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Archives of Microbiology (v.171, #2)

A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds by Patricia Spiekermann; Bernd H. A. Rehm; Rainer Kalscheuer; Dirk Baumeister; A. Steinbüchel (pp. 73-80).

The oxazine dye Nile blue A and its fluorescent oxazone form, Nile red, were used to develop a simple and highly sensitive staining method to detect poly(3-hydroxybutyric acid) and other polyhydroxyalkanoic acids (PHAs) directly in growing bacterial colonies. In contrast to previously described methods, these dyes were directly included in the medium at concentrations of only 0.5 μg/ml, and growth of the cells occurred in the presence of the dyes. This allowed an estimation of the presence of PHAs in viable colonies at any time during the growth experiment and a powerful discrimination between PHA-negative and PHA-positive strains. The presence of Nile red or Nile blue A did not affect growth of the bacteria. This viable-colony staining method was in particular applicable to gram-negative bacteria such as Azotobacter vinelandii, Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. It was less suitable for discriminating between PHA-negative and PHA-positive strains of gram-positive bacteria such as Bacillus megaterium or Rhodococcus ruber, but it could also be used to discriminate between wax-ester- and triacylglycerol-negative and -positive strains of Acinetobacter calcoaceticus or Rhodococcus opacus. The potential of this new method and its application to further investigations of PHA synthases and PHA biosynthesis pathways are discussed.

Keywords: Key wordsAcinetobacter calcoaceticus; Azotobacter vinelandii; Pseudomonas putida; Ralstonia eutropha; Rhodococcus opacus; Nile red; Nile blue A; Viable colony staining; Polyhydroxyalkanoic acid; Triacylglycerols; Lipid inclusions; Poly(3-hydroxybutyric acid); Poly(4-hydroxybutyric acid); Poly(3-hydroxyoctanoic acid)

Anaerobic transformation of quercetin-3-glucoside by bacteria from the human intestinal tract by Heiko Schneider; Andreas Schwiertz; Matthew David Collins; M. Blaut (pp. 81-91).

From human feces two phenotypically different types of bacteria were isolated on quercetin-3-glucoside as carbon and energy source. Isolates of one type were identified as strains of Enterococcus casseliflavus. They utilized the sugar moiety of the glycoside, but did not degrade the aglycon further. The sugar moiety (4 mM) was fermented to 5.5 ± 2.1 mM formate, 2.1 ± 0.7 mM acetate, 1.6 ± 0.3 mM l-lactate, and 1.3 ± 0.4 mM ethanol. The second type of isolate was identified as Eubacterium ramulus. This organism was capable of degrading the aromatic ring system. Growing cultures of Eubacterium ramulus converted 5 mM quercetin-3-glucoside to 1.7 ± 0.6 mM 3,4-dihydroxyphenylacetic acid, 7.6 ± 1.0 mM acetate, and 4.0 ± 0.4 mM butyrate. Molecular hydrogen, 3,4-dihydroxybenzaldehyde, and ethanol were detected in small amounts. Phloroglucinol was a transient intermediate in the breakdown of quercetin-3-glucoside. Eubacterium ramulus did not grow on the aglycon quercetin or the ring-fission intermediate phloroglucinol, but cleaved the flavonoid ring system when glucose was present as a cosubstrate. The most probable number of quercetin-3-glucoside-degrading bacteria determined in nine human fecal samples was 10⁷–10⁹/g dry mass. Isolates from these experiments were all identified as Eubacterium ramulus.

Keywords: Key words Human intestinal bacteria; Flavonoid; degradation; Quercetin-3-glucoside; Ring cleavage; 3; 4-Dihydroxyphenylacetic acid; Phloroglucinol

Characterization of the Escherichia coli CcmH protein reveals new insights into the redox pathway required for cytochrome c maturation by Renata Annette Fabianek; Thomas Hofer; L. Thöny-Meyer (pp. 92-100).

The CcmH protein of Escherichia coli is encoded by the last gene of the ccm gene cluster required for cytochrome c maturation. A mutant in which the entire ccmH gene was deleted failed to synthesize both indigenous and foreign c-type cytochromes. However, deletion of the C-terminal hydrophilic domain homologous to CycH of other gram-negative bacteria affected neither the biogenesis of indigenous c-type cytochromes nor that of the Bradyrhizobium japonicum cytochrome c ₅₅₀. This confirmed that only the N-terminal domain containing a conserved CXXC motif is required in E. coli. PhoA fusion analysis showed that this domain is periplasmic. Site-directed mutagenesis of the cysteines of the CXXC motif revealed that both cysteines are required for cytochrome c maturation during aerobic growth, whereas only the second cysteine is required for cytochrome c maturation during anaerobic growth. The deficiency of the point mutants was complemented when 2-mercapto-ethanesulfonic acid was added to growing cells; other thiol compounds did not stimulate cytochrome c formation in these strains. We propose a model for the reaction sequence in which CcmH keeps the heme binding site of apocytochrome c in a reduced form for subsequent heme ligation.

Keywords: Key words CcmH; Cytochrome c maturation; Dithiol reduction pathway; Escherichia coli

Glucosylglycerol-phosphate synthase: target for ion-mediated regulation of osmolyte synthesis in the cyanobacterium Synechocystis sp. strain PCC 6803 by A. Schoor; Martin Hagemann; Norbert Erdmann (pp. 101-106).

The response of cyanobacteria to a changing osmotic environment includes the accumulation of organic osmolytes such as glucosylglycerol. The activation of the enzymes involved in glucosylglycerol synthesis [glucosylglycerol-phosphate synthase (GGPS) and glucosylglycerol-phosphate phosphatase (GGPP)] in Synechocystis sp. strain PCC 6803 by various salts and salt concentrations was investigated in vitro. GGPS seemed to be the target for salt-mediated regulation of glucosylglycerol synthesis in vitro. GGPS activation was dependent on the concentration of NaCl, and a sigmoidal plot was obtained. Sensitivity to NaCl was markedly enhanced by low Mg⁺² concentrations (optimal at 4 mM), but Mg²⁺ was not absolutely necessary for the Na⁺ stimulation. As in the case of NaCl, other salts (including MgCl₂) stimulated GGPS. The relative order of GGPS activation in the presence of chloride by the cations at constant ionic strength was Li⁺ > Na⁺ > K⁺, Mg²⁺ Mn²⁺. No absolute dependence on ionic strength was observed in Mg²⁺/Na⁺-exchange experiments. The degree of activation by ions at various concentrations was positively related to the increasing destabilizing properties of the cations according to the Hofmeister rule, where chaotropic cations are most efficient. Cations were responsible for activation since chaotropic anions counteracted the activating effect of cations.

Keywords: Key words Cyanobacteria; Glucosylglycerol-phosphate; synthase/phosphatase; Ions; Osmolytes; Salt activation; Synechocystis

Syntrophus aciditrophicus sp. nov., a new anaerobic bacterium that degrades fatty acids and benzoate in syntrophic association with hydrogen-using microorganisms by Bradley E. Jackson; V. K. Bhupathiraju; Ralph S. Tanner; Carl R. Woese; M. J. McInerney (pp. 107-114).

Strain SB^T is a new, strictly anaerobic, gram-negative, nonmotile, non-sporeforming, rod-shaped bacterium that degrades benzoate and certain fatty acids in syntrophic association with hydrogen/formate-using microorganisms. Strain SB^T produced approximately 3 mol of acetate and 0.6 mol of methane per mol of benzoate in coculture with Methanospirillum hungatei strain JF1. Saturated fatty acids, some unsaturated fatty acids, and methyl esters of butyrate and hexanoate also supported growth of strain SB^T in coculture with Desulfovibrio strain G11. Strain SB^T grew in pure culture with crotonate, producing acetate, butyrate, caproate, and hydrogen. The molar growth yield was 17 ± 1 g cell dry mass per mol of crotonate. Strain SB^T did not grow with fumarate, iron(III), polysulfide, or oxyanions of sulfur or nitrogen as electron acceptors with benzoate as the electron donor. The DNA base composition of strain SB^T was 43.1 mol% G+C. Analysis of the 16 S rRNA gene sequence placed strain SB^T in the δ-subdivision of the Proteobacteria, with sulfate-reducing bacteria. Strain SB^T was most closely related to members of the genus Syntrophus. The clear phenotypic and genotypic differences between strain SB^T and the two described species in the genus Syntrophus justify the formation of a new species, Syntrophus aciditrophicus.

Keywords: Key words Syntrophic metabolism; Aromatic biodegradation; Benzoate; Volatile fatty acids; Methanogenesis; Syntrophus

Membrane-bound F420H2-dependent heterodisulfide reduction in Methanococcus voltae by Jens Brodersen; Gerhard Gottschalk; U. Deppenmeier (pp. 115-121).

Washed membranes prepared from H₂+CO₂- or formate-grown cells of Methanococcus voltae catalyzed the oxidation of coenzyme F₄₂₀H₂ and the reduction of the heterodisulfide (CoB–S–S–CoM) of 2-mercaptoethanesulfonate and 7-mercaptoheptanoylthreonine phosphate, which is the terminal electron acceptor of the methanogenic pathway. The reaction followed a 1:1 stoichiometry according to the equation: F₄₂₀H₂ + COB–S–S–CoM → F₄₂₀ + CoM–SH + CoB–SH. These findings indicate that the reaction depends on a membrane-bound F₄₂₀H₂-oxidizing enzyme and on the heterodisulfide reductase, which remains partly membrane-bound after cell lysis. To elucidate the nature of the F₄₂₀H₂-oxidizing protein, washed membranes were solubilized with detergent, and the enzyme was purified by sucrose density centrifugation, anion-exchange chromatography, and gel filtration. Several lines of evidence indicate that F₄₂₀H₂ oxidation is catalyzed by a membrane-associated F₄₂₀-reducing hydrogenase. The purified protein catalyzed the H₂-dependent reduction of methyl viologen and F₄₂₀. The apparent molecular mass and the subunit composition (43, 37, and 27 kDa) are almost identical to those of the F₄₂₀-reducing hydrogenase that has already been purified from Mc. voltae. Moreover, the N-terminus of the 37-kDa subunit is identical to the amino acid sequence deduced from the fruG gene of the operon encoding the selenium-containing F₄₂₀-reducing hydrogenase from Mc. voltae. A distinct F₄₂₀H₂ dehydrogenase, which is present in methylotrophic methanogens, was not found in this organism.

Keywords: Key words Methanogenic archaea; Methanococcus; Methanosarcina; Electron transport; Hydrogenases; Coenzyme F420; F42OH2 dehydrogenase; Heterodisulfide reductase

Integration of minitransposons for expression of the Escherichia coli elt genes at a preferred site in Salmonella typhimurium identifies a novel putative fimbrial locus by M. Brocchi; M. Giuseppina Covone; Emanuela Palla; C. L. Galeotti (pp. 122-126).

An asd-complementing mini-Tn5 transposon was constructed for random insertion of the Escherichia coli LT enterotoxin genes (elt) into the genome of Δasd attenuated strains of Salmonella typhimurium. Transfer of the minitransposon to different S. typhimurium strains resulted in random integration only in strain χ4072, while in strain χ3987, which harbours the virulence plasmid, over 20% of the insertions occurred at the same site. Expression of elt was found to be highest in Salmonella isolates carrying the mini-Tn5 integrated at the preferred site, which was mapped to an uncharacterised region of the virulence plasmid. Sequence analysis of the integration site showed that it lies within an open reading frame with sequence similarity to E. coli leuO and contiguous to a novel fimbrial locus.

Keywords: Key wordsSalmonella typhimurium; Minitransposons; LT enterotoxin; Expression; Fimbriae

Expression of bvgAS of Bordetella pertussis represses flagellar biosynthesis of Escherichia coli by Y. W. Han; M. A. Uhl; Seog Joon Han; W. Shi (pp. 127-130).

BvgAS is a two-component system of Bordetella pertussis involved in the reciprocal regulation of the virulence genes and the flagellar biosynthesis. In this study, we found that expression of bvgAS in Escherichia coli also results in reduced motility. The repression was relieved by the addition of known chemical modulators of BvgAS such as MgSO₄ and nicotinic acid, indicating that functional BvgAS proteins are required for the negative control of E. coli motility. In addition, BvgAS repressed the transcription of the flhDC master operon of E. coli, which consequently caused non-flagellation on the cell surface. However, expression of BvgAS had no effect on stress-resistant motile mutants of E. coli. These data suggest that E. coli may have BvgA-like protein(s) involved in the regulatory interactions between the stress response and the flagellar biosynthesis.

Keywords: Key words Motility; Chemotaxis; Gene regulation

Accumulation of ppGpp in symbiotic and free-living nitrogen-fixing bacteria following amino acid starvation by Samantha M. Howorth; R. R. England (pp. 131-134).

Following amino acid or ammonium starvation, ppGpp is accumulated by Rhizobium meliloti strain 1021 but not by R. meliloti strain 41 or Rhizobium tropici. Azorhizobium caulinodans ORS571 produced ppGpp following amino acid deprivation; however, the free-living nitrogen-fixing bacteria Azotobacter vinelandii and Azomonas agilis did not produce ppGpp. Western blot analysis using anti-RelA antibody demonstrated that R. meliloti strain 1021, Azotobacter vinelandii and Azorhizobium caulinodans cross-reacted under conditions that detected RelA in Escherichia coli CF1648. Cross-reaction was not observed in R. meliloti strain 41, R. tropici, or Azomonas agilis. All strains that accumulated ppGpp also produced high intracellular levels of ATP.

Keywords: Key words Nitrogen-fixing bacteria; Stringent response; ppGpp; RelA; ATP

Cold shock proteins CspB and CspC are major stationary-phase-induced proteins in Bacillus subtilis by P. L. Graumann; Mohamed A. Marahiel (pp. 135-138).

Shortly after the transition from exponential growth to stationary phase, the pattern of protein synthesis in Bacillus subtilis changes markedly. Among the most profoundly induced proteins are two homologous small acidic proteins, CspB and CspC, which are also major cold-shock-induced proteins. The third cold shock protein (CSP) in B. subtilis, CspD, is not induced following entry into stationary phase. Deletion of both cspB and cspC genes has been previously shown to lead to lysis of cells during stationary phase. These findings reveal that CSPs in B. subtilis are induced under several stress conditions, and that an increase in the synthesis of CspB and CspC is needed for efficient adaptation to stationary phase. Enhanced synthesis of CspB occurs through a combination of transcriptional and post-transcriptional activation, indicating a mechanism similar to that mediating cold shock induction of CSPs. Induction of CSPs in bacteria may be triggered by a common signal, the inactivation of ribosomes, occurring under both cold shock and stationary-phase conditions.

Keywords: Key wordsBacillus subtilis; Cold shock proteins; Stationary phase; Post-transcriptional activation; Stress response; Ribosome inactivation

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Archives of Microbiology (v.171, #2)