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Archives of Microbiology (v.170, #4)

Methylglyoxal production in bacteria: suicide or survival? by G. P. Ferguson; S. Tötemeyer; M. J. MacLean; I. R. Booth (pp. 209-218).

Methylglyoxal is a toxic electrophile. In Escherichia coli cells, the principal route of methylglyoxal production is from dihydroxyacetone phosphate by the action of methylglyoxal synthase. The toxicity of methylglyoxal is believed to be due to its ability to interact with the nucleophilic centres of macromolecules such as DNA. Bacteria possess an array of detoxification pathways for methylglyoxal. In E. coli, glutathione-based detoxification is central to survival of exposure to methylglyoxal. The glutathione-dependent glyoxalase I-II pathway is the primary route of methylglyoxal detoxification, and the glutathione conjugates formed can activate the KefB and KefC potassium channels. The activation of these channels leads to a lowering of the intracellular pH of the bacterial cell, which protects against the toxic effects of electrophiles. In addition to the KefB and KefC systems, E. coli cells are equipped with a number of independent protective mechanisms whose purpose appears to be directed at ensuring the integrity of the DNA. A model of how these protective mechanisms function will be presented. The production of methylglyoxal by cells is a paradox that can be resolved by assigning an important role in adaptation to conditions of nutrient imbalance. Analysis of a methylglyoxal synthase-deficient mutant provides evidence that methylglyoxal production is required to allow growth under certain environmental conditions. The production of methylglyoxal may represent a high-risk strategy that facilitates adaptation, but which on failure leads to cell death. New strategies for antibacterial therapy may be based on undermining the detoxification and defence mechanisms coupled with deregulation of methylglyoxal synthesis.

Keywords: Key words Methylglyoxal; Cytoplasmic pH; Potassium; Glyoxalase; Glycolysis; Metabolism

Tetramethylammonium:coenzyme M methyltransferase system from Methanococcoides sp. by S. Asakawa; Karin Sauer; Werner Liesack; R. K. Thauer (pp. 220-226).

A methanogen (strain NaT1) that belongs to the family of Methanosarcinaceae and that can grow on tetramethylammonium as the sole energy source has recently been isolated. We report here that cell extracts of the archaeon catalyze the formation of methyl-coenzyme M from coenzyme M and tetramethylammonium. The activity was dependent on the presence of Ti(III) citrate and ATP, and was rapidly lost under oxic conditions. Anoxic chromatography on DEAE-Sepharose revealed that two fractions, fractions 3 and 4, were required for activity. A 50-kDa protein that together with fraction 3 catalyzed methyl-coenzyme M formation from tetramethylammonium and coenzyme M was purified from fraction 4. From fraction 3, a 22-kDa corrinoid protein and a 40-kDa protein exhibiting methylcobalamin:coenzyme M methyltransferase (MT2) activity were purified. The N-terminal amino acid sequences of these purified proteins were determined. The 40-kDa protein showed sequence similarity to MT2 isoenzymes from Methanosarcina barkeri. Cell extract of strain NaT1 grown on trimethylamine rather than on tetramethylammonium did not exhibit tetramethylammonium:coenzyme M methyltransferase activity. The strain was identified as belonging to the genus of Methanococcoides, its closest relative being Methanococcoides methylutens.

Keywords: Key words Methylamine metabolism; Methyltransferases; Corrinoid proteins; Methanogenic; archaea; Methanosarcina; Methanococcoides

Nucleoids and coated vesicles of “Epulopiscium” spp. by C. Robinow; Esther R. Angert (pp. 227-235).

We describe here aspects of the anatomy of two “Epulopiscium” morphotypes, unusually large bacteria that are not yet cultured and that reproduce by the internal generation of two or more vegetative daughter cells. Two morphotypes, A and B, which are enteric symbionts of several species of herbivorous surgeonfish (Acanthuridae), were collected around the Great Barrier Reef of Australia, preserved there, and later stained for light microscopy. Some samples were examined by electron microscopy. In both morphotypes, countless discrete nucleoplasms or nucleoids were found to occupy a single shallow layer just beneath the surface all around these organisms. At each end of the morphotype B cells, a membrane-bound compartment containing dense cords of chromatin was observed. When these were found at each end of growing daughter cells, no polar compartments were then found in their mother organism. Electron micrographs of sections of morphotype A symbionts show that their outermost region is composed of tightly packed coated vesicles, each surrounded by a thin, dense, spacious capsule. Near the surface of type A organisms the remains of broken vesicles, broken capsules, and a finely fibrous matrix fuse to form a fabric that serves as the cell wall. Morphotype B organisms, however, were observed to have a distinct, morphologically continuous outer wall.

Keywords: Key words ”Epulopiscium”; Nucleoids; Polar; chromatin; Coated vesicles; Unusual composite wall; Daughter cells

The Serratia marcescens hemolysin is secreted but not activated by stable protoplast-type L-forms of Proteus mirabilis by Stefan Sieben; Ralf Hertle; Johannes Gumpert; V. Braun (pp. 236-242).

The outer-membrane protein ShlB of Serratia marcescens activates and secretes hemolytic ShlA into the culture medium. Without ShlB, inactive ShlA (termed ShlA*) remains in the periplasm. Since Proteus mirabilis L-form cells lack an outer membrane and a periplasm, it was of interest to determine in which compartment recombinant ShlA* and ShlB are localized and whether ShlB activates ShlA*. The cloned shlB and shlA genes were transcribed in P. mirabilis stable L-form cells by the temperature-inducible phage T7 RNA polymerase. Radiolabeling, Western blotting, and complementation with C-terminally truncated ShlA (ShlA255) identified inactive ShlA* in the culture supernatant. ShlB remained cell-bound and did not activate ShlA without integration in an outer membrane. Although hemolytic ShlA added to L-form cells had access to the cytoplasmic membrane, it did not affect L-form cells. Synthesis of the large ShlA protein (165 kDa) in P. mirabilis L-form cells under phage T7 promoter control demonstrates that L-form cells are suitable for the synthesis and secretion of large recombinant proteins. This property and the easy isolation of released proteins make L-form cells suitable for the biotechnological production of proteins.

Keywords: Key words Hemolysin; Activation; Secretion; L-forms; Protoplasts; Serratia marcescens; Proteus mirabilis

High genetic and physiological diversity of sulfate-reducing bacteria isolated from an oligotrophic lake sediment by Henrik Sass; Elze Wieringa; Heribert Cypionka; Hans-Dietrich Babenzien; J. Overmann (pp. 243-251).

The community structure of sulfate-reducing bacteria in littoral and profundal sediments of the oligotrophic Lake Stechlin (Germany) was investigated. A collection of 32 strains was isolated from the highest positive dilutions of most-probable-number series, and their partial 16S rRNA gene sequences and genomic fingerprints based on ERIC (enterobacterial repetitive intergenic consensus)-PCR were analyzed. The strains fell into eight distinct phylogenetic lineages, and the majority (70%) showed a close affiliation to the genus Desulfovibrio. Most of the remaining strains (22%) were related to the gram-positive Sporomusa and Desulfotomaculum groups. A high redundancy of 16S rRNA gene sequences was found within several of the phylogenetic lineages. This low phylogenetic diversity was most pronounced for the subset of strains isolated from oxic sediment layers. ERIC-PCR revealed that most of the strains with identical 16S rRNA gene sequences were genetically different. Since strains with identical 16S rRNA gene sequences but different genomic fingerprints also differed considerably with respect to their physiological capabilities, the high diversity detected in the present work is very likely of ecological relevance. Our results indicate that a high diversity of sulfate-reducing bacterial strains can be recovered from the natural environment using the established cultivation media.

Keywords: Key words Sulfate-reducing bacteria; Molecular; fingerprinting; Bacterial diversity; Culturability

Environmental and physiological factors affecting the uptake of phosphate by Chlorobium limicola by L. Bañeras; L. J. Garcia-Gil (pp. 252-258).

The uptake of soluble phosphate by the green sulfur bacterium Chlorobium limicola UdG6040 was studied in batch culture and in continuous cultures operating at dilution rates of 0.042 or 0.064 h^–1. At higher dilution rates, washout occurred at phosphate concentrations below 7.1 μM. This concentration was reduced to 5.1 μM when lower dilution rates were used. The saturation constant for growth on phosphate (K _μ) was between 2.8 and 3.7 μM. The specific rates of phosphate uptake in continuous culture were fitted to a hyperbolic saturation model and yielded a maximum rate (Va _max) of 66 nmol P (mg protein)^–1 h^–1 and a saturation constant for transport (K _t) of 1.6 μM. In batch cultures specific rates of phosphate uptake up to 144 nmol P (mg protein)^–1 h^–1 were measured. This indicates a difference between the potential transport of cells and the utilization of soluble phosphate for growth, which results in a significant change in the specific phosphorus content. The phosphorus accumulated within the cells ranged from 0.4 to 1.1 μmol P (mg protein)^–1 depending on the growth conditions and the availability of external phosphate. Transport rates of phosphate increased in response to sudden increases in soluble phosphate, even in exponentially growing cultures. This is interpreted as an advantage that enables Chl. limicola to thrive in changing environments.

Keywords: Key wordsChlorobium limicola; Green sulfur bacteria; Phosphate limitation; Continuous culture; Phosphate; accumulation

Mutations in cell division proteins FtsZ and FtsA inhibit φX174 protein-E-mediated lysis of Escherichia coli by A. Witte; Edith Brand; Peter Mayrhofer; Frank Narendja; Werner Lubitz (pp. 259-268).

Electron microscopic studies emphasized that the protein-E-specific transmembrane tunnel structure, which permeabilizes Escherichia coli, is not randomly distributed over the cell envelope but is restricted to areas of potential division sites. These sites were located predominantly in the middle of the cell, but approximately one-third of these structures are found at the polar sites. Therefore, E. coli mutant strains with defects in cell division components were tested for their sensitivity to protein-E-mediated lysis. The ftsZ84 and the ftsA12 cell division mutant strains of E. coli were tolerant to protein-E-mediated lysis, whereas the ftsA3 mutant strain was lysed by protein E under conditions nonpermissive for division. The protein-E-tolerant phenotype of ftsZ84 and ftsA12 and the lysis-sensitive phenotype of other components of the septosome (e.g., ftsA3, ftsQ, and ftsI) suggest that initiation of cell division – rather than specific functions of cell division – plays an essential role in protein-E-mediated lysis. SulA-overproducing cells had a lysis-positive phenotype, the ring structure – but not the GTPase function - of FtsZ was impaired.

Keywords: Key words Lysis protein E; φX174; Escherichia coli; Cell division; FtsZ; FtsA

Identification of phototrophic sulfur bacteria through the analysis of lmwRNA band patterns by E. O. Casamayor; Juan I. Calderón-Paz; Jordi Mas; Carlos Pedrós-Alió (pp. 269-278).

Several phototrophic sulfur bacteria were identified preliminarily through the analysis of the low-molecular-weight RNA fraction (lmwRNA) of bacterial cells. This fraction includes the ribosomal 5S RNA and several transfer RNAs. These molecules were separated by high-resolution electrophoresis in polyacrylamide gels, and the resulting band patterns were used as fingerprints for the identification of the organisms. We examined a large number of well-characterized reference strains together with a broad range of purple sulfur bacterial isolates from freshwater and marine environments. A cluster analysis was run using the similarity matrix calculated from the band patterns. Despite the shortcomings of the method, close relatives were clustered together yielding a number of groups consistent with the phylogenetic arrangement established through the analyses of a few available 16S rRNA gene sequences. Thus, the classification obtained gives further support to rearrangement of the group as the analyses of 16S rRNA gene sequences had previously suggested. We conclude that the analysis of lmwRNA band patterns is a rapid and simple tool for grouping and preliminarily identifying new isolates of phototrophic sulfur bacteria.

Keywords: Key words Phototrophic sulfur bacteria; Chromatiaceae; Identification; lmwRNA; 5S RNA; tRNA; Taxonomic relationships

Metronidazole resistance and microaerophily in Campylobacter species by M. A. Smith; Margaret A. Jorgensen; George L. Mendz; Stuart L. Hazell (pp. 279-284).

Metronidazole is active against most anaerobic organisms and is also used in the treatment of the microaerophilic bacterium Helicobacter pylori. Resistance to metronidazole is uncommon in most anaerobic organisms, but it is increasingly prevalent in H. pylori. Previously we have suggested that metronidazole resistance in H. pylori is inherent in the microaerophilic nature of the organism and therefore would be present in other microaerophiles such as Campylobacter. Short periods of anaerobiosis caused metronidazole-resistant (Mtr^R) strains of Campylobacter spp. to become sensitive to metronidazole. Under microaerophilic conditions, cultures of the Mtr^R mutant Campylobacter coli R1 at bacterial cell densities of greater than 10⁸ cfu/ml lost viability, whereas no loss in viability was observed in cultures at cell densities of less than 10⁸. The Mtr^S C. coli strain lost viability at all cell densities. Comparisons of NAD(P)H oxidase activity between MtrS and Mtr^R strains indicated that the Mtr^S C. coli strain contained fourfold higher NADH oxidase activity and twofold higher NADPH oxidase activity than did the Mtr^R Campylobacter strains. These results show that Mtr^R Campylobacter spp. display resistance characteristics similar to those of H. pylori, suggesting that the resistance mechanism is a phenomenon of the microaerophilic nature of these bacteria.

Keywords: Key words Metronidazole resistance; Campylobacter; Microaerophily; NAD(P)H oxidase

Characterization of the group 1 and group 2 sigma factors of the green sulfur bacterium Chlorobium tepidum and the green non-sulfur bacterium Chloroflexus aurantiacus by T. M. Gruber; D. A. Bryant (pp. 285-296).

The group 1 and group 2 σ⁷⁰-type sigma factors of the green sulfur bacterium Chlorobium tepidum and of the green nonsulfur bacterium Chloroflexus aurantiacus were cloned and characterized. Cb. tepidum was found to contain one σ⁷⁰-type sigma factor; the expression of the gene was analyzed by Northern blot hybridization and primer-extension mapping. Cf. aurantiacus has genes encoding four sigma factors of groups 1 and 2. The expression of these genes was examined in cells grown aerobically and anaerobically. The sigC gene was expressed at approximately equal levels under both conditions, resulting in its designation as the group 1 sigma factor of this organism. The only other detectable transcripts arose from the sigB gene, which was expressed at higher levels during aerobic growth. A phylogenetic tree was obtained using the group 1 sigma factors of Cb. tepidum, Cf. aurantiacus, and diverse eubacteria as the molecular marker. The resulting phylogenetic tree shows that Cb. tepidum and Cf. aurantiacus are related to each other and to the cyanobacteria. The relationship of the group 2 sigma factors of Cf. aurantiacus and the cyanobacteria was more specifically examined phylogenetically. The group 2 sigma factors of Cf. aurantiacus probably arose by gene duplication events after the split of the green nonsulfur bacteria from other photosynthetic eubacteria.

Keywords: Key words Chloroflexus aurantiacus; Chlorobium tepidum; Green non-sulfur bacteria; Green sulfur; bacteria; Sigma factor; RNA polymerase; Phylogenetic studies

Membrane-associated redox activities in Thermotoga neapolitana by Sandra A. Käslin; Susan E. Childers; K. M. Noll (pp. 297-303).

Elemental sulfur reduction by the hyperthermophilic bacterium Thermotoga neapolitana provides an alternative to hydrogen evolution during fermentation. Electrons are transferred from reduced cofactors (ferredoxin and NADH) to sulfur by a series of unknown steps. One enzyme that may be involved is an NADH:methyl viologen oxidoreductase (NMOR), an activity that in other fermenting organisms is associated with NADH:ferredoxin oxidoreductase. We found that 83% of NMOR activity was contained in the pellet fraction of cell extracts subjected to ultracentrifugation. This pellet fraction, presumably containing cell membranes, was required for electron transfer to NAD⁺ from ferredoxin-dependent pyruvate oxidation. However, the NMOR activity in this fraction used neither Thermotoga nor clostridial ferredoxins as substrates. NMOR activity was also detected in aerobically prepared vesicles. By comparison with ATPase activities, NMOR was found primarily on the cytoplasmic face of these vesicles. During these studies, an extracytoplasmic hydrogenase activity was discovered. In contrast to the soluble hydrogenase, this hydrogenase activity was completely inhibited when intact cells were treated with cupric chloride and was present on the extracytoplasmic face of vescides. In contrast to a soluble hydrogenase reported in Thermotoga maritima, this activity was air-stable and was inhibited by low concentrations of nitrite.

Keywords: Key words Hyperthermophile; Hydrogenase; Oxidoreductase; Ferredoxin; Membrane

Expression of the succinate dehydrogenase genes (sdhCAB) from the facultatively anaerobic Paenibacillus macerans during aerobic growth by J. Schirawski; Thomas Hankeln; G. Unden (pp. 304-308).

Paenibacillus (formerly Bacillus) macerans is capable of succinate oxidation under oxic conditions and fumarate reduction under anoxic conditions. The reactions are catalyzed by different enzymes, succinate dehydrogenase (Sdh) and fumarate reductase (Frd). The genes encoding Sdh (sdhCAB) were analyzed. The gene products of sdhA and sdhB were similar to the subunits of known Sdh and Frd enzymes. The hydrophobic subunit SdhC showed close sequence similarity to the class of Sdh/Frd enzymes containing diheme cytochrome b. From the sdhCAB gene cluster two transcripts were produced, one comprising sdhCAB, the other sdhAB. The transcripts were found only during aerobic growth, and the amount was directly proportional to Sdh activity, but inversely proportional to Frd activity.

Keywords: Key words Succinate dehydrogenase; sdhCAB genes; Fumarate reductase; Menaquinone; Paenibacillus (Bacillus) macerans

Effects of pisatin on Dictyostelium discoideum: its relationship to inducible resistance to nystatin and extension to other isoflavonoid phytoalexins by T. Bhavani Prasanna; M. Vairamani; D. P. Kasbekar (pp. 309-312).

Dictyostelium discoideum amoebae can acquire resistance to otherwise inhibitory concentrations of pisatin, an isoflavonoid phytoalexin of pea, and nystatin, a polyene antibiotic, following pretreatment with sublethal concentrations of these compounds. Additionally, growth on medium containing pisatin can induce nystatin resistance. We show here that distinct mechanisms mediate the inducible resistance to these two compounds because it is possible to isolate mutations that specifically block the induction of nystatin resistance but do not affect the induction of pisatin resistance. Pisatin did not affect wild-type sterol biosynthesis; therefore, the induction of nystatin resistance by pisatin is probably not via an alteration of membrane sterols. The inducible pisatin resistance phenotype was shown to extend to the isoflavonoid phytoalexins maackiain and biochanin A, and all three compounds inhibited the aggregation of amoebae that is normally triggered by starvation.

Keywords: Key words Legume rhizosphere; Isoflavonoid; phytoalexins; Pisatin; Maackiain; Biochanin A; Nystatin resistance

Isolation and characterization of Desulfovibrio senezii sp. nov., a halotolerant sulfate reducer from a solar saltern and phylogenetic confirmation of Desulfovibrio fructosovorans as a new species by I.-Hsien Tsu; C.-Y. Huang; J.-L. Garcia; Bharat K. C. Patel; Jean-Luc Cayol; Larry Baresi; Robert A. Mah (pp. 313-317).

A new halotolerant Desulfovibrio, strain CVL^T (T = type strain), was isolated from a solar saltern in California. The curved, gram-negative, nonsporeforming cells (0.3 × 1.0–1.3 μm) occurred singly, in pairs, or in chains, were motile by a single polar flagellum and tolerated up to 12.5% NaCl. Strain CVL^T had a generation time of 60 min when grown in lactate-yeast extract medium under optimal conditions (37°C, pH 7.6, 2.5% NaCl). It used lactate, pyruvate, cysteine, or H₂/CO₂ + acetate as electron donors, and sulfate, sulfite, thiosulfate, or fumarate as electron acceptors. Elemental sulfur, nitrate, or oxygen were not used. Sulfite and thiosulfate were disproportionated to sulfate and sulfide. The G+C content of the DNA was 62 mol%. Phylogenetic analysis revealed that Desulfovibrio fructosovorans was the nearest relative. Strain CVL^T is clearly different from other Desulfovibrio species, and is designated Desulfovibrio senezii sp. nov. (DSM 8436).

Keywords: Key words Desulfovibrio senezii; Desulfovibrio; fructosovorans; Desulfovibrionaceae; Halotolerant; Sulfate reduction

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Archives of Microbiology (v.170, #4)