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Archives of Microbiology (v.185, #6)
Characterization of a mutant strain of Rhodovulum sulfidophilum lacking the pufA and pufB genes encoding the polypeptides for the light-harvesting complex 1 (B 870) by Laura J. Raiger-Iustman; Norma L. Kerber; Norma L. Pucheu; Marc J. Bornmann; Simon Kohler; Andreas Labahn; Monier Tadros; Gerhart Drews; Augusto F. García (pp. 407-415).
Contradictory results on the effectiveness of energy transfer from the light harvesting complex 2 (LH2) directly to the reaction center (RC) in mutant strains lacking the core light-harvesting complex 1 (LH1) have been obtained with cells of Rhodobacter capsulatus and Rhodobacter sphaeroides. A LH1− mutant of Rhodovulum sulfidophilum, named rsLRI, was constructed by deletion of the pufBA genes, resulting in a kanamycin resistant photosynthetically positive clone. To restore the wild type phenotype, a complemented strain C2 was constructed by inserting in trans a DNA segment containing the pufBA genes. Light-induced FTIR difference spectra indicate that the RC in the rsLRI mutant and in the C2 complemented strains are functionally and structurally identical with those in the wild type strain, demonstrating that the assembly and the function of the RC is not impaired by the LH1 deletion. The photosynthetic growth rate of the rsLRI strain increased with decreasing light intensity. At 50 W m−2 no photosynthetic growth was observed. These results indicate that the light energy harvested by the LH2 complex was not or inefficiently transferred to the RC; thus most of the energy necessary for photosynthetic growth is in the LH1− strain directly absorbed by the RC. It is supposed that in the mutant strain, RC and LH2 cannot interact in an efficient way.
Keywords: Photosynthetic bacteria; Light-harvesting polypeptides; Energy transfer; Deletion mutation; Reaction center
Cell aggregation of Pseudomonas aeruginosa strain PAO1 as an energy-dependent stress response during growth with sodium dodecyl sulfate by Janosch Klebensberger; Oliver Rui; Eva Fritz; Bernhard Schink; Bodo Philipp (pp. 417-427).
Pseudomonas aeruginosa strain PAO1 grew with the detergent sodium dodecyl sulfate (SDS). The growth started with the formation of macroscopic cell aggregates which consisted of respiring cells embedded in an extracellular matrix composed of acidic polysaccharides and DNA. Damaged and uncultivable cells accumulated in these aggregates compared to those cells that remained suspended. We investigated the response of suspended cells to SDS under different conditions. At high energy supply, the cells responded with a decrease in optical density and in viable counts, release of protein and DNA, and formation of macroscopic aggregates. This response was not observed if the energy supply was reduced by inhibiting respiration with KCN, or if cells not induced for SDS degradation were exposed to SDS. Exposure to SDS caused cell lysis without aggregation if cells were completely deprived of energy, either by applying anoxic conditions, by addition of CCCP, or by addition of KCN to a mutant defective in cyanide-insensitive respiration. Aggregated cells showed a more than 100-fold higher survival rate after exposure to SDS plus CCCP than suspended cells. Our results demonstrate that cell aggregation is an energy-dependent response of P. aeruginosa to detergent stress which might serve as a survival strategy during growth with SDS.
Keywords: Cell aggregation; Pseudomonas aeruginosa ; SDS; Detergent degradation; Stress response
Effect of anaerobic and stationary phase growth conditions on the heat shock and oxidative stress responses in Escherichia coli K-12 by Alondra Díaz-Acosta; María L. Sandoval; Luis Delgado-Olivares; Jorge Membrillo-Hernández (pp. 429-438).
The natural living style of Escherichia coli occurs in the gastrointestinal tract, where most of its existence is spent under anaerobic conditions and in stationary phase of growth. Here we report on the heat shock response of E. coli K-12 cells growing in the presence or absence of oxygen. An rpoH mutant (impaired in the synthesis of the σ32 transcriptional factor) exhibited an increased sensitivity to heat shock but only in the exponential phase of aerobic growth, suggesting that in anaerobic growth conditions, and in aerobic stationary phase, σ32-independent mechanisms are playing a prime role in protecting cells from heat stress. Our results demonstrated that σS is not involved in this protection system. Studies on the kinetics of synthesis of Heat shock proteins (Hsp) after an abrupt rise in temperature demonstrated that in the absence of oxygen, the synthesis of Hsp is triggered faster and is sustained for a longer period of time compared to aerobic growth conditions. Finally, the heated cells in the exponential phase of aerobic growth displayed a high concentration of oxidatively damaged proteins in the presence of 4 mM H2O2, in sharp contrast to cultures of stationary phase or anaerobic growth.
Keywords: E. coli ; rpoS ; rpoH ; Heat shock; Bacteria; Oxygen; Oxidative stress
A neutral protease from Bacillus nematocida, another potential virulence factor in the infection against nematodes by Qiuhong Niu; Xiaowei Huang; Lin Zhang; Yunxia Li; Juan Li; Jinkui Yang; Keqin Zhang (pp. 439-448).
A neutral protease (npr) (designated Bae16) toxic to nematodes was purified to homogeneity from the strain Bacillus nematocida. The purified protease showed a molecular mass of approximately 40 kDa and displayed optimal activity at 55°C, pH 6.5. Bioassay experiments demonstrated that this purified protease could destroy the nematode cuticle and its hydrolytic substrates included gelatin and collagen. The gene encoding Bae16 was cloned, and the deduced amino acid sequence showed 94% sequence identity with npr gene from B. amyloliquefaciens, but had low similarity (13–43%) with the previously reported virulence serine proteases from fungi or bacteria, which reflected their differences. Recombinant mature Bae16 (rm-Bae16) was expressed in Escherichia coli BL21 using pET30 vector system, and its nematicidal activity confirmed that Bae16 could be involved in the infection process. Our present study revealed that the npr besides the known alkaline serine protease could serve as a potential virulence factor in the infection against nematodes, furthermore, the two proteases with different characteristics produced by the same strain co-ordinated efforts to kill nematodes. These data helped to understand the interaction between this bacterial pathogen and its host.
Keywords: Neutral protease; Nematicidal activity; Virulence factor; Bacillus nematocida
Detection of a Phage Genome Carrying a Zonula Occludens like Toxin Gene (zot) in clinical isolates of Stenotrophomonas maltophilia by Martin Hagemann; Dirk Hasse; Gabriele Berg (pp. 449-458).
During a study of the genetic diversity of Stenotrophomonas strains, we found an autonomous replicating DNA molecule in chromosomal DNA preparations of the clinical Stenotrophomonas maltophilia strain c5. The entire sequence of 6,907 bp of the isolated DNA molecule was determined, which was called φSMA9. Seven ORFs, which code for proteins with considerable similarity to proteins in databases, were identified in the DNA sequence. The largest ORF shows high sequence similarities to the pI protein of the filamentous phage φLf, which was later shown to be identical to toxin Zot of Vibrio cholerae. Beside the Zot-like protein, six other proteins with similarities to known phage proteins such as a phage replication protein RstA and phage absorption or coat protein are encoded on φSMA9, which indicate that this circular DNA molecule represents the replicative form of a linear phage genome. A PCR-based screening showed that only five from the totally investigated 47 Stenotrophomonas strains of clinical and environmental origin harbor these genes. Altogether, we describe the first genome of a phage for the nosocomial pathogen Stenotrophomonas, which contains a Zot toxin like gene and might be regarded as the first Stenotrophomonas virulence factor.
Keywords: Clinical isolates; Cluster analysis; Phage sequence
Oenococcus oeni preference for peptides: qualitative and quantitative analysis of nitrogen assimilation by Fabienne Remize; Aurélie Gaudin; Yu Kong; Jean Guzzo; Hervé Alexandre; Sibylle Krieger; Michèle Guilloux-Benatier (pp. 459-469).
Optimization of malolactic fermentation in wine depends mainly on better understanding of nitrogen nutritional requirements of Oenococcus oeni. Four widely used starter strains and the reference ATCC BAA-1163 strain were grown in media containing different N sources: free amino acids, oligopeptides (0.5–10 kDa) or polypeptides (> 10 kDa). Amino acid auxotrophies were determined by the single omission technique. The tested strains were indifferent to only two to four amino acids and two of the starter strains appeared to be particularly demanding. Nitrogen consumption was investigated and a significant level of nitrogen was consumed by O. oeni only in the free amino acid medium. In media containing complex nitrogen sources, a global balance above 5 mg N l−1 was enough to ensure biomass formation of all tested strains. Moreover, for all strains, bacterial growth yield was higher in the presence of nitrogen from peptides than that from free amino acids. However, no direct relationship between the bacterial growth level and the amount of nitrogen metabolized could be established. These findings were discussed in relation to the physiology of wine malolactic bacteria.
Keywords: Lactic acid bacteria; Oenococcus ; Wine; Metabolism; Nitrogen; Peptide; Amino acid
Roles for heme–copper oxidases in extreme high-light and oxidative stress response in the cyanobacterium Synechococcus sp. PCC 7002 by Christopher T. Nomura; Toshio Sakamoto; Donald A. Bryant (pp. 471-479).
The ctaCIDIEI and ctaCIIDIIEII gene clusters that encode heme–copper cytochrome oxidases have been characterized in the marine cyanobacterium Synechococcus sp. PCC 7002 and the inactivation of ctaDI was shown to affect high-light adaptation. In this study, Synechococcus sp. PCC 7002 wild-type, ctaDI, ctaDII, and ctaDI–ctaDII double mutants were grown under extreme high-light and oxidative stress to further assess the roles of cytochrome oxidases in cyanobacteria. Cells of the ctaDI mutant strain barely grew under extreme high-light illumination of 4.5 mE m−2 s−1, suggesting that CtaDI is required for high-light acclimation in Synechococcus sp. PCC 7002. The ctaDI–ctaDII double mutant cells unexpectedly tolerated extreme high-light intensity, indicating that the disruption of ctaDII gene suppresses the high-light sensitivity phenotype of the ctaDI single mutant. The ctaDII mutant cells also exhibited higher tolerance to the oxidative stress compound, methyl viologen, in the growth media. The ctaDII mutant and the ctaDI–ctaDII double mutant cells had approximately twofold higher levels of superoxide dismutase (SOD) activity, indicating that the disruption of ctaDII gene increased the capacity to decompose active oxygen species. These results suggest that the CtaII cytochrome oxidase may be involved with the oxidative stress response, including the control of SOD expression.
Keywords: Cyanobacteria; Heme–copper oxidases; High-light stress; Oxidative stress
3-Methylglutaconyl-CoA hydratase from Acinetobacter sp
by Matthias Mack; Michael Liesert; Johannes Zschocke; Verena Peters; Dietmar Linder; Wolfgang Buckel (pp. 481-481).