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Archives of Microbiology (v.182, #2-3)
Identification of a regulatory pathway that controls the heavy-metal resistance system Czc via promoter czcNp in Ralstonia metallidurans by Cornelia Große; Andreas Anton; Toni Hoffmann; Sylvia Franke; Grit Schleuder; Dietrich H. Nies (pp. 109-118).
The CzcCBA cation-proton-antiporter is the most complicated and efficient heavy-metal resistance system known today and is essential for survival of Ralstonia metallidurans at high cobalt, zinc, or cadmium concentrations. Expression of Czc is tightly controlled by the complex interaction of several regulators. Double- and multiple-deletion studies demonstrated that four regulators encoded downstream of the czcCBA operon, CzcD, CzcS, CzcR and the newly identified CzcE, are involved in, but not essential for metal-dependent induction of czc. These proteins control expression of the czcNICBA region from the promoter czcNp. Northern analysis showed that czcDRS was transcribed as czcDR-specific and czcDRS-specific mRNAs. Transcription of czcE occurred independently of czcDRS transcription and was induced by zinc. CzcE is a periplasmic protein as indicated by phoA fusions. CzcE was purified and identified as a metal-binding protein. These data demonstrate that the transport protein CzcD, the two-component regulatory system CzcR, CzcS, and the periplasmic metal-binding protein CzcE exert metal-dependent control of czcNICBA expression via regulation of czcNp activity.
Keywords: RND systems; Two-component regulatory systems; Heavy-metal resistance; Heavy-metal homeostasis; Alcaligenes eutrophus ; Metal-dependent regulation; Ralstonia metallidurans
Overproduction of NAD+ and 5′-inosine monophosphate in the presence of 10 μM Mn2+ by a mutant of Corynebacterium ammoniagenes with thermosensitive nucleotide reduction (nrd ts) after temperature shift by Bouziane Abbouni; Hesham M. Elhariry; Georg Auling (pp. 119-125).
Corynebacterium ammoniagenes strain CH31 is thermosensitive due to a mutation in nucleotide reduction (nrd ts). The strain was examined for nucleotide overproduction upon shifting the culture temperature to a range of elevated temperatures. No overproduction of NAD+ was detected in the control maintained at 27°C whereas NAD+ was accumulated extracellularily by strain CH31 at 37°C and at 40°C. As a result of the temperature shift, division-inhibited cells displayed only limited elongation. This is a characteristic morphological feature of cell-cycle-arrested coryneform bacteria. Ribonucleotide reductase (RNR) activity was inactivated immediately after the temperature shift in the NAD+-proficient cultures, leading presumably to an exhaustion of deoxyribonucleotide pools and impairment of DNA replication. In contrast to the low extracellular accumulation of NAD+, at the non-permissive temperature of 35°C a distinct capacity for intracellular nucleotide overproduction was revealed by a new method using nucleotide-permeable cells. The approach of shifting the culture temperature was applied successfully to the overproduction of taste-enhancing nucleotides in the presence of 10 μM Mn2+. Concomitant with a dramatic loss of viability, the thermosensitive mutant CH31 accumulated 5.3 g 5′-inosine monophosphate per liter following the addition of hypoxanthine as precursor for the salvage pathway.
Keywords: Corynebacterium ammoniagenes ; NAD+ ; IMP; Manganese-ribonucleotide reductase; Thermosensitive mutation; Arrest of cell cycle
F420H2 oxidase (FprA) from Methanobrevibacter arboriphilus, a coenzyme F420-dependent enzyme involved in O2 detoxification by Henning Seedorf; Annette Dreisbach; Reiner Hedderich; Seigo Shima; Rudolf K. Thauer (pp. 126-137).
Cell suspensions of Methanobrevibacter arboriphilus catalyzed the reduction of O2 with H2 at a maximal specific rate of 0.4 U (μmol/min) per mg protein with an apparent K m for O2 of 30 μM. The reaction was not inhibited by cyanide. The oxidase activity was traced back to a coenzyme F420-dependent enzyme that was purified to apparent homogeneity and that catalyzed the oxidation of 2 F420H2 with 1 O2 to 2 F420 and 2 H2O. The apparent K m for F420 was 30 μM and that for O2 was 2 μM with a V max of 240 U/mg at 37°C and pH 7.6, the pH optimum of the oxidase. The enzyme did not use NADH or NADPH as electron donor or H2O2 as electron acceptor and was not inhibited by cyanide. The 45-kDa protein, whose gene was cloned and sequenced, contained 1 FMN per mol and harbored a binuclear iron center as indicated by the sequence motif H–X–E–X–D–X62–H–X18–D–X60–H. Sequence comparisons revealed that the F420H2 oxidase from M. arboriphilus is phylogenetically closely related to FprA from Methanothermobacter marburgensis (71% sequence identity), a 45-kDa flavoprotein of hitherto unknown function, and to A-type flavoproteins from bacteria (30–40%), which all have dioxygen reductase activity. With heterologously produced FprA from M. marburgensis it is shown that this protein is also a highly efficient F420H2 oxidase and that it contains 1 FMN and 2 iron atoms. The presence of F420H2 oxidase in methanogenic archaea may explain why some methanogens, e.g., the Methanobrevibacter species in the termite hindgut, cannot only tolerate but thrive under microoxic conditions.
Keywords: Type A flavoprotein; F420H2 oxidase; Rubredoxin oxidase; Oxygen detoxification; Methanobrevibacter ; Methanothermobacter ; Moorella ; Desulfovibrio ; Methanogenic archaea
The role of the active site-coordinating cysteine residues in the maturation of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16 by Gordon Winter; Thorsten Buhrke; Anne K. Jones; Bärbel Friedrich (pp. 138-146).
The H2-splitting active site of [NiFe] hydrogenases is tightly bound to the protein matrix via four conserved cysteine residues. In this study, the nickel-binding cysteine residues of HoxC, the large subunit of the H2-sensing regulatory hydrogenase (RH) from Ralstonia eutropha, were replaced by serine. All four mutant proteins, C60S, C63S, C479S, and C482S, were inactive both in H2 sensing and H2 oxidation and did not adopt the native oligomeric structure of the RH. Nickel was bound only to the C482S derivative. The assembly of the [NiFe] active site is a complex process that requires the function of at least six accessory proteins. Among these proteins, HypC has been shown to act as a chaperone for the large subunit during the maturation process. Immunoblot analysis revealed the presence of a strong RH-dependent HypC-specific complex in extracts containing the C60S, C63S, and C482S derivatives, pointing to a block in maturation for these mutant proteins. The lack of this complex in the extract containing C479S indicates that this specific cysteine residue might be crucial for the interaction between HoxC and HypC.
Keywords: Ralstonia eutropha ; [NiFe] hydrogenase; H2 sensor; Nickel metabolism; Metal center assembly
Microorganisms degrading chlorobenzene via a meta-cleavage pathway harbor highly similar chlorocatechol 2,3-dioxygenase-encoding gene clusters by Markus Göbel; Oliver H. Kranz; Stefan R. Kaschabek; Eberhard Schmidt; Dietmar H. Pieper; Walter Reineke (pp. 147-156).
Pseudomonas putida GJ31 harbors a degradative pathway for chlorobenzene via meta-cleavage of 3-chlorocatechol. Pseudomonads using this route for chlorobenzene degradation, which was previously thought to be generally unproductive, were isolated from various contaminated environments of distant locations. The new isolates, Pseudomonas fluorescens SK1 (DSM16274), Pseudomonas veronii 16-6A (DSM16273), Pseudomonas sp. strain MG61 (DSM16272), harbor a chlorocatechol 2,3-dioxygenase (CbzE). The cbzE-like genes were cloned, sequenced, and expressed from the isolates and a mixed culture. The chlorocatechol 2,3-dioxygenases shared 97% identical amino acids with CbzE from strain GJ31, forming a distinct family of catechol 2,3-dioxygenases. The chlorocatechol 2,3-dioxygenase, purified from chlorobenzene-grown cells of strain SK1, showed an identical N-terminal sequence with the amino acid sequence deduced from cloned cbzE. In all investigated chlorobenzene-degrading strains, cbzT-like genes encoding ferredoxins are located upstream of cbzE. The sequence data indicate that the ferredoxins are identical (one amino acid difference in CbzT of strain 16-6A compared to the others). In addition, the structure of the operon downstream of cbzE is identical in strains GJ31, 16-6A, and SK1 with genes cbzX (unknown function) and the known part of cbzG (2-hydroxymuconic semialdehyde dehydrogenase) and share 100% nucleotide sequence identity with the entire downstream region. The current study suggests that meta-cleavage of 3-chlorocatechol is not an atypical pathway for the degradation of chlorobenzene.
Keywords: Degradation of chloroaromatic compounds; Meta-cleavage pathway
Thermotolerant poly(3-hydroxybutyrate)-degrading bacteria from hot compost and characterization of the PHB depolymerase of Schlegelella sp. KB1a by Fabian Romen; Simone Reinhardt; Dieter Jendrossek (pp. 157-164).
Eighteen gram-negative thermotolerant poly(3-hydroxybutyrate) (PHB)-degrading bacterial isolates (T max≈60°C) were obtained from compost. Isolates produced clearing zones on opaque PHB agar, indicating the presence of extracellular PHB depolymerases. Comparison of physiological characteristics and determination of 16S rRNA gene sequences of four selected isolates revealed a close relatedness of three isolates (SA8, SA1, and KA1) to each other and to Schlegelella thermodepolymerans and Caenibacterium thermophilum. The fourth strain, isolate KB1a, showed reduced similarities to the above-mentioned isolates and species and might represent a new species of Schlegelella. Evidence is provided that S. thermodepolymerans and C. thermophilum are only one species. The PHB depolymerase gene, phaZ, of isolate KB1a was cloned and functionally expressed in Escherichia coli. Purified PHB depolymerase was most active around pH 10 and 76°C. The DNA-deduced amino acid sequence of the mature protein (49.4 kDa) shared significant homologies to other extracellular PHB depolymerases with a domain substructure: catalytic domain type 2—linker domain fibronectin type 3—substrate-binding domain type 1. A catalytic triad consisting of S20, D104, and H138 and a pentapeptide sequence (GLS20AG) characteristic for PHB depolymerases (PHB depolymerase box, GLSXG) and for other serine hydrolases (lipase box, GXSXG) were identified.
Keywords: Thermophile PHB depolymerase; PHA depolymerase; Schlegelella thermodepolymerans ; Caenibacterium thermophilum
The role of the sulfur globule proteins of Allochromatium vinosum: mutagenesis of the sulfur globule protein genes and expression studies by real-time RT-PCR by Alexander Prange; Harald Engelhardt; Hans G. Trüper; Christiane Dahl (pp. 165-174).
During oxidation of reduced sulfur compounds, the purple sulfur bacterium Allochromatium vinosum stores sulfur in the periplasm in the form of intracellular sulfur globules. The sulfur in the globules is enclosed by a protein envelope that consists of the homologous 10.5-kDa proteins SgpA and SgpB and the smaller 8.5-kDa SgpC. Reporter gene fusions of sgpA and alkaline phosphatase showed the constitutive expression of sgpA in A. vinosum and yielded additional evidence for the periplasmic localization of the sulfur globules. Expression analysis of the wild-type sgp genes by quantitative RT-PCR using the LightCycler system showed the constitutive expression of all three sgp genes. The expression of sgpB and sgpC is significantly enhanced under photolithotrophic conditions. Interestingly, sgpB is expressed ten times less than sgpA and sgpC implying that SgpA and SgpC are the “main proteins” of the sulfur globule envelope. Mutants with inactivated sgpA or sgpB did not show any differences in comparison with the wild-type, i.e., the encoded proteins can replace each other, whereas inactivation of sgpC leads to the formation of considerably smaller sulfur globules. This indicates a role of SgpC for globule expansion. A sgpBC double mutant was unable to grow on sulfide and could not form sulfur globules, showing that the protein envelope is indispensible for the formation and deposition of intracellular sulfur.
Keywords: Allochromatium vinosum ; Sulfur globule proteins; Sulfide oxidation; Sulfur globules; Phototrophic sulfur bacteria
Ferrihydrite reduction by Geobacter species is stimulated by secondary bacteria by Kristina L. Straub; Bernhard Schink (pp. 175-181).
Geobacter species such as G. bremensis, G. pelophilus, and G. sulfurreducens are obligately anaerobic and grow in anoxic, non-reduced medium by fast reduction of soluble ferric citrate. In contrast, insoluble ferrihydrite was either only slowly or not reduced when supplied as electron acceptor in similar growth experiments. Ferrihydrite reduction was stimulated by addition of a reducing agent or by concomitant growth of secondary bacteria that were physiologically and phylogenetically as diverse as Escherichia coli, Lactococcus lactis, or Pseudomonas stutzeri. In control experiments with heat-inactivated Geobacter cells and viable secondary bacteria, no (E. coli, P. stutzeri) or only little (L. lactis) ferrihydrite was reduced. Redox indicator dyes showed that growing E. coli, P. stutzeri, or L. lactis cells lowered the redox potential of the medium in a similar way as a reducing agent did. The lowered redox potential was presumably the key factor that stimulated ferrihydrite reduction by all three Geobacter species. The observed differences in anoxic non-reduced medium with ferric citrate versus ferrihydrite as electron acceptor indicated that reduction of these electron acceptors involved different cellular components or different biochemical strategies. Furthermore, it appears that redox-sensitive components are involved, and/or that gene expression of components needed for ferrihydrite reduction is controlled by the redox state.
Keywords: Ferrihydrite reductionGeobacter bremensisGeobacter pelophilusGeobacter sulfurreducensSecondary bacteriaEscherichia coliLactococcus lactisPseudomonas stutzeriRedox potential; Redox indicator dyes
Sporulation genes in members of the low G+C Gram-type-positive phylogenetic branch (Firmicutes) by Rob U. Onyenwoke; Julia A. Brill; Kamyar Farahi; Juergen Wiegel (pp. 182-192).
Endospore formation is a specific property found within bacteria belonging to the Gram-type-positive low G+C mol% branch (Firmicutes) of a phylogenetic tree based on 16S rRNA genes. Within the Gram-type-positive bacteria, endospore-formers and species without observed spore formation are widely intermingled. In the present study, a previously reported experimental method (PCR and Southern hybridization assays) and analysis of genome sequences from 52 bacteria and archaea representing sporulating, non-spore-forming, and asporogenic species were used to distinguish non-spore-forming (void of the majority of sporulation-specific genes) from asporogenic (contain the majority of sporulation-specific genes) bacteria. Several sporulating species lacked sequences similar to those of Bacillus subtilis sporulation genes. For some of the genes thought to be sporulation specific, sequences with weak similarity were identified in non-spore-forming bacteria outside of the Gram-type-positive phylogenetic branch and in archaea, rendering these genes unsuitable for the intended classification into sporulating, asporogenic, and non-spore-forming species. The obtained results raise questions regarding the evolution of sporulation among the Firmicutes.
Keywords: Phylogeny; Sporulation genes; Dipicolinic acid synthase; Small acid soluble protein; Asporogenic and non-spore-forming bacteria; Lactic acid bacteria; Genome sequences; Gene evolution
Escherichia coli Hmp, an “oxygen-binding flavohaemoprotein”, produces superoxide anion and self-destructs by Guanghui Wu; Hazel Corker; Yutaka Orii; Robert K Poole (pp. 193-203).
Escherichia coli Hmp is a homologue of Ralstonia eutropha FHP, the first reported bacterial flavohaemoglobin, and functions in NO detoxification. Photolysis of CO-ligated Hmp in the presence of oxygen gave a photodissociable oxy species with kon 2.82×107 M−1 s−1 and koff 4.49×103 s−1. The dissociation constant of the primary O2 compound was 160 μM (25°C, pH 7.0). In order to detect superoxide formation, ferric horseradish peroxidase was used. Hmp formed the oxy compound within milliseconds, followed by formation of compound III, arising from superoxide formation. The rate of superoxide formation was independent of oxygen concentration between 0.05 and 0.7 mM oxygen, suggesting a Km <0.05 mM. During prolonged oxidation of NADH, the spectral signals of Hmp decayed and iron was released in a process prevented by superoxide dismutase or catalase. NADH oxidation by purified Hmp was characterised by progressive slowing of oxygen uptake. Inclusion of NO, superoxide dismutase or catalase during NADH oxidation partially protected oxygen uptake, consistent with the formation, in the absence of NO, of reactive oxygen species that inhibit Hmp function. The results are discussed in relation to the tight control exerted on Hmp synthesis in vivo.
Keywords: Haemoglobin (bacterial); Escherichia coli; Hmp; Oxygenated form; Superoxide; Horseradish peroxidase
Desulfosporomusa polytropa gen. nov., sp. nov., a novel sulfate-reducing bacterium from sediments of an oligotrophic lake by Henrik Sass; Jörg Overmann; Heike Rütters; Hans-Dietrich Babenzien; Heribert Cypionka (pp. 204-211).
Five strains of sulfate-reducing bacteria were isolated from the highest positive dilutions of a most probable number (MPN) series supplemented with lactate and inoculated with sediments from the oligotrophic Lake Stechlin. The isolates were endospore-forming and were motile by means of laterally inserted flagella. They stained Gram-negative and contained b-type cytochromes. CO difference spectra indicated the presence of P582 as a sulfite reductase. Phylogenetic analyses of the 16S rDNA sequences revealed that the isolates were very closely affiliated with the genus Sporomusa. However, sulfate and amorphous Fe(OH)3, but not sulfite, elemental sulfur, MnO2, or nitrate were used as terminal electron acceptors. Homoacetogenic growth was found with H2/CO2 gas mixture, formate, methanol, ethanol, and methoxylated aromatic compounds. The strains grew autotrophically with H2 plus CO2 in the presence or absence of sulfate. Formate, butyrate, several alcohols, organic acids, carbohydrates, some amino acids, choline, and betaine were also utilized as substrates. The growth yield with lactate and sulfate as substrate was 7.0 g dry mass/mol lactate and thus two times higher than in sulfate-free fermenting cultures. All isolates were able to grow in a temperature range of 4–37°C. Physiologically and by the presence of a Gram-negative cell wall, the new isolates resemble known Desulfosporosinus species. However, phylogenetically they are affiliated with the Gram-negative genus Sporomusa belonging to the Selenomonas subgroup of the Firmicutes. Therefore, the new isolates reveal a new phylogenetic lineage of sulfate-reducing bacteria. A new genus and species, Desulfosporomusa polytropa gen. nov., sp. nov. is proposed.
Keywords: Sulfate-reducing bacteriaSporomusaHomoacetogen; Growth yield; Ferric iron reduction; Spore formation; Sediment bacteria; Anoxic sediments
Studies on the biodegradability of polythioester copolymers and homopolymers by polyhydroxyalkanoate (PHA)-degrading bacteria and PHA depolymerases by Khaled Elbanna; Tina Lütke-Eversloh; Dieter Jendrossek; Heinrich Luftmann; Alexander Steinbüchel (pp. 212-225).
The biodegradability of microbial polythioesters (PTEs), a novel class of biopolymers which were discovered recently and can be produced by polyhydroxyalkanoate (PHA)-accumulating bacteria, was studied. Using poly(3-hydroxybutyrate-co-3-mercaptopropionate) [poly(3HB-co-3MP)] as sole carbon source for screening, 22 new bacterial strains were isolated and characterized. Interestingly, none of the PHA-degrading bacteria was able to utilize the homopolymer poly(3MP) as a carbon source for growth or to form clear zones on poly(3MP)-containing agar plates. The extracellular PHA depolymerases from two strains ( Schlegelella thermodepolymerans, Pseudomonas indica K2) were purified to electrophoretic homogeneity and biochemically characterized. The PHA depolymerase of S. thermodepolymerans exhibited a temperate optimum of about 75°C to 80°C and was stable at 70°C for more than 24 h. Regarding the substrate specificities of the PHA depolymerase of S. thermodepolymerans, enzyme activities decreased significantly with increasing 3MP content of the copolymer substrates. Interestingly, no activity could be detected with homoPTEs consisting only of 3MP or of 3-mercaptobutyrate. Similar results were obtained with the PHA depolymerases PhaZ2, PhaZ5 and PhaZ7 of Paucimonas lemoignei which were also investigated. The PHA depolymerase of Ps. indica K2 did not cleave any of the investigated polymers containing 3MP. Gas chromatography, infrared and 1H-NMR spectrometry and matrix-assisted laser desorption/ionization time-of-flight analysis revealed that 3MPs containing oligomers were enriched in the water-insoluble fraction remaining after partial digestion of poly(3HB-co-3MP) by purified poly(3HB) depolymerase of S. thermodepolymerans. In contrast, 3HB was enriched in the water-soluble fraction, which also contained 3HB-co-3MP dimer obtained by partial digestion of this copolymer by the enzyme. This study clearly indicates that PHA depolymerases are specific for oxoester linkages of PHAs and that the thioester bonds of PTEs cannot be cleaved by this type of enzyme.
Keywords: Polythioester; PTE; Poly(3-mercaptopropionate); Polyhydroxyalkanoate; PHA; Biodegradation; PHA depolymerase
Characterisation of a thermoalkali-stable cyclodextrin glycosyltransferase from the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii by Volker Thiemann; Catharina Dönges; Steffen G. Prowe; Reinhard Sterner; Garabed Antranikian (pp. 226-235).
The thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii produces an extracellular CGTase when grown on starch at 55°C and pH 9.0. The gene encoding this CGTase was cloned and successfully expressed in Escherichia coli. It encodes a protein consisting of 721 amino acids with a signal sequence of 34 amino acids. On SDS–polyacrylamide gels, the purified CGTase from A. gottschalkii displayed the expected molecular mass of 78 kDa. The recombinant enzyme was purified with a yield of 13.5% and displayed a specific activity of 210 units/mg. This CGTase, which represents the first report of a CGTase from an anaerobic thermoalkaliphile, was active at a broad range of temperature and pH, namely 55–70°C and pH 5–10. It completely converted amylose, amylopectin and native starch to cyclodextrins, preferentially α-cyclodextrin. With a longer incubation period, the α-cyclodextrin to β-cyclodextrin ratio declined. Variations in substrate type and concentration influenced the product pattern. Increasing the substrate concentration (0.5–20.0%) and glucans containing branching points (α-1,6 glycosidic linkages) shifted the product pattern to: β-cyclodextin > α-cyclodextrin > γ-cyclodextrin. In addition to these cyclodextrins, larger cyclodextrins (>8 glucose units) were formed in the initial reaction period. The CGTase was stabilised against thermal inactivation by calcium ions and high substrate concentrations; and 5 mM of CaCl2 shifted the apparent melting point of the enzyme from 60°C to 69°C.
Keywords: Thermoalkaliphile; Starch metabolism; CGTase; Cyclodextrin
A gas vesiculate planktonic strain of the purple non-sulfur bacterium Rhodoferax antarcticus isolated from Lake Fryxell, Dry Valleys, Antarctica by Deborah O. Jung; Laurie A. Achenbach; Elizabeth A. Karr; Shinichi Takaichi; Michael T. Madigan (pp. 236-243).
A moderately psychrophilic purple non-sulfur bacterium, Rhodoferax antarcticus strain Fryx1, is described. Strain Fryx1 was isolated from the water column under the ice of the permanently frozen Lake Fryxell, Antarctica. Cells of Fryx1 are long thin rods and contain gas vesicles, the first report of such structures in purple non-sulfur bacteria. Gas vesicles are clustered at 2–4 sites per cell. Surprisingly, the 16S rRNA gene sequence of strain Fryx1 is nearly identical to that of Rfx. antarcticus strain AB, a short, vibrio-shaped phototroph isolated from an Antarctic microbial mat. Although showing physiological parallels, strains AB and Fryx1 differ distinctly in their morphology and absorption spectra. DNA–DNA hybridization shows that the genomes of strains AB and Fryx1 are highly related, yet distinct. We conclude that although strains AB and Fryx1 may indeed be the same species, their ecologies are quite different. Unlike strain AB, strain Fryx1 has adapted to a planktonic existence in the nearly freezing water column of Lake Fryxell.
Keywords: Purple anoxygenic phototrophic bacteria; Lake Fryxell; AntarcticaRhodoferax antarcticusGas vesicles; Psychrophily
Alkaliflexus imshenetskii gen. nov. sp. nov., a new alkaliphilic gliding carbohydrate-fermenting bacterium with propionate formation from a soda lake by Tatyana N. Zhilina; Ramona Appel; Christina Probian; Enrique Llobet Brossa; Jens Harder; Friedrich Widdel; Georgii A. Zavarzin (pp. 244-253).
Anaerobic saccharolytic bacteria thriving at high pH values were studied in a cellulose-degrading enrichment culture originating from the alkaline lake, Verkhneye Beloye (Central Asia). In situ hybridization of the enrichment culture with 16S rRNA-targeted probes revealed that abundant, long, thin, rod-shaped cells were related to Cytophaga. Bacteria of this type were isolated with cellobiose and five isolates were characterized. Isolates were thin, flexible, gliding rods. They formed a spherical cyst-like structure at one cell end during the late growth phase. The pH range for growth was 7.5–10.2, with an optimum around pH 8.5. Cultures produced a pinkish pigment tentatively identified as a carotenoid. Isolates did not degrade cellulose, indicating that they utilized soluble products formed by so far uncultured hydrolytic cellulose degraders. Besides cellobiose, the isolates utilized other carbohydrates, including xylose, maltose, xylan, starch, and pectin. The main organic fermentation products were propionate, acetate, and succinate. Oxygen, which was not used as electron acceptor, impaired growth. A representative isolate, strain Z-7010, with Marinilabilia salmonicolor as the closest relative, is described as a new genus and species, Alkaliflexus imshenetskii. This is the first cultivated alkaliphilic anaerobic member of the Cytophaga/Flavobacterium/Bacteroides phylum.
Keywords: Soda lakes; Alkaliphiles; Anaerobes; Polysaccharide degradation; Gliding bacteriaCytophaga/Flavobacterium/Bacteroides phylum; CFB bacteria; Propionate fermentationAlkaliflexus imshenetskii
Sulfoacetate generated by Rhodopseudomonas palustris from taurine by Karin Denger; Sonja Weinitschke; Klaus Hollemeyer; Alasdair M. Cook (pp. 254-258).
Genes thought to encode (a) the regulator of taurine catabolism under carbon-limiting or nitrogen-limiting conditions and (b) taurine dehydrogenase were found in the genome of Rhodopseudomonas palustris. The organism utilized taurine quantitatively as a sole source of nitrogen (but not of carbon) for aerobic and photoheterotrophic growth. No sulfate was released, and the C-sulfonate bond was recovered stoichiometrically as sulfoacetate, which was identified by mass spectrometry. An inducible sulfoacetaldehyde dehydrogenase was detected. R. palustris thus contains a pathway to generate a natural product that was previously believed to be formed solely from sulfoquinovose.
Keywords: Sulfoacetate formation; Taurine deamination; Taurine dehydrogenase; Sulfoacetaldehyde dehydrogenase; Rhodopseudomonas palustris
Characterization of a heme oxygenase of Clostridium tetani and its possible role in oxygen tolerance by Holger Brüggemann; Rosalie Bauer; Stéphanie Raffestin; Gerhard Gottschalk (pp. 259-263).
In order to colonize mammalian wounds, the anaerobic bacterium Clostridium tetani must presumably cope with temporary oxic conditions. Therefore, the recently decoded genome sequence was searched for genes which could confer oxygen tolerance. A few identified systems such as superoxide dismutases and peroxidases are probably responsible for this protection against toxic oxygen species. Another system was detected, a heme oxygenase which could have a role in establishing or maintaining an anoxic microenvironment in the process of wound colonization. The hemT gene encoding the heme oxygenase is expressed in C. tetani, as shown by reverse transcription–PCR. When overexpressed in Escherichia coli, the enzyme converts heme to biliverdin under strict oxic conditions.
Keywords: Clostridium tetaniTetanus; Heme oxygenase; Oxidative stress; Aerotolerance; Iron acquisition