potential to provide valuable insight for understanding mechanosensing mechanisms in higher organisms. This review summarizes recent and future research trends aimed at understanding the dynamic effects of changes in the mechanical forces that occur in microgravity and other low-shear environments on a wide variety of important microbial parameters.     

Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities

ITSF and ITSReub, constituting a new primer set designed for the amplification of the 16S-23S rRNA intergenic transcribed spacers, have been compared with primer sets consisting of 1406F and 23Sr (M. M. Fisher and E. W. Triplett, Appl. Environ. Microbiol. 65:4630-4636, 1999) and S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 (L. Ranjard et al., Appl. Environ. Microbiol. 67:4479-4487, 2001), previously proposed for automated ribosomal intergenic spacer analysis (ARISA) of complex bacterial communities. An agricultural soil and a polluted soil, maize silage, goat milk, a small marble sample from the facade of the Certosa of Pavia (Pavia, Italy), and brine from a deep hypersaline anoxic basin in the Mediterranean Sea were analyzed with the three primer sets. The number of peaks in the ARISA profiles, the range of peak size (width of the profile), and the reproducibility of results were used as indices to evaluate the efficiency of the three primer sets. The overall data showed that ITSF and ITSReub generated the most informative (in term of peak number) and reproducible profiles and yielded a wider range of spacer sizes (134 to 1,387) than the other primer sets, which were limited in detecting long fragments. The minimum amount of DNA template and sensitivity in detection of minor DNA populations were evaluated with artificial mixtures of defined bacterial species. ITSF and ITSReub amplified all the bacteria at DNA template concentrations from 280 to 0.14 ng microl(-1), while the other primer sets failed to detect the spacers of one or more bacterial strains. Although the primer set consisting of ITSF and ITSReub and that of S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 showed similar sensitivities for the DNA of Allorhizobium undicula mixed with the DNA of other species, the S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 primer set failed to detect the DNA of Pseudomonas stutzeri.