The chimærical structure of a somatic solanum mutant revealed by ionizing irradiation

The aim of the present study was to investigate whether it is possible to reveal the chimaerical structure of somatic mutants inSolanum tuberosum by means of ionizing irradiation. A somatic subdivided-leaf mutant of the variety Prosident was chosen as subject. This mutant was isolated in 1957 and has proved to keep permanent and stable during a five years' propagation. In spite of a two years' eye-excision experiment in which, totally, 50 tuber-halves were tested, it has been impossible to demonstrate the chimærical structure of this type. Im May 1961, 50 tuber-halves of the subdivided-leaf type were X-irradiated with a dose of 4,000 r. Five of the surviving 40 plants showed from one to three normal shoots. Furthermore, in one of these plants, a few leaves were observed one half of which was subdivided and the other normal. In the corresponding 50 tuber-halves, which served as a control, no morphological changes could be found. It is concluded that X-irradiation is an efficient and easy method for revealing the chimærical structure of somatic mutants inSolanum tuberosum.     

Generation of a stable non-reverting Leu− mutant of Kluyveromyces lactis by gene disruption

The LEU2 gene coding for -isopropylmalate dehydrogenase of the yeast Kluyveromyces lactis strain AWJ137 was disrupted. In the resulting Leu^– strain a 0.57 × 10³-base pairs PstI/BglII fragment of the LEU2 coding region was replaced by the TRP1 gene of Saccharomyces cerevisiae. The mutant strain was characterized by stability tests and a physical map of the disrupted region was established by restriction-enzyme analysis combined with hybridization experiments. The usefulness of the mutant strain as a recipient was shown by transformation experiments.     

Functional analysis of the eyespot in Chlamydomonas reinhardtii mutant ey 627, mt −

The function of the eyespot in phototaxis of the flagellate green alga Chlamydomonas reinhardtii Dangeard was studied using quantitative reflection confocal laser scanning microscopy and photoelectric measurements. The reflective properties of the eyespot and the photoreceptor current of the C. reinhardtii eyespot mutant ey 627, mt ^– were compared with those of Chlamydomonas strains possessing a well-developed eyespot. Under growth conditions in which strongly disorganized eyespots were observed in the mutant by electron microscopy, there was a significant reduction in the reflection intensity of the eyespot and in the amplitude ratio (500440 nm) of photoreceptor currents induced by flashes of 500- and 440-nm light in non-oriented cells. Photoelectrical responses of pre-oriented cells revealed that the latter effect is caused by an altered directional sensitivity of the antenna complex, whereas the functional state of the photoreceptor pigment is not strongly affected in mutant cells. Both the reflection intensity and the amplitude ratio of photoreceptor currents increased to the level of reference strains under conditions supporting the development of a well-organized eyespot in the mutant. Furthermore, incubation of the mutant with high concentrations of all-trans-retinal (10 M), independent of whether carotenoid biosynthesis was inhibited or not, was found to increase the reflection intensity of the eyespot. An increase in the rate of photoorientation of the mutant occurred concomitant with the increase in the reflective properties of the mutant eyespot. These observations demonstrate the importance of an intact eyespot for interference reflection and absorption of phototactically active light, and thus for the directional sensitivity of the eyespot apparatus.Abbreviations HSM high-salt medium This study was supported by the Deutsche Forschungsgemeinschaft. O. A. Sineshchekov was supported by a Research Fellowship from the Alexander von Humboldt Foundation. The authors wish to thank U. Powalowski (Botanisches Institut, Universität zu Köln) for help with electron microscopy.     

Removal of nitrogen by heterotrophic nitrification–aerobic denitrification of a novel halotolerant bacterium Pseudomonas mendocina TJPU04

Excess inorganic nitrogen in water poses a severe threat to enviroment. Removal of inorganic nitrogen by heterotrophic nitrifying–aerobic denitrifying microorganism is supposed to be a promising and applicable technology only if the removal rate can be maintained sufficiently high in real wastewater under various conditions, such as high concentration of salt and wide range of different nitrogen concentrations. Here, a new heterotrophic nitrifying–aerobic denitrifying bacterium was isolated and named as Pseudomonas mendocina TJPU04, which removes NH₄⁺-N, NO₃⁻-N and NO₂⁻-N with average rate of 4.69, 5.60, 4.99 mg/L/h, respectively. It also maintains high nitrogen removal efficiency over a wide range of nitrogen concentrations. When concentration of NH₄⁺-N, NO₃⁻-N and NO₂⁻-N was up to 150, 150 and 50 mg/L, 98%, 93%, and 100% removal efficiency could be obtained, respectively, after 30-h incubation under sterile condition. When it was applied under non-sterile condition, the ammonia removal efficiency was slightly lower than that under sterile condition. However, the nitrate and nitrite removal efficiencies under non-sterile condition were significantly higher than those under sterile condition. Strain TJPU04 also showed efficient nitrogen removal performance in the presence of high concentration of salt and nitrogen. In addition, the removal efficiencies of NH₄⁺-N, NO₃⁻-N and TN in real wastewater were 91%, 52%, and 75%, respectively. These results suggest that strain TJPU04 is a promising candidate for efficient removal of inorganic nitrogen in wastewater treatment.

     

Does Streptococcus mitis,a neonatal oropharyngeal bacterium,influence the pathogenicity of Pseudomonas aeruginosa?

Microorganisms in a biofilm might promote or suppress each other. We previously found that Pseudomonas aeruginosa (P. aeruginosa) and the normal colonized bacteria in the oropharynx, Streptococcus mitis (S. mitis), were the most common bacteria in the biofilm found on newborns' endotracheal tubes. Here, we found that S. mitis enhanced the adhesion and biofilm formation of P. aeruginosa. Furthermore, it alleviated the immune response induced by P. aeruginosa. These findings remind us that we should not ignore the role of traditionally viewed non-pathogenic bacteria in biofilms and provide new insights into exploring bacterial interaction mechanisms in biofilm related infections.     

Enhancement of bacteriophage λ stability using a λQ − S − mutant in the continuous culture of Escherichia coli

In this study, we used a bacteriophage λQ ⁻ S ⁻ mutant that increased the stability of recombinant Escherichia coli during continuous culture. The operation was conducted in two stages: the first stage was carried out to promote cell growth, and the second stage was performed for product formation. The productivity of recombinant proteins depends on the substrate concentration of the fresh medium supplied to the second stage (S ₃) and dilution rate of the second stage (D ₂). With the optimal value of S ₃ and D ₂, the first and second stages were stably maintained for 170 and 80 h, respectively. To further improve this process, a three-stage continuous process was conducted with an additional induction stage between the growth and production stages. Compared with the two-stage operation, the stable production period was extended by 1.7 fold, and the recombinant protein production increased by 1.3 fold.     

Molecular basis of α1-antitrypsin deficiency revealed by the structure of a domain-swapped trimer

α(1)-Antitrypsin (α1AT) deficiency is a disease with multiple manifestations, including cirrhosis and emphysema, caused by the accumulation of stable polymers of mutant protein in the endoplasmic reticulum of hepatocytes. However, the molecular basis of misfolding and polymerization remain unknown. We produced and crystallized a trimeric form of α1AT that is recognized by an antibody specific for the pathological polymer. Unexpectedly, this structure reveals a polymeric linkage mediated by domain swapping the carboxy-terminal 34 residues. Disulphide-trapping and antibody-binding studies further demonstrate that runaway C-terminal domain swapping, rather than the s4A/s5A domain swap previously proposed, underlies polymerization of the common Z-mutant of α1AT in vivo.     

Novel adaptive responses revealed by transcription profiling of a Synechocystis sp. PCC 6803 ΔisiA mutant in the presence and absence of hydrogen peroxide

The isiAB genes have proven to be highly stress-responsive under a variety of environmental conditions, including iron deficiency, high salt and oxidative stress. In order to understand the function of IsiA and its importance in oxidative stress, we constructed a knock out mutant of the isiA gene and compared differential gene expression of the DeltaisiA strain in the presence and absence of H2O2. We used the full genome microarray for the cyanobacterium Synechocystis sp. PCC 6803 as previously described [Postier BL, Wang HL, Singh A, Impson L, Andrews, HL, Klahn J, Li H, Risinger G, Pesta D, Deyholos M, Galbraith DW, Sherman LA and Burnap RL (2003) BMC Genenomics 4: 23-34]. We determined that one of the main differences in DeltaisiA compared to wild-type (in the absence of peroxide) was the induction of a gene cluster (sll1693-sll1696) that encoded genes resembling pilins or general secretory proteins (Gsp). These proteins are targeted to the cytoplasmic membrane and we suggest that they may be involved in the assembly of membrane complexes, including pigment-protein complexes. The DeltaisiA strain was more resistant to H2O2 compared to the wild-type. In the presence of 1.5 mM H2O2 for 30 min, a cluster of genes that includes a peroxiredoxin was induced 7- to 8-fold and we suggest that this peroxide scavenging enzyme is responsible for the increased peroxide resistance of the DeltaisiA strain.     

Resistance to the third-generation cephalosporin ceftazidime by a deacylation-deficient mutant of the TEM β-lactamase by the uncommon covalent-trapping mechanism

The Glu166Arg/Met182Thr mutant of Escherichia coli TEM(pTZ19-3) β-lactamase produces a 128-fold increase in the level of resistance to the antibiotic ceftazidime in comparison to that of the parental wild-type enzyme. The single Glu166Arg mutation resulted in a dramatic decrease in both the level of enzyme expression in bacteria and the resistance to penicillins, with a concomitant 4-fold increase in the resistance to ceftazidime, a third-generation cephalosporin. Introduction of the second amino acid substitution, Met182Thr, restored enzyme expression to a level comparable to that of the wild-type enzyme and resulted in an additional 32-fold increase in the minimal inhibitory concentration of ceftazidime to 64 μg/mL. The double mutant formed a stable covalent complex with ceftazidime that remained intact for the entire duration of the monitoring, which exceeded a time period of 40 bacterial generations. Compared to those of the wild-type enzyme, the affinity of the TEM(pTZ19-3) Glu166Arg/Met182Thr mutant for ceftazidime increased by at least 110-fold and the acylation rate constant was augmented by at least 16-fold. The collective experimental data and computer modeling indicate that the deacylation-deficient Glu166Arg/Met182Thr mutant of TEM(pTZ19-3) produces resistance to the third-generation cephalosporin ceftazidime by an uncommon covalent-trapping mechanism. This is the first documentation of such a mechanism by a class A β-lactamase in a manifestation of resistance.     

Characteristics and flocculating mechanism of a novel bioflocculant HBF-3 produced by deep-sea bacterium mutant Halomonas sp. V3a’

A novel bioflocculant HBF-3 produced by deep-sea bacterium mutant Halomonas sp. V3a’ was investigated with regard to its flocculating characteristics and mechanism. 4.0 m g l⁻¹ HBF-3 showed the maximum flocculating activity of 96.9% in 5.0 g l⁻¹ Kaolin suspension containing 11.25 mM CaCl₂, and that its flocculating activity was more than 90% within 5–40°C and over 80% in a wide pH range (3.0–11.0). Chemical analyses indicated that the biopolymer HBF-3 was mainly a polysaccharide, including neutral sugar residues (20.6%), uronic acid residues (7.6%), amino sugar residues (1.6%) and sulfate groups (5.3%). Fourier transform infrared (FTIR) spectrum showed the presence of carboxyl and hydroxyl groups in HBF-3 molecular. The average molecular mass of HBF-3, as determined by gel filtration chromatography (GFC), was approximately 590 kDa. Flocculation of Kaolin suspension with HBF-3 acted as a model to explore the flocculating mechanism in which bridging mediated by Ca²⁺ was proposed as the primary action based upon the experimental observations.     

Liquid-chromatographic analysis of the depolymerization of (1→4)-β-d-mannuronan by an extracellular alginate lyase from a marine bacterium

The extracellular alginate lyase activity from a fermentative marine bacterium isolated from actively growing tissues of Sargassum fluitans has been purified and studied with respect to substrate specificity and mechanism. The enzyme endolytically depolymerizes (1→4)-β-d-mannuronan derived from alginate to oligomeric products possessing 4,5-unsaturated, nonreducing termini. Reversed-phase liquid chromatography has established that early in the reaction the tri-, tetra-, and pentameric oligomers are the predominant species. The pentamer and larger products that at first accumulate in the reaction are later degraded to smaller products. The trimer is the major product late in the reaction, at which time the dimer and tetramer are also present in significant amounts. By incubating purified oligomers with enzyme, the trimer is shown to be completely refractory to further depolymerization and therefore represents a limit product of the reaction catalyzed by this enzyme. The tetramer is slowly converted into trimer and monomer, whereas the pentamer is readily converted into trimer and dimer.     

Retion of human apolipoprotein A-II by a mutant of Saccharomyces cerevisiæ

Summary A modified cDNA of mature human apolipoprotein A-II (apoA-II) was expressed by a sterol-uptake yeast strain (erg 10) of Saccharomyces cerevisiæ. ApoA-II cDNA was fused with a modified yeast alpha factor leader peptide coding sequence under phosphoglycerate kinase promoter control in a 2 micron-based plasmid construction. The use of cholesterol allowed apoA-II secretion in the culture medium. The mono and dimeric forms of apoA-II (approximately 3 micrograms per ml) were detected.     

Genome evolution trend of common carp （Cyprinus carpio L.） as revealed by the analysis of microsatellite loci in a gynogentic family

Genome evolution arises from two main ways of duplication and reduction. Fish specific genome duplication （FSGD） may have occurred before the radiation of the teleosts. Common carp （Cyprinus carpio L.） has been considered to be a tetraploid species, because of its chromosome numbers （2n=100） and its high DNA content. Using 69 microsatellite primer pairs, the variations were studied to better understand the genome evolution （genome duplication and diploidization） of common carp from a gynogenetic family. About 48% of primer pairs were estimated to amplify duplicates based on the number of PCR amplification per individual. Segregation patterns in the family suggested a partially duplicated genome structure and disomic inheritance. This indicates that the common carp is tetraploid and polyploidy occurred by allotetraploidy. Two primer pairs （HLJ021 and HLJ332） were estimated to amplify reduction based on the number of PCR amplification per individual. One allele in HLJ002 locus and HLJ332 locus was clearly lost in the gynogenetic family and the same as in six wild populations. Segregation patterns in the family suggested a partially diplodization genome structure. A hypothesis transition （dynamic） and equilibrium （static） were proposed to explain the common carp genome evolution between genome duplication and diploidization.     

Effect of laminar flow velocity on the kinetics of surface recolonization by Mot+ and Mot− Pseudomonas fluorescens

Computer-enhanced microscopy (CEM) was used to monitor bacteria colonizing the inner surfaces of a 1×3 mm glass flow cell. Image analysis provided a rapid and reliable means of measuring microcolony count, microcolony area, and cell motility. The kinetics of motile and nonmotilePseudomonas fluorescens surface colonization were compared at flow velocities above (120m sec^–1) and below (8m sec^–1) the strain's maximum motility rate (85m sec^–1). A direct attachment assay confirmed that flagellated cells undergo initial attachment more rapidly than nonflagellated cells at high and low flow. During continuous-flow slide culture, neither the rate of growth nor the timing of recolonization (cell redistribution within surface microenvironments) were influenced by flow rate or motility. However, the amount of reattachment of recolonizing cells was both flow and motility dependent. At 8m sec^–1 flow, motility increased reattachment sixfold, whereas at 120m sec^–1 flow, motility increased reattachment fourfold. The spatial distribution of recolonizing cells was also influenced by motility. Motile cells dispersed over surfaces more uniformly (mean distance to the nearest neighbor was 47.0m) than nonmotile cells (mean distance was 14.2m) allowing uniform biofilm development through more effective redistribution of cells over the surface during recolonization. In addition, motile cell backgrowth (where cells colonize against laminar flow) occurred four times more rapidly than nonmotile cell backgrowth at low flow (where rate of motility exceeded flow), and twice as rapidly at high flow (where flow exceeded the rate of motility). The observed backgrowth of Mot⁺ cells against high flow could only have occurred as the result of motile attachment behavior. These results confirm the importance of motility as a behavioral mechanism in colonization and provides an explanation for enhanced colonization by motile cells in environments lacking concentration gradients necessary for chemotactic behavior.     

Kinetics of improved productivity of β-galactosidase by a cycloheximide-resistant mutant of Kluyveromyces marxianus

The maximum volumetric productivity of beta-galactosidase by a Kluyveromyces marxianus mutant, grown on lactose/corn steep liquor medium for 3 d, was 150 IU l(-1) h(-1) which is twice that of the parent organism. During product formation, mutated cells provided more resistance against thermal inactivation.     

Antigenic similarity between the β-subunits of the ATPases of a bacterium,a yeast and a higher plant

Antiserum raised against the β-subunit of wheat (Triticum aestivum) chloroplast ATPase cross-reacts with a 51000 protein located in the membrane fraction of Escherichia coli. The differential solubility of this polypeptide after chloroform treatment of unc⁺ and uncD409 strains indicates that this cross-reacting polypeptide is the bacterial β-subunit of ATPase. Thus a high degree of conservation of antigenic determinant sites exists between a bacterial β-subunit and the β-subunit of a monocot. This conservation also seems to extend to the β-subunit of mitochondrial ATPase of yeast (Saccharomyces cerevisiae).     

Volatile compounds associated to the loss of astringency in persimmon fruit revealed by untargeted GC–MS analysis

High resolution volatile profiling (67 compounds identified) of fruits from 12 persimmon cultivars was established and used to characterize the different astringency types of persimmon fruit before and after deastringency treatment. Analysis of the volatile profile of fruit enables us to differentiate between cultivars that at the moment of harvest produced non-astringent fruit (Pollination Constant Non Astringent—PCNA-type) from astringent ones (non-PCNA-type). Fruit failing to accumulate astringent compounds naturally (PCNA fruit) showed high levels of 3(2H)-benzofuranone, while this compound was not detected in any astringent type fruit (non-PCNA). In addition to this, PCNA cultivars also showed at harvest higher accumulation of benzeneacetaldehyde and lipid-derived aldehydes (hexanal, heptanal, octanal and decanal) than non-PCNA fruit. The application of postharvest deastringency treatment to all non-PCNA cultivars resulted on an important insolubilization of tannins. In general the CO₂-treatment enhanced the levels of acetaldehyde, however those cultivars showing high levels of dihydrobenzofuran at harvest did not present an increment of acetaldehyde. In contrast, all non-PCNA cultivars exhibited an important accumulation of lipid-derived aldehydes due to CO₂-treatment. Therefore, we propose that lipid-derived aldehydes (mainly decanal, octanal and heptanal) may be playing a role in the astringency loss. Our results suggest that 3(2H)-benzofuranone, benzeneacetaldehyde and lipid-derived aldehydes could be used as markers for both natural and artificial loss of astringency.