Showing papers in "Archives of Microbiology in 2002"

Molecular biology and biochemistry of ammonia oxidation by Nitrosomonas europaea.

Abstract: Nitrosomonas europaea uses only NH3, CO2 and mineral salts for growth and as such it is an obligate chemo-lithoautotroph. The oxidation of NH3 is a two-step process catalyzed by ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO). AMO catalyzes the oxidation of NH3 to NH2OH and HAO catalyzes the oxidation of NH2OH to NO2–. AMO is a membrane-bound enzyme composed of three subunits. HAO is located in the periplasm and is a homotrimer with each subunit containing eight c-type hemes. The electron flow from HAO is channeled through cytochrome c554 to cytochrome cm552, where it is partitioned for further utilization. Among the ammonia-oxidizing bacteria, the genes for AMO, these cytochromes, and HAO are present in up to three highly similar copies. Mutants with mutations in the copies of amoCAB and hao in N. europaea have been isolated. All of the amoCAB and hao gene copies are functional. N. europaea was selected by the United States Department of Energy for a whole-genome sequencing project. In this article, we review recent research on the molecular biology and biochemistry of NH3 oxidation in nitrifiers.

Diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria.

Abstract: Autotrophic ammonia-oxidizing bacteria use the essential enzyme ammonia monooxygenase (AMO) to transform ammonia to hydroxylamine. The amo operon consists of at least three genes, amoC, amoA, and amoB; amoA encodes the subunit containing the putative enzyme active site. The use of the amo genes as functional markers for ammonia-oxidizing bacteria in environmental applications requires knowledge of the diversity of the amo operon on several levels: (1) the copy number of the operon in the genome, (2) the arrangement of the three genes in an individual operon, and (3) the primary sequence of the individual genes. We present a database of amo gene sequences for pure cultures of ammonia-oxidizing bacteria representing both the β- and the γ-subdivision of Proteobacteria in the following genera: Nitrosospira (6 strains), Nitrosomonas (5 strains) and Nitrosococcus (2 strains). The amo operon was found in multiple (2–3) nearly identical copies in the β-subdivision representatives but in single copies in the γ-subdivision ammonia oxidizers. The analysis of the deduced amino acid sequence revealed strong conservation for all three Amo peptides in both primary and secondary structures. For the amoA gene within the β-subdivision, nucleotide identity values are approximately 85% within the Nitrosomonas or the Nitrosospira groups, but approximately 75% when comparing between these groups. Conserved regions in amoA and amoC were identified and used as primer sites for PCR amplification of amo genes from pure cultures, enrichments and the soil environment. The intergenic region between amoC and amoA is variable in length and may be used to profile the community of ammonia-oxidizing bacteria in environmental samples. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00203-001-0369-z.

Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria.

Abstract: A highly efficient method of transposon mutagenesis was developed for genetic analysis of Xanthobacter autotrophicus Py2. The method makes use of a transposon delivery vector that encodes a hyperactive Tn 5 transposase that is 1,000-fold more active than the wild-type transposase. In this construct, the transposase is expressed from the promoter of the tetA gene of plasmid RP4, which is functional in a wide variety of organisms. The transposon itself contains a kanamycin resistance gene as a selectable marker and the origin of replication from plasmid R6K to facilitate subsequent cloning of the resulting insertion site. To test the effectiveness of this method, mutants unable to produce the characteristic yellow pigment (zeaxanthin dirhamnoside) of X. autotrophicus Py2 were isolated and analyzed. Transposon insertions were obtained at high frequency: approximately 1 x 10(-3) per recipient cell. Among these, pigment mutants were observed at a frequency of approximately 10(-3). Such mutants were found to have transposon insertions in genes homologous to known carotenoid biosynthetic genes previously characterized in other pigmented bacteria. Mutants were also isolated in Pseudomonas stutzeri and in an Alcaligenes faecalis, demonstrating the effectiveness of the method in diverse Proteobacteria. Preliminary results from other laboratories have confirmed the effectiveness of this method in additional phylogenetically diverse species.

Factors influencing methicillin resistance in staphylococci.

Abstract: Methicillin resistance in staphylococci is due to an acquired penicillin-binding protein, PBP2' (PBP2a). This additional PBP, encoded by mecA, confers an intrinsic resistance to all beta-lactams and their derivatives. Resistance levels in methicillin-resistant Staphylococcus aureus (MRSA) depend on efficient PBP2' production and are modulated by chromosomal factors. Depending on the genetic background of the strain that acquired mecA, resistance levels range from phenotypically susceptible to highly resistant. Characteristic for most MRSA is the heterogeneous expression of resistance, which is due to the segregation of a more highly resistant subpopulation upon challenge with methicillin. Maximal expression of resistance by PBP2' requires the efficient and correct synthesis of the peptidoglycan precursor. Genes involved in cell-wall precursor formation and turnover, regulation, transport, and signal transduction may determine the level of resistance that is expressed. At this stage, however, there is no information available on the functionality or efficacy of such factors in clinical isolates in relation to methicillin resistance levels.

Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe

Abstract: A native-feather-degrading thermophilic anaerobe was isolated from a geothermal hot stream in Indonesia. Isolate AW-1, identified as a member of the species Fervidobacterium islandicum, was shown to degrade native feathers (0.8%, w/v) completely at 70 °C and pH 7 with a maximum specific growth rate (0.14 h–1) in Thermotoga-Fervidobacterium (TF) medium. After 24 h of culture, feather degradation led to an increase in free amino acids such as histidine, cysteine and lysine. Moreover, nutritionally essential amino acids such as tryptophan and methionine, which are rare in feather keratin, were also produced as microbial metabolites. A homomultimeric membrane-bound keratinolytic protease (>200 kDa; 97 kDa subunits) was purified from a cell extract of F. islandicum AW-1. The enzyme exhibited activity toward casein and soluble keratin optimally at 100 °C and pH 9, and had a half-life of 90 min at 100 °C. The enzyme showed higher specific activity for the keratinous substrates than other proteases and catalyzed the cleavage of peptide bonds more rapidly following the reduction of disulfide bridges in feather keratin by 10 mM dithiothreitol. Therefore, the enzyme from F. islandicum AW-1 is a novel, thermostable keratinolytic serine protease.

Phylogenetic and metabolic diversity of bacteria degrading aromatic compounds under denitrifying conditions, and description of Thauera phenylacetica sp. nov., Thauera aminoaromatica sp. nov., and Azoarcus buckelii sp. nov.

Abstract: Six strains of denitrifying bacteria isolated from various oxic and anoxic habitats on different monocyclic aromatic substrates were characterized by sequencing 16S rRNA genes, determining physiological and morphological traits, and DNA-DNA hybridization. According to these criteria, strains S100, SP and LG356 were identified as members of Thauera aromatica. Strains B5–1 and B5–2 were tentatively affiliated to the species Azoarcus tolulyticus. Strains B4P and S2 were only distantly related to each other and to other described Thauera species. These two strains are proposed as the type strains of two new species, Thauera phenylacetica sp. nov. and Thauera aminoaromatica sp. nov., respectively. By 16S rRNA gene analysis, strain U120 was highly related to the type strains of Azoarcus evansii and Azoarcus anaerobius, whereas corresponding DNA-DNA reassociation values indicated only a low degree of genomic relatedness. Based upon a low DNA similarity value and the presence of distinguishing physiological properties, strain U120 is proposed as the type strain of a new species, Azoarcus buckelii sp. nov. Almost all of the new isolates were obtained with different substrates. The highly varied substrate spectra of the isolates indicates that an even higher diversity of denitrifying bacteria degrading aromatic compounds would be discovered in the different habitats by using a larger spectrum of aromatic substrates for enrichment and isolation.

Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria.

Abstract: Methylotrophic bacteria can grow on a number of substrates as energy source with only one carbon atom, such as methanol, methane, methylamine, and dichloromethane These compounds are metabolized via the cytotoxin formaldehyde The formaldehyde consumption pathways, especially the pathways for the oxidation of formaldehyde to CO2 for energy metabolism, are a central and critical part of the metabolism of these aerobic bacteria Principally, two main types of pathways for the conversion of formaldehyde to CO2 have been described: (1) a cyclic pathway initiated by the condensation of formaldehyde with ribulose monophosphate, and (2) distinct linear pathways that involve a dye-linked formaldehyde dehydrogenase or C1 unit conversion bound to the cofactors tetrahydrofolate (H4F), tetrahydromethanopterin (H4MPT), glutathione (GSH), or mycothiol (MySH) The pathways involving the four cofactors have in common the following sequence of events: the spontaneous or enzyme-catalyzed condensation of formaldehyde and the respective C1 carrier, the oxidation of the cofactor-bound C1 unit and its conversion to formate, and the oxidation of formate to CO2 However, the H4MPT pathway is more complex and involves intermediates that were previously known solely from the energy metabolism of methanogenic archaea The occurrence of the different formaldehyde oxidation pathways is not uniform among different methylotrophic bacteria The pathways are in part also used by other organisms to provide C1 units for biosynthetic reactions (eg, H4F-dependent enzymes) or detoxification of formaldehyde (eg, GSH-dependent enzymes)

Sequence and phylogenetic analyses of the twin-arginine targeting (Tat) protein export system.

Abstract: Twin-arginine targeting (Tat) protein secretion systems consist of two protein types, members of the TatA and TatC families. Homologues of these proteins are found in many archaea, bacteria, chloroplasts and mitochondria. Every prokaryotic organism with a fully sequenced genome exhibits either neither family member, or between one and three paralogues of these two family members. The Arabidopsis thaliana genome encodes three of each. Although many mitochondrially encoded TatC homologues have been identified, corresponding TatA homologues have not been found in this organelle. Phylogenetic analyses reveal that most prokaryotic Tat systems consist of one TatC homologue and two sequence-divergent TatA homologues (TatA and TatB). When only one TatA homologue is present, TatB is missing, and when three TatA homologues are present, the third one arose by duplication of TatA, not TatB. Further, homologues most resembling TatB are more sequence-divergent than those more closely resembling TatA. In contrast to the TatA family, the TatC family shows phylogenetic clustering in strict accordance with organismal type. These results are discussed in terms of their probable structural, functional and evolutionary significance.

Genes involved in the anaerobic degradation of toluene in a denitrifying bacterium, strain EbN1

Abstract: The organization of all genes required for the anaerobic conversion of toluene to benzoyl-CoA was investigated in denitrifying Azoarcus-like strain EbN1. All of these genes are clustered within 25.3 kb of contiguous DNA sequence, which includes only a few intervening sequences. The toluene-catabolic genes are organized in two apparent operons. One contains the genes (bssCAB) for the three subunits of benzylsuccinate synthase, which initiates anaerobic toluene degradation by converting toluene to (R)-benzylsuccinate. The BssCAB proteins of strain EbN1 are most similar to those of Thauera aromatica strain K172. The bssCAB genes are part of a larger putative operon (bssDCABEFGH), which contains the gene bssD, encoding the activase for benzylsuccinate synthase, and four genes (bssEFGH) encoding proteins of unknown function. RT-PCR experiments showing continuation of transcription over the three largest intergenic regions of the bss operon support the assumed structure. Moreover, BssG was identified as toluene-induced protein. Downstream of the bss genes, another large putative operon (bbsA–H) was identified that contains all genes required for β-oxidation of benzylsuccinate to benzoyl-CoA, e.g. bbsEF, encoding succinyl-CoA:(R)-benzylsuccinate CoA-transferase. Immediately upstream of the bss operon, genes for a two-component regulatory system were identified; their products may sense toluene and induce the expression of both catabolic operons. The order and sequences of the bss and bbs genes are highly similar among toluene-degrading denitrifiers. The bss and bbs genes of the FeIII-reducing Geobacter metallireducens display less sequence similarity and are organized differently. The genes between the bss and bbs operons and in the flanking regions differ between strain EbN1 and the other strains.

Cellular differentiation in the cyanobacterium Nostoc punctiforme

Abstract: Nostoc punctiforme is a phenotypically complex, filamentous, nitrogen-fixing cyanobacterium, whose vegetative cells can mature in four developmental directions. The particular developmental direction is determined by environmental signals. The vegetative cell cycle is maintained when nutrients are sufficient. Limitation for combined nitrogen induces the terminal differentiation of heterocysts, cells specialized for nitrogen fixation in an oxic environment. A number of unique regulatory events and genes have been identified and integrated into a working model of heterocyst differentiation. Phosphate limitation induces the transient differentiation of akinetes, spore-like cells resistant to cold and desiccation. A variety of environmental changes, both positive and negative for growth, induce the transient differentiation of hormogonia, motile filaments that function in dispersal. Initiation of the differentiation of heterocysts, akinetes and hormogonia are hypothesized to depart from the vegetative cell cycle, following separate and distinct events. N. punctiforme also forms nitrogen-fixing symbiotic associations; its plant partners influence the differentiation and behavior of hormogonia and heterocysts. N. punctiforme is genetically tractable and its genome sequence is nearly complete. Thus, the regulatory circuits of three cellular differentiation events and symbiotic interactions of N. punctiforme can be experimentally analyzed by functional genomics.

Biology of extremophilic and extremotolerant methanotrophs.

Abstract: This review summarizes recent findings on the biology of obligate methanotrophic bacteria living in various extreme environments. By using molecular ecology techniques, it has become clear that obligate methanotrophs are ubiquitous in nature and well adapted to high or low temperature, pH and salinity. The isolation and characterization of pure cultures has led to the discovery of several new genera and species of extremophilic/tolerant methanotrophs. Their major physiological role is participation in the methane cycle and supplying C(1) intermediates and various metabolites to other members of microbial communities in extreme ecosystems. To survive under extreme conditions, methanotrophs have developed diverse structure-function adaptive mechanisms including cell-surface layer formation, changes in cellular phospholipid composition and de novo synthesis of organic osmolytes such as ectoine, 5-oxoproline and sucrose. However, despite the above advances, basic knowledge of other stress protectants, as well as bioenergetic and genetic aspects of methanotroph adaptation, is still lacking. This information is necessary for better understanding the molecular mechanisms underlying the versatility of methanotrophs and for the development of novel biotechnological processes.

Anaerobic degradation of n -hexane in a denitrifying bacterium: Further degradation of the initial intermediate (1-methylpentyl)succinate via C-skeleton rearrangement

Abstract: The anaerobic degradation pathway of the saturated hydrocarbon n-hexane in a denitrifying strain (HxN1) was examined by gas chromatography-mass spectrometry of derivatized extracts from cultures grown with unlabeled and deuterated substrate; several authentic standard compounds were included for comparison. The study was focused on possible reaction steps that follow the initial formation of (1-methylpentyl)succinate from n-hexane and fumarate. 4-Methyloctanoic, 4-methyloct-2-enoic, 2-methylhexanoic, 2-methylhex-2-enoic and 3-hydroxy-2-methylhexanoic acids (in addition to a few other methyl-branched acids) were detected in n-hexane-grown but not in n-hexanoate-grown cultures. Labeling indicated preservation of the original carbon chain of n-hexane in these acids. Tracing of the deuterium label of 3-d 1-(1-methylpentyl)succinate in tentative subsequent products indicated a deuterium/carboxyl carbon exchange in the succinate moiety. This suggests that the metabolism of (1-methylpentyl)succinate employs reactions analogous to those in the established conversion of succinyl-CoA via methylmalonyl-CoA to propionyl-CoA. Accordingly, a pathway is proposed in which (1-methylpentyl)succinate is converted to the CoA-thioester, rearranged to (2-methylhexyl)malonyl-CoA and decarboxylated (perhaps by a transcarboxylase) to 4-methyloctanoyl-CoA. The other identified fatty acids match with a further degradation of 4-methyloctanoyl-CoA via rounds of conventional β-oxidation. Such a pathway would also allow regeneration of fumarate (for n-hexane activation) from propionyl-CoA formed as intermediate and hence present a cyclic process.

Effect of environmental factors on the synthesis of scytonemin, a UV-screening pigment, in a cyanobacterium (Chroococcidiopsis sp.).

Abstract: The UV-screening pigment scytonemin is found in many species of ensheathed cyanobacteria. Past work has shown that the pigment is synthesized in response to exposure to UV-A irradiance. This study investigated the effect of other correlated stress factors including heat, osmotic and oxidative stress on the synthesis of scytonemin in a clonal cyanobacterial isolate (Chroococcidiopsis sp.) from an epilithic desert crust. Stress experiments were carried out both in conjunction with UV-A irradiance and in isolation. Increases in both temperature and photooxidative conditions in conjunction with UV-A caused a synergistic increase in the rate of scytonemin production. In contrast, increased salt concentration under UV-A irradiance inhibited scytonemin synthesis. However, unlike the responses to temperature and oxidative stress, cells synthesized low levels of scytonemin under osmotic stress in the absence of scytonemin-inducing irradiance. These results suggest that scytonemin induction may be regulated as a part of a complex stress response pathway in which multiple environmental signals affect its synthesis.

Different effectors of dimorphism in Yarrowia lipolytica.

Abstract: Yarrowia lipolytica is an ascomycete with biotechnological potential. In common media, the fungus grows as a mixture of yeast-like and short mycelial cells. The environmental factors that affect dimorphism in the wild-type strain, W29, and its auxotrophic derivative, PO1a, were analyzed. In both strains, pH was the most important factor regulating the dimorphic transition. Mycelium formation was maximal at pH near neutrality and decreased as pH was lowered to become almost null at pH 3. Carbon and nitrogen sources, namely glucose and ammonium, were also important for mycelium formation; and their effect was antagonized by some alternative carbon and nitrogen sources. Citrate was an important positive effector of mycelium growth. Anaerobic stress induced formation of mycelial cells. The importance of the protein kinase A pathway was suggested by the inhibition of mycelium growth by cAMP. We propose that the interplay of these factors regulates the adaptation of the fungus, to better exploit its natural ecological niches.

Genetics and control of CO(2) assimilation in the chemoautotroph Ralstonia eutropha.

Abstract: The nutritional versatility of facultative autotrophs requires efficient overall control of their metabolism. Most of these organisms are Proteobacteria that assimilate CO2 via the highly energy-demanding Calvin-Benson-Bassham reductive pentose-phosphate cycle. The enzymes of the cycle are encoded by cbb genes organized in cbb operons differing in size and composition, although conserved features are apparent. Transcription of the operons, which may form regulons, is strictly controlled, being induced during autotrophic but repressed to varying extents during heterotrophic growth of the bacteria. The chemoautotroph Ralstonia eutropha is one of the organisms studied extensively for the mechanisms involved in the expression of cbb gene systems. CbbR is a LysR-type transcriptional regulator and the key activator protein of cbb operons. The cbbR gene is typically located adjacent and in divergent orientation to its cognate operon. The activating function of CbbR seems to be modulated by metabolites signaling the nutritional state of the cell to the cbb system. Phosphoenolpyruvate is such a signal metabolite acting as a negative effector of R. eutropha CbbR, whereas NADPH has been proposed to be a coactivator of the protein in two other chemoautotrophs, Xanthobacter flavus and Hydrogenophilus thermoluteolus. There is evidence for the participation of additional regulators in cbb control. In the photoautotrophs Rhodobacter capsulatus and Rhodobacter sphaeroides, response regulator RegA of the global two-component signal transduction system RegBA serves this function. It is conceivable that specific variants of cbb control systems have evolved to ensure their optimal integration into regulatory networks operating in the diverse autotrophs characterized by different metabolic capabilities.

Genetic groups of the insect-pathogenic fungus Beauveria bassiana are associated with habitat and thermal growth preferences.

Abstract: A persistent paradigm in insect pathology is one that relates the insect host to certain genetic groups of insect-pathogenic fungi. This paradigm assumes that the genotype of an insect-pathogenic fungus coevolves with a certain taxon of insect host that it infects. The insect-pathogenic fungus Beauveria bassiana shows a wide host range and is considered to be a facultative insect pathogen. In this study, a population genetics analysis of B. bassiana from forested and agricultural habitats as well as from the Canadian Arctic showed distinct genetic groups associated with the three different habitats. Within each group, recombining population structures and clonally reproducing lineages were observed. The B. bassiana isolates were also assessed for their abilities to grow at 8, 15, 25 and 37 °C and for their tolerances to UV exposure. The genetic groups from the Arctic and from the forested habitats grew at lower temperatures, while the genetic group from the agricultural habitat grew at 37 °C and was tolerant to UV exposure. There were no clear associations between the genetic group and the ability to infect coleopteran or lepidopteran insect larvae. There is increasing evidence that such studies represent a significant paradigm shift; habitat selection, not insect host selection, drives the population structure of deuteromycetous insect-pathogenic fungi. We suggest that adaptation to a certain habitat type is an important criterion for identifying insect-pathogenic fungal strains for use in insect biocontrol efforts.

Isolation of an anaerobic intestinal bacterium capable of cleaving the C-ring of the isoflavonoid daidzein.

Abstract: Colonic bacteria were screened for bacteria involved in the conversion of phytoestrogens. A gram-positive anaerobic bacterium, strain HGH 136, capable of conversion of the isoflavonoid daidzein, was isolated and identified as a Clostridium sp. The bacterium cleaved the C-ring of daidzein to produce O-demethylangolensin (O-Dma). This compound was identified by comparison of the HPLC retention time and UV spectrum of the metabolite with chemically synthesized O-Dma. The identity of the metabolite was confirmed by liquid chromatography-mass spectrometry and NMR using synthetic O-Dma as a standard. The bacterium incubated with synthetic dihydrodaidzein also produced O-Dma. After 3 days of incubation, 28% of added daidzein and 12% of added dihydrodaidzein were converted to O-Dma. This is the first study in which an anaerobic bacterium involved in the ring cleavage of daidzein to produce O-Dma has been identified.

Evaluation of non-cyanobacterial genome sequences for occurrence of genes encoding proteins homologous to cyanophycin synthetase and cloning of an active cyanophycin synthetase from Acinetobacter sp. strain DSM 587.

Abstract: All publicly accessible microbial genome databases were searched for the occurrence of genes encoding proteins homologous to the cyanophycin synthetase (CphA) of Synechocystis sp. strain PCC 6803 in order to reveal the capability of microorganisms not belonging to the cyanobacteria to synthesize cyanophycin. Among 65 genome sequences, genes homologous to cphA were found in Acinetobacter sp. strain ADP1 (encoding a protein homologous to CphA with 40% amino acid identity), Bordetella bronchiseptica strain RB50 (39%), Bordetella pertussis strain Tohama I (39%), Bordetella parapertussis strain 12822 (39%), Clostridium botulinum strain ATCC 3502 (39%), Desulfitobacterium hafniense strain DCB-2 (38%) and Nitrosomonas europaea strain ATCC 25978 (37%). The gene homologous to cphA from Acinetobacter sp. strain DSM 587 was amplified by PCR, ligated to the vector pBluescript SK– downstream of the lac promoter and introduced into Escherichia coli. The recombinant strain of E. coli expressed CphA activity at up to 1.2 U/mg protein and accumulated cyanophycin to up to 7.5% of the cellular dry matter, indicating that CphA of Acinetobacter sp. strain DSM 587 is functionally active. In Acinetobacter sp. strain DSM 587 itself, cyanophycin accumulated to up to 1.4% of the total protein under phosphate-limited conditions, and cyanophycin synthetase activity was detected, which indicated the function of cyanophycin as a storage compound in this strain.

New genes involved in chromate resistance in Ralstonia metallidurans strain CH34.

Abstract: Chromate resistance inRalstonia metallidurans CH34 is based on chromate efflux catalyzed by ChrA efflux pumps. The bacterium harbors two chromate resistance determinants, the previously knownchr 1 on plasmid pMOL28 (geneschrI, chrB 1,chrA 1,chrC, chrE, chrF 1) andchr 2 on the chromosome (geneschrB 2,chrA 2,chrF 2). Deletion of the geneschrl, chrC, chrA 2,chrB 2 andchrF 2 influenced chromate resistance and transcription from achrBp 1::lacZ fusion. Deletion of the plasmid-encoded genechrBx did not change chromate resistance orchrBp 1 regulation. Northern hybridization and primer-extension experiments were used to study transcription of the plasmid-encodedchr 1 determinant. Transcription ofchrB 1,chrA 1 andchrC was induced by chromate. The presence of sulfate influenced transcription positively. ThechrBp 1,chrAp 1 andchrCp promoters showed some similarity to heat-shock promoters. Transcription of the generpoH encoding a putative heat-shock sigma factor was also induced by chromate, butrpoH was not essential for chromate resistance. The ChrC protein was purified as a homotetramer and exerted superoxide dismutase activity. Thus, possible regulators for chromate resistance (Chrl, ChrB1, ChrB2, ChrF1, and ChrF2) and an additional detoxification system (ChrC) were newly identified as parts of chromate resistance inR. metallidurans. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00203-002-0492-5.

Cysteine-mediated electron transfer in syntrophic acetate oxidation by cocultures of Geobacter sulfurreducens and Wolinella succinogenes.

Abstract: Syntrophic cocultures of Geobacter sulfurreducens and Wolinella succinogenes oxidize acetate with nitrate as terminal electron acceptor. It has been postulated earlier that electrons are transferred in these cocultures not via hydrogen, but via a different carrier, e.g., a small c-type cytochrome that is detected in the supernatant of growing cultures. In the present study, L-cysteine, which was provided as a reducing agent, was found to mediate the electron transfer between the two partners. Low concentrations of L-cysteine or L-cystine (10–100 µM) supported syntrophic growth, and no acetate oxidation was observed in the absence of cysteine or cystine. Cell suspensions of G. sulfurreducens or coculture cell suspensions reduced cystine to cysteine, and suspensions of W. succinogenes or coculture suspensions oxidized cysteine with nitrate, as measured by the formation or depletion of free thiol groups. Added cysteine was rapidly oxidized by the coculture during growth, but the formed cystine was not entirely rereduced even under acceptor-limited conditions. The redox potential prevailing in acetate-oxidizing cocultures was –160 to –230 mV. Sulfide at low concentrations supported syntrophic growth as well and could replace cysteine. Neither growth nor acetate degradation was found with D-cysteine, homocysteine, cysteamine, 3-mercaptopropionate, dithiothreithol, thioglycolate, glutathione, coenzyme M, dimethylsulfoxide, trimethylamine-N-oxide, anthraquinone-2,6-disulfonate, or ascorbate.

Desulfobulbus mediterraneus sp. nov., a sulfate-reducing bacterium growing on mono- and disaccharides.

Abstract: A new sulfate-reducing bacterium, strain 86FS1, was isolated from a deep-sea sediment in the western Mediterranean Sea with sodium lactate as electron and carbon source. Cells were ovoid, gram-negative and motile. Strain 86FS1 contained b- and c-type cytochromes. The organism was able to utilize propionate, pyruvate, lactate, succinate, fumarate, malate, alanine, primary alcohols (C(2)-C(5)), and mono- and disaccharides (glucose, fructose, galactose, ribose, sucrose, cellobiose, lactose) as electron donors for the reduction of sulfate, sulfite or thiosulfate. The major products of carbon metabolism were acetate and CO(2), with exception of n-butanol and n-pentanol, which were oxidized only to the corresponding fatty acids. The growth yield with sulfate and glucose or lactate was 8.3 and 15 g dry mass, respectively, per mol sulfate. The temperature limits for growth were 10 degrees C and 30 degrees C with an optimum at 25 degrees C. Growth was observed at salinities ranging from 10 to 70 g NaCl l(-1). Sulfide concentrations above 4 mmol l(-1) inhibited growth. The fatty acid pattern of strain 86FS1 resembled that of Desulfobulbus propionicus with n-14:0, n-16:1omega7, n-16:1 omega5, n-17:1 omega6 and n-18:1 omega7 as dominant fatty acids. On the basis of its phylogenetic position and its phenotypic properties, strain 86FS1 affiliates with the genus Desulfobulbus and is described as a new species, Desulfobulbus mediterraneus sp. nov.

Polyphasic characterization of benthic, moderately halophilic, moderately thermophilic cyanobacteria with very thin trichomes and the proposal of Halomicronema excentricum gen. nov., sp. nov.

Abstract: A new genus of moderately halophilic, moderately halotolerant and moderately thermophilic cyanobacteria with very thin trichomes is described The four strains included in this genus were isolated from benthic microbial mats in a man-made hypersaline pond Trichomes were around 1 µm thick, with small constrictions at the cross-walls and diffluent colorless sheaths Thylakoids were parallel to the cell wall, but thylakoids and nucleoid were often excentrically arranged within the cytoplasm with respect to the main trichome axis Strains grew at between 32 and 12–15% (w/v) salinity with optima between 32 and 12% They showed lower temperature limits around 20 °C and upper limits between 45 and 50 °C, with optima between 28 and 45–50 °C Carotenoid and mycosporine amino-acid complements were identical among strains Phylogenetic analyses based on 16S rRNA gene sequence showed that all strains were closely related (99% or higher similarity) and distantly related to other cyanobacteria (91% or lower similarity) We propose the new genus and species Halomicronema excentricum for these strains The type strain is TFEP1

Characterisation, genome size and genetic manipulation of the myxobacterium Sorangium cellulosum So ce56.

Abstract: In this study, Sorangium cellulosum So ce56 was phenotypically and genotypically analysed in order to evaluate whether this strain can be used in a comprehensive genome project as a representative of the secondary metabolite-producing myxobacteria. In contrast to many other strains of S. cellulosum, strain So ce56 was found to have various advantageous features, including fast and homogeneous growth in submerged cultures and the ability to complete its morphological differentiation cycle on agar, even when the inoculant originates from a liquid culture. Two groups of secondary metabolites isolated from the culture broth were identified, the polyketides etnangien and chivosazole. The presence of polyketide synthase-encoding genes in the genome of strain So ce56 was demonstrated via PCR. The phenotypic classification was confirmed by comparison of 16S rDNA sequences which showed that S. cellulosum So ce56 clusters within a separate lineage together with S. cellulosum ATCC 25531 and the epothilone producer S. cellulosum So ce90. The genome of S. cellulosum So ce56 belongs to the largest bacterial genomes described so far. It is estimated to be 12.2 Mb in size, by pulsed-field gel electrophoresis. In order to demonstrate that S. cellulosum So ce56 is a convenient strain for molecular biological studies, a genetic manipulation system was developed. Using triparental mating, polyketide synthase-encoding genes were inactivated, leading to chivosazole-negative mutants.

Identification of the mycothiol synthase gene (mshD) encoding the acetyltransferase producing mycothiol in actinomycetes.

Abstract: Mycothiol is the predominant thiol in most actinomycetes, including Mycobacterium tuberculosis, and appears to play a role analogous to glutathione, which is not found in these bacteria. The enzymes involved in mycothiol biosynthesis are of interest as potential targets for new drugs directed against tuberculosis. In this work we describe the isolation and characterization of a Tn5 transposon mutant of Mycobacterium smegmatis that is blocked in the production of mycothiol and accumulates its precursor, 1D-myo-inosityl 2-L-cysteinylamido-2-deoxy-α-D-glucopyranoside (Cys-GlcN-Ins). Cys-GlcN-Ins isolated from this mutant was used to assay for acetyl-CoA:Cys-GlcN-Ins acetyltransferase (mycothiol synthase, MshD) activity, which was found in wild-type cells, but not in the mutant. Sequencing outward of the DNA of the mutant strain from the site of insertion permitted identification of the mshD gene in the M. smegmatis genome, as well as the orthologous gene Rv0819 in the M. tuberculosis genome. Cloning and expression of mshD from M. tuberculosis (Rv0819) in Escherichia coli gave a transformant with MshD activity, demonstrating that Rv0819 is the mshD mycothiol biosynthesis gene.

Tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans : substrate specificity of the native enzyme and its corrinoid cofactor

Abstract: The substrate specificity of the tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans and its corrinoid cofactor were studied. Besides reduced methyl viologen, titanium(III) citrate could serve as electron donor for reductive dehalogenation of tetrachloroethene (PCE) and trichloroethene to cis-1,2-dichloroethene. In addition to chlorinated ethenes, chlorinated propenes were reductively dechlorinated solely by the native enzyme. trans-1,3-Dichloropropene, 1,1,3-trichloropropene and 2,3-dichloropropene were reduced to a mixture of mono-chloropropenes, 1,1-dichloropropene, and 2-chloropropene, respectively. Other halogenated compounds that were rapidly reduced by the enzyme were also dehalogenated abiotically by the heat-inactivated enzyme and by commercially available cyanocobalamin. The rate of this abiotic reaction was dependent on the number and type of halogen substituents and on the type of catalyst. The corrinoid cofactor purified from the tetrachloroethene dehalogenase of D. multivorans exhibited an activity about 50-fold higher than that of cyanocobalamin (vitamin B12) with trichloroacetate as electron acceptor, indicating that the corrinoid cofactor of the PCE dehalogenase is not cyanocobalamin. Corrinoids catalyzed the rapid dehalogenation of trichloroacetic acid. The rate was proportional to the amount of, e.g. cyanocobalamin; therefore, the reductive dehalogenation assay can be used for the sensitive and rapid quantification of this cofactor.

Localization of Mn(II)-oxidizing activity and the putative multicopper oxidase, MnxG, to the exosporium of the marine Bacillus sp. strain SG-1

Abstract: Dormant spores of the marine Bacillus sp. strain SG-1 catalyze the oxidation of manganese(II), thereby becoming encrusted with insoluble Mn(III,IV) oxides. In this study, it was found that the Mn(II)-oxidizing activity could be removed from SG-1 spores using a French press and recovered in the supernatant following centrifugation of the spores. Transmission electron microscopy of thin sections of SG-1 spores revealed that the ridged outermost layer was removed by passage through the French press, leaving the remainder of the spore intact. Comparative chemical analysis of this layer with the underlying spore coats suggested that this outer layer is chemically distinct from the spore coat. Taken together, these results indicate that this outer layer is an exosporium. Previous genetic analysis of strain SG-1 identified a cluster of genes involved in Mn(II) oxidation, the mnx genes. The product of the most downstream gene in this cluster, MnxG, appears to be a multicopper oxidase and is essential for Mn(II) oxidation. In this study, MnxG was overexpressed in Escherichia coli and used to generate polyclonal antibodies. Western blot analysis demonstrated that MnxG is localized to the exosporium of wild-type spores but is absent in the non-oxidizing spores of transposon mutants within the mnx gene cluster. To our knowledge, Mn(II) oxidation is the first oxidase activity, and MnxG one of the first gene products, ever shown to be associated with an exosporium.

Assembly of membrane-bound respiratory complexes by the Tat protein-transport system.

Abstract: The Tat protein-export system serves to translocate folded proteins, often containing redox cofactors, across the bacterial inner membrane. Substrate proteins are directed to the Tat apparatus by distinctive N-terminal signal peptides containing a consensus SRRxFLK 'twin-arginine' motif. Here we review recent studies of the Tat system with particular emphasis on the assembly of membrane-bound respiratory complexes. We discuss the connection between Tat targeting and topological organisation of the complexes and consider the role of chaperone proteins in cofactor insertion and Tat targeting. The crystal structure of Escherichia coli formate dehydrogenase-N demonstrates that some Tat substrates are integral membrane proteins. Sequence analysis suggests that one-quarter of all traffic on the E. coli Tat pathway is inner-membrane proteins.

Desulfotignum phosphitoxidans sp. nov. : a new marine sulfate reducer that oxidizes phosphite to phosphate

Abstract: A new sulfate-reducing bacterium was isolated from marine sediment with phosphite as sole electron donor and CO2 as the only carbon source. Strain FiPS-3 grew slowly, with doubling times of 3–4 days, and oxidized phosphite, hydrogen, formate, acetate, fumarate, pyruvate, glycine, glutamate, and other substrates nearly completely, with concomitant reduction of sulfate to sulfide. Acetate was formed as a side product to a small extent. Glucose, arabinose, and proline were partly oxidized and partly fermented to acetate plus propionate. Growth with phosphite, hydrogen, or formate was autotrophic. Also, in the presence of sulfate, CO dehydrogenase was present, and added acetate did not increase growth rates or growth yields. In the absence of sulfate, phosphite oxidation was coupled to homoacetogenic acetate formation, with growth yields similar to those in the presence of sulfate. Cells were small rods, 0.6–0.8×2–4 µm in size, and gram-negative, with a G+C content of 53.9 mol%. They contained desulforubidin, but no desulfoviridin. Based on sequence analysis of the 16S rRNA gene and the sulfite reductase genes dsrAB, strain FiPS-3 was found to be closely related to Desulfotignum balticum. However, physiological properties differed in many points from those of D. balticum. These findings justify the establishment of a new species, Desulfotignum phosphitoxidans.

The role of the fatty acid beta-oxidation multienzyme complex from Pseudomonas oleovorans in polyhydroxyalkanoate biosynthesis: molecular characterization of the fadBA operon from P. oleovorans and of the enoyl-CoA hydratase genes phaJ from P. oleovorans and Pseudomonas putida.

Abstract: In order to investigate the role of the putative epimerase function of the beta-oxidation multienzyme complex (FadBA) in the provision of (R)-3-hydroxyacyl-CoA thioesters for medium-chain-length polyhydroxyalkanoate (PHA(MCL)) biosynthesis, the fadBA(Po) operon of Pseudomonas oleovorans was cloned and characterized. The fadBA(Po) operon and a class-II PHA synthase gene of Pseudomonas aeruginosa were heterologously co-expressed in Escherichia coli to determine whether the putative epimerase function of FadBA(Po) has the ability to provide precursors for PHA accumulation in a non-PHA-accumulating bacterium. Cultivation studies with fatty acids as carbon source revealed that FadBA(Po) did not mediate PHA(MCL) biosynthesis in the E. coli wild-type strain harboring a PHA synthase gene. However, PHA accumulation was strongly impaired in a recombinant E. coli fadB mutant, which harbored a PHA synthase gene. These data indicate that in pseudomonads FadBA does not possess the inherent property, based on a putative epimerase function, to provide the ( R)-enantiomer of 3-hydroxyacyl-CoA efficiently and that other linking enzymes are required to efficiently channel intermediates of beta-oxidation towards PHA(MCL) biosynthesis. However, the phaJ gene from P. oleovorans and from Pseudomonas putida, both of which encoded a 3- Re enoyl-CoA hydratase, was identified. The co-expression of phaJ(Po/Pp) with either a class-II PHA synthase gene or the PHA synthase gene from Aeromonas punctata in E. coli revealed that PhaJ(Po/Pp) mediated biosynthesis of either PHA(MCL), contributing to about 1% of cellular dry mass, or of poly(3-hydroxybutyrate- co-3-hydroxyhexanoate), contributing to 3.6% of cellular dry mass, when grown on decanoate. These data indicate that FadBA(Po)does not mediate the provision of (R)-3-hydroxyacyl-CoA, which resembles FadBA of non-PHA-accumulating bacteria, and that 3- Re enoyl-CoA hydratases are required to divert intermediates of fatty acid beta-oxidation towards PHA biosynthesis in P. oleovorans.

Genetic and biochemical characterization of exopolysaccharide biosynthesis by Lactobacillus delbrueckii subsp. bulgaricus.

Abstract: Lactobacillus delbrueckii subsp. bulgaricus produces exopolysaccharides (EPSs), which play a role in the rheological properties of fermented food products. Lb. bulgaricus Lfi5 produces a high-molecular-weight EPS composed of galactose, glucose, and rhamnose in the molar ratio 5:1:1. An 18-kb DNA region containing 14 genes, designated epsA to epsN, was isolated by genomic DNA library screening and inverted PCR. The predicted gene products are homologous to proteins involved in the biosynthesis of other bacterial polysaccharides and the genetic organization was found to be similar to that of other eps clusters from lactic acid bacteria. Transcriptional analysis revealed that the 14 eps genes are co-ordinately expressed and transcribed as a single mRNA of 15–16 kb. The transcription start site of the promoter was mapped upstream of the first gene, epsA. Genes encoding glycosyltranferases were further studied by heterologous expression and functional assays. We showed that the epsE gene product is a phospho-glucosyltransferase initiating the biosynthesis of EPS. Heterologous expression of epsE in a LactococcuslactisepsD mutant restored EPS production, demonstrating its role and importance in EPS biosynthesis. Functional assays of other glycosyltransferases allowed their sugar specificity to be elucidated and an overall biosynthetic pathway for EPS synthesis by Lb. bulgaricus to be proposed.