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Archives of Microbiology (v.180, #5)
Poly β-hydroxybutyrate depolymerase (PhaZ) in Azospirillum brasilense and characterization of a phaZ mutant by Daniel Kadouri; Edouard Jurkevitch; Yaacov Okon (pp. 309-318).
Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly-β-hydroxybutyrate (PHB) under sub-optimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments. PHB depolymerase (PhaZ) is an essential enzyme in PHB degradation. The phaZ gene was identified in Azospirillum brasilense, cloned, sequenced, and shown to be located on the chromosome. Insertion of a kanamycin-resistant cassette within phaZ of A. brasilense resulted in a phaZ mutant that was unable to degrade PHB; however, carbon source utilization was similar in both the wild-type and the mutant strain. The ability of the wild-type to endure starvation conditions, ultraviolet irradiation, heat, and osmotic shock, and to grow in the presence of hydrogen peroxide was higher than that of the mutant strain. By contrast, the ability of the phaZ mutant strain to endure desiccation was higher than that of the wild-type strain. No differences between the strains were seen in their ability to endure sonication, or to survive in carrier materials used for soil inoculants. In addition, motility was the same between the two strains, whereas cell aggregation and exopolysaccharide production were higher in the wild-type than in the phaZ mutant strain.
Keywords: Poly-β-hydroxybutyrate (PHB); PHA depolymerase (Phaz); Azospirillum brasilense
An additional regulator, TsaQ, is involved with TsaR in regulation of transport during the degradation of p-toluenesulfonate in Comamonas testosteroni T-2 by Tewes Tralau; Alasdair M. Cook; Jürgen Ruff (pp. 319-326).
The degradation of p-toluenesulfonate (TSA) by Comamonas testosteroni T-2 is initiated by a transport system (TsaST) and enzymes (TsaMBCD) encoded on the tsa transposon, Tntsa, on the TSA plasmid (pTSA). Tntsa comprises an insert of 15 kb between two IS1071 elements. The left-hand 6 kb and the right-hand 6 kb are nearly mirror images. The regulator of the tsaMBCD 1 genes (right-hand side) is the centrally located LysR-type TsaR, which is encoded upstream of tsaMBCD 1 on the reverse strand. The other centrally located genes are tsaS and tsaT, encoded downstream of tsaR and on the same strand as both tsaR and tsaMBCD 2. The latter four genes are not expressed. Downstream of tsaD 1 (tsaD 2) is tsaQ 1 (tsaQ 2) and another open reading frame of unknown function. The tsaQ genes have identical sequences. Sequence analysis indicated that TsaQ could be an IclR-type regulator, whose expression during degradation of TSA was proven by data from RT-PCR. Both copies of tsaQ could be knocked-out by homologous recombination. Double mutants failed to grow with TSA but grew with p-toluenecarboxylate (TCA), which is also degraded via TsaMBCD. This showed TsaQ to be essential for the degradation of TSA but not TCA. We attributed this to regulation of the transport of TSA, especially to regulation of the expression of tsaT, which was expressed solely during growth with TSA. Seven independently isolated bacteria containing the tsa operon were available. Those six which contained tsaT on Tntsa also contained tsaQ. The promoter region of tsaT was found to be a target of the regulator TsaR. Band-shift data indicate that TsaR is required for the expression of tsaT, which suggests that tsaR and tsaQ 1,2, together with tsaMBCD 1, belong to a common regulatory unit.
Keywords: Regulation; Degradation; Transport; Toluene sulfonate; IclR family; LysR family
Isolation and characterization of Erythrobacter sp. strains from the upper ocean by Michal Koblížek; Oded Béjà; Robert R. Bidigare; Stephanie Christensen; Bryan Benitez-Nelson; Costantino Vetriani; Marcin K. Kolber; Paul G. Falkowski; Zbigniew S. Kolber (pp. 327-338).
Seven strains of marine aerobic anoxygenic phototrophs belonging to the genus Erythrobacter were isolated. The strains were characterized regarding their physiological and biochemical properties, 16S rDNA and pufM gene sequences, morphological features, substrate preference, as well as pigment and lipid composition. All strains had functional type-2 reaction centers containing bacteriochlorophyll, served by small, light-harvesting complex 1, and were photosynthetically competent. In addition, large pools of carotenoids were found, but only some of the accessory pigments transfer energy to the reaction centers. All of the isolates were facultative photoheterotrophs. They required an organic carbon substrate for growth; however, they are able to supplement a significant fraction of their metabolic requirements with photosynthetically derived energy.
Keywords: Aerobic anoxygenic phototrophs; Aerobic photosynthetic bacteria; Bacteriochlorophyll a ; Erythrobacter ; Photoheterotrophy
Chromosomally encoded gyrase inhibitor GyrI protects Escherichia coli against DNA-damaging agents by Monalisa Chatterji; Sugopa Sengupta; Valakunja Nagaraja (pp. 339-346).
DNA gyrase, a type II topoisomerase, is the sole supercoiling activity in the cell and is essential for cell survival. There are two proteinaceous inhibitors of DNA gyrase that are plasmid-borne and ensure maintenance of the plasmids in bacterial populations. However, the physiological role of GyrI, an inhibitor of DNA gyrase encoded by the Escherichia coli genome, has been elusive. Previously, we have shown that GyrI imparts resistance against microcin B17 and CcdB. Here, we find that GyrI provided partial/limited protection against the quinolone class of gyrase inhibitors but had no effect on inhibitors that interfere with the ATPase activity of the enzyme. Moreover, GyrI negated the effect of alkylating agents, such as mitomycin C and N-methyl-N-nitro-N-nitrosoguanidine, that act independently of DNA gyrase. Hence, in vivo, GyrI appears to be involved in reducing DNA damage from many sources. In contrast, GyrI is not effective against lesions induced by ultraviolet radiation. Furthermore, the expression of GyrI does not significantly alter the topology of DNA. Thus, although isolated as an inhibitor of DNA gyrase, GyrI seems to have a broader role in vivo than previously envisaged.
Keywords: DNA gyrase; Microcin B17; GyrI; SbmC; CcdB
Influence of arsenic on antimony methylation by the aerobic yeast Cryptococcus humicolus by Louise M. Hartmann; Peter J. Craig; Richard O. Jenkins (pp. 347-352).
The anamorphic basidomycetous yeast Cryptococcus humicolus was shown by hydride generation-gas chromatography-atomic absorption spectrometry to methylate inorganic antimony compounds to mono-, di-, and trimethylantimony species under oxic growth conditions. Methylantimony levels were positively correlated with initial substrate concentrations up to 300 mg Sb l−1 as potassium antimony tartrate (K-Sb-tartrate). Increasing concentrations of K-Sb-tartrate increased the ratio of di- to trimethylantimony species, indicating that methylation of dimethylantimony was rate limiting. Antimony methylation capability in C. humicolus was developed after the exponential growth phase and was dependent upon protein synthesis in the early stationary phase. Inclusion of inorganic arsenic (III) or (V) species alongside antimony in culture incubations enhanced antimony methylation. Pre-incubation of cells with inorganic arsenic (III) further induced antimony methylation capability, whereas pre-incubation with inorganic antimony (III) did not. Exposure of cells to inorganic arsenic—either through pre-incubation or provision during cultivation—influenced the antimony speciation; involatile trimethylantimony species was the sole methylated antimony species detected, i.e. mono- and dimethylantimony species were not detected. Competitive inhibition of antimony methylation was observed at high arsenic loadings. These data indicate that antimony methylation is a fortuitous process, catalysed at least in part by enzymes responsible for arsenic methylation.
Keywords: Arsenic; Antimony; Trimethylantimony; Cryptococcus humicolus ; Yeast; Methylation; Competitive inhibition; Induction
PduP is a coenzyme-a-acylating propionaldehyde dehydrogenase associated with the polyhedral bodies involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar Typhimurium LT2 by Nicole A. Leal; Gregory D. Havemann; Thomas A. Bobik (pp. 353-361).
Salmonella enterica forms polyhedral bodies involved in coenzyme-B12-dependent 1,2-propanediol degradation. Prior studies showed that these bodies consist of a proteinaceous shell partly composed of the PduA protein, coenzyme-B12-dependent diol dehydratase, and additional unidentified proteins. In this report, we show that the PduP protein is a polyhedral-body-associated CoA-acylating aldehyde dehydrogenase important for 1,2-propanediol degradation by S. enterica. A PCR-based method was used to construct a precise nonpolar deletion of the gene pduP. The resulting pduP deletion strain grew poorly on 1,2-propanediol minimal medium and expressed 105-fold less propionaldehyde dehydrogenase activity (0.011 μmol min−1 mg−1) than did wild-type S. enterica grown under similar conditions (1.15 μmol min−1 mg−1). An Escherichia coli strain was constructed for high-level production of His8-PduP, which was purified by nickel-affinity chromatography and shown to have 15.2 μmol min−1 mg−1 propionaldehyde dehydrogenase activity. Analysis of assay mixtures by reverse-phase HPLC and mass spectrometry established that propionyl-CoA was the product of the PduP reaction. For subcellular localization, purified His8-PduP was used as antigen for the preparation of polyclonal antiserum. The antiserum obtained was shown to have high specificity for the PduP protein and was used in immunogold electron microscopy studies, which indicated that PduP was associated with the polyhedral bodies involved in 1,2-propanediol degradation. Further evidence for the localization of the PduP enzyme was obtained by showing that propionaldehyde dehydrogenase activity co-purified with the polyhedral bodies. The fact that both Ado-B12-dependent diol dehydratase and propionaldehyde dehydrogenase are associated with the polyhedral bodies is consistent with the proposal that these structures function to minimize propionaldehyde toxicity during the growth of S. enterica on 1,2-propanediol.
Keywords: 1,2-Propanediol; B12; Polyhedral organelles; Aldehyde dehydrogenase; Carboxysomes
The acrAB locus is involved in modulating intracellular acetyl coenzyme A levels in a strain of Escherichia coli CM2555 expressing the chloramphenicol acetyltransferase (cat) gene by Joanna Potrykus; Grzegorz Wegrzyn (pp. 362-366).
Recently, an Escherichia coli CM2555 strain was described as sensitive to chloramphenicol when expressing the chloramphenicol resistance gene (cat) from a multicopy plasmid. This sensitivity was linked to dysfunction of the acrA gene, which encodes a component of the AcrAB-TolC multidrug efflux pump. Preliminary data indicate that the sensitivity phenotype might be due to a decline in intracellular acetyl coenzyme A concentration accompanying the reaction catalyzed by chloramphenicol acetyltransferase, the cat-encoded resistance protein. Here, we demonstrate that the acrA dysfunction is the factor impairing the intracellular acetyl coenzyme A levels in the cat-expressing CM2555 strain. This effect might be alleviated by the interplay of proteins constituting two homologous efflux systems: AcrAB-TolC and AcrEF-TolC. However, our results show also that this is a genetic background-specific phenomenon, as the decrease in acetyl coenzyme A level is not evident in a cat-bearing ΔacrAB derivative of the commonly used strain C600.
Keywords: Chloramphenicol resistance; Efflux pumps; AcrAB; AcrEF; Acetyl coenzyme A
Conjugative plasmid pIP501 undergoes specific deletions after transfer from Lactococcus lactis to Oenococcus oeni by Manuel Zúñiga; Isabel Pardo; Sergi Ferrer (pp. 367-373).
Conjugal transfer of plasmids pIP501 and its derivative pVA797 from Lactococcus lactis to Oenococcus oeni was assayed by filter mating. Plasmid pIP501 was transferred to a number of O. oeni strains whereas a single transconjugant of O. oeni M42 was recovered when pVA797 was used. Physical analysis of the transconjugant plasmids revealed that pIP501 and pVA797 underwent extensive deletions in O. oeni that affected the tra region (conjugal transfer) and SegB region (stability). All derivatives showed segregational instability in O. oeni, but were stably maintained in L. lactis. These differences correlated with the different plasmid copy numbers and the extent of deletions within the SegB region.
Keywords: Oenococcus oeni ; Lactococcus lactis ; pIP501; pVA797; Conjugation.
Transcriptome analysis of the Pseudomonas aeruginosa response to iron by Marco Palma; Stefan Worgall; Luis E. N. Quadri (pp. 374-379).
To successfully infect humans, Pseudomonas aeruginosa (Pa) must overcome the low iron availability in host tissues. A transcriptome comparison was carried out between iron-starved cells of Pa treated with iron and untreated controls. The present study is the first global analysis of the early transcriptional response of exponentially growing Pa to iron. Approximately 1.3% of the Pa genes displayed ≥5.0-fold changes in mRNA levels in iron-treated cells. Treatment affected the mRNA levels of many genes required for iron acquisition as well as several genes with relevance to virulence previously known to be regulated by iron. More importantly, the analysis permitted identification of 107 Pa genes whose mRNA levels were not previously known to be affected by iron. These genes are good candidates for mutagenesis studies aimed at identifying novel functions relevant to iron metabolism in Pa. Some of these genes encode predicted siderophore receptors, iron transport systems, TonB-dependent receptors, regulatory proteins, and proteins relevant to virulence. Notably, 49 genes encode hypothetical or conserved hypothetical proteins of unknown function, suggesting that they are involved directly or indirectly in iron metabolism or metabolic adaptation to different iron-availability conditions.
Keywords: Pseudomonas ; Microarray; Gene expression; Iron; Transcriptome; Virulence; Genomics
The ssgB gene, encoding a member of the regulon of stress-response sigma factor σH, is essential for aerial mycelium septation in Streptomyces coelicolor A3(2) by Beatrica Sevcikova; Jan Kormanec (pp. 380-384).
The Streptomyces coelicolor A3(2) gene ssgB belongs to the regulon of stress-response sigma factor σH. By integrative transformation via double cross-over, a stable null mutant of ssgB was obtained. This mutation had no obvious effect on vegetative growth, but critically affected aerial mycelium septation. The S. coelicolor ssgB mutant produced aerial hyphae without any signs of septation into spore compartments. The mutation was complemented in trans by wild-type ssgB including the σH-dependent ssgBp promoter. The results proved that ssgB belongs a developmental branch of the σH regulon.
Keywords: Differentiation; Gene disruption; Recombinant DNA; RNA polymerase; Sigma factor; Stress response; Streptomyces coelicolor A3(2)