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Archives of Microbiology (v.178, #6)

Professor Dr. phil. Achim Kröger by Bernhard Schink (pp. 387-387).

Mycothiol biochemistry by Gerald L. Newton; Robert C. Fahey (pp. 388-394).

Mycothiol (MSH) is a novel thiol comprised of N-acetylcysteine amide-linked to GlcN-α(1–1)-Ins. It is the major thiol in most actinomycetes and is produced at millimolar levels in mycobacteria and streptomycetes. MSH biosynthesis occurs by linkage of GlcNAc to Ins, deacetylation to GlcN-Ins, ligation of the latter to L-cysteine, and transacetylation of the cysteinyl residue by CoASAc to produce MSH. The genes encoding the respective enzymes have been designated mshA, mshB, mshC, and mshD; all but mshA have been identified. Mycobacterium smegmatis mutants deficient in mshA, mshC, and mshD have been characterized. MSH plays a significant role in the detoxification of thiol-reactive substances, including formaldehyde, various electrophiles, and antibiotics. Mycothiol S-conjugates derived from electrophiles and antibiotics are cleaved by mycothiol S-conjugate amidase to release GlcN-Ins, used to resynthesize MSH, and a mercapturic acid which is excreted from the cell. A mycothiol-disulfide-selective reductase has been identified and likely helps to maintain cellular MSH in the reduced state. Mycothiol biochemistry has characteristics similar to those of glutathione but also has a variety of unique features. Electronic Supplementary Material is available if you access this article at http://dx.doi.org/10.1007/s00203-002-0469-4. On that page (frame on the left side), a link takes you directly to the supplementary material.

Keywords: Mycothiol mshA, mshB mshC mshD mca mtr Mycobacterium tuberculosis Antibiotic detoxification Mercapturic acid

Cellular differentiation in the cyanobacterium Nostoc punctiforme by John C. Meeks; Elsie L. Campbell; Michael L. Summers; Francis C. Wong (pp. 395-403).

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.

Keywords: Akinetes Cellular differentiation Cyanobacteria Heterocysts Hormogonia Nostoc punctiforme Symbiosis

mRNA-mediated detection of environmental conditions by Franz Narberhaus (pp. 404-410).

Bacteria inhabit an amazing variety of ecological niches and readily adapt to changing environmental conditions. Precise monitoring of external and internal parameters is a prerequisite for the induction of appropriate stress responses. Proteins are often used as sensing devices. However, there is now accumulating evidence that the 5′ end of mRNA can be responsive to environmental stimuli. The folding of certain mRNA species is thermally controlled. RNA-based thermosensors modulate the efficiency of translation by controlling the accessibility of translation initiation signals that are captured in hairpin structures under repressing conditions. Recently, it has been recognized that mRNA can also sense physiological signals other than temperature. The overall structure of several vitamin-regulated leader mRNAs depends on the availability of the respective effector molecule. Binding of the vitamin or its precursor to the transcript impairs ribosome binding. Most likely, detection of environmental conditions by sensory RNAs is an emerging concept that is likely to extend much beyond the presently known examples.

Keywords: Heat shock Cold shock Starvation Stress Virulence RNA folding Sensing Signal transduction Regulation

Identification of Salmonella enterica serovar Typhimurium genes associated with growth suppression in stationary-phase nutrient broth cultures and in the chicken intestine by Ivan Rychlik; Gerald Martin; Ulrich Methner; Margaret Lovell; Lenka Cardova; Alena Sebkova; Mojmor Sevcik; Jiri Damborsky; Paul A. Barrow (pp. 411-420).

Over 2,800 Tn5 insertion mutants of Salmonella enterica sv. Typhimurium were screened for the loss of ability to suppress the multiplication of a spectinomycin-resistant (Spc^r) but otherwise isogenic S. enterica sv. Typhimurium strain, when the Spc^r mutant was added to 24-h LB broth cultures of the mutants. Selected "growth non-suppressive" (GNS) mutants were defective in respiration (insertions in arcA and fnr), amino acid biosynthesis (aroA and aroD), nutrient uptake and its regulation (tdcC and crp), and chemotaxis (fliD). In the last GNS mutant, the transposon inactivated yhjH, an ORF with unknown function which shows sequence similarity to di-guanylate cyclase and to novel two-component signal transduction proteins. In newly hatched chickens, all of the mutants, with the exception of the fliD mutant, were also unable to suppress colonization of the alimentary tract by the parent strain inoculated 1 day later. Defined mutations in luxS or sdiA, genes which contribute to quorum sensing in S. enterica sv. Typhimurium, had no effect on the stationary-phase growth suppression. Analysis of a transcriptional fusion construct indicated that yhjH was moderately expressed in the exponential phase of growth and up-regulated upon entry into stationary phase. Expression of yhjH was also considerably suppressed by the addition of supernatant from a 24-h stationary-phase S. enterica sv. Typhimurium culture, suggesting that the gene belongs to a new sensing and signaling regulatory pathway in S. enterica sv. Typhimurium.

Keywords: Salmonella enterica sv. Typhimurium Growth inhibition Quorum-sensing system Stationary phase yhjH

Detection of hepatotoxic Microcystis strains by PCR with intact cells from both culture and environmental samples by Hui Pan; Lirong Song; Yongding Liu; Thomas Börner (pp. 421-427).

Microcystins are small hepatotoxic peptides produced by a number of cyanobacteria. They are synthesized non-ribosomally by multifunctional enzyme complex synthetases encoded by the mcy genes. Primers deduced from mcy genes were designed to discriminate between toxic microcystin-producing strains and non-toxic strains. Thus, PCR-mediated detection of mcy genes could be a simple and efficient means to identify potentially harmful genotypes among cyanobacterial populations in bodies of water. We surveyed the distribution of the mcyB gene in different Microcystis strains isolated from Chinese bodies of water and confirmed that PCR can be reliably used to identify toxic strains. By omitting any DNA purification steps, the modified PCR protocol can greatly simplify the process. Cyanobacterial cells enriched from cultures, field samples, or even sediment samples could be used in the PCR assay. This method proved sensitive enough to detect mcyB genes in samples with less than 2,000 Microcystis cells per ml. Its accuracy, specificity and applicability were confirmed by sequencing selected DNA amplicons, as well as by HPLC, ELISA and mouse bioassay as controls for toxin production of every strain used.

Keywords: Cyanobacteria Microcystis Microcystin Toxin Peptide synthetase Whole-cell PCR

Validation of Cdc68p as a novel antifungal target by Ed T. Buurman; Weidong Jiang; Melissa McCoy; Devron R. Averett; Craig M. Thompson; Richard C. Wobbe (pp. 428-436).

Candida albicans is the main cause of systemic fungal infections for which there is an urgent need for novel antifungal drugs. The CP (Cdc68p-Pob3p) complex, which is involved in transcription elongation, was evaluated as a putative antifungal target. In order to predict the consequences of inhibition of this complex, the largest CP subunit in Saccharomyces cerevisiae, Cdc68p, was the first novel target to be tested in GATE, a recently described, quantitative target inactivation system. Depletion of the cell's pool of Cdc68p led to rapid cell death. Subsequently, the C. albicans orthologue of CDC68, CaCDC68, was cloned. Attempts to disrupt both alleles were unsuccessful, thus suggesting an essential role of CaCDC68 in this fungus also. Furthermore, CDC68 was proven to be present in Neurospora crassa and Aspergillus nidulans, thus suggesting that the CP complex is widespread among fungi and could serve as a broad range antifungal target. Analysis of Cdc68p and Pob3p sequences indicated significant structural differences between fungal CP complexes and those present in higher eukaryotes. These results predict that, in principle, fungal-specific ligands of CP complexes could be identified that could subsequently serve as chemical starting points towards the development of new antifungal therapeutic agents.

Keywords: CDC68 POB3 Saccharomyces cerevisiae Candida albicans Antifungal target

Effect of redox potential on activity of hydrogenase 1 and hydrogenase 2 in Escherichia coli by Tatyana V. Laurinavichene; Nikolai A. Zorin; Anatoly A. Tsygankov (pp. 437-442).

This report elucidates the distinctions of redox properties between two uptake hydrogenases in Escherichia coli. Hydrogen uptake in the presence of mediators with different redox potential was studied in cell-free extracts of E. coli mutants HDK103 and HDK203 synthesizing hydrogenase 2 or hydrogenase 1, respectively. Both hydrogenases mediated H₂ uptake in the presence of high-potential acceptors (ferricyanide and phenazine methosulfate). H₂ uptake in the presence of low-potential acceptors (methyl and benzyl viologen) was mediated mainly by hydrogenase 2. To explore the dependence of hydrogen consumption on redox potential of media in cell-free extracts, a chamber with hydrogen and redox (E _h) electrodes was used. The mutants HDK103 and HDK203 exhibited significant distinctions in their redox behavior. During the redox titration, maximal hydrogenase 2 activity was observed at the E _h below –80 mV. Hydrogenase 1 had maximum activity in the E _h range from +30 mV to +110 mV. Unlike hydrogenase 2, the activated hydrogenase 1 retained activity after a fast shift of redox potential up to +500 mV by ferricyanide titration and was more tolerant to O₂. Thus, two hydrogenases in E. coli are complementary in their redox properties, hydrogenase 1 functioning at higher redox potentials and/or at higher O₂ concentrations than hydrogenase 2.

Keywords: Escherichia coli Hydrogenase 1 Hydrogenase 2 Redox titration Hydrogen consumption

A non-dechlorinating strain of Dehalospirillum multivorans: evidence for a key role of the corrinoid cofactor in the synthesis of an active tetrachloroethene dehalogenase by Anke Siebert; Anke Neumann; Torsten Schubert; Gabriele Diekert (pp. 443-449).

A strain of Dehalosprillum multivorans, designated strain N, was isolated from the same source as the formerly described tetrachloroethene (PCE)-dechlorinating D. multivorans, herein after referred to as strain K. Neither growing cells nor cell extracts of strain N were able to dechlorinate PCE. The pceA and pceB genes encoding for the PCE-reductive dehalogenase were detected in cells of strain N; and they were 100% homologous to the corresponding genes of strain K. Since the PCE dehalogenase of D. multivorans strain K contains a corrinoid cofactor, the corrinoids of strain N cells were extracted. Analysis of the corrinoids revealed the absence of the specific corrinoid, which is the cofactor of the PCE dehalogenase of strain K cells. RT-PCR of mRNA indicated that the pceA gene was transcribed in strain N cells to a far lower extent than the pceA gene of strain K under the same experimental conditions. Western blot analysis of crude extracts of strain N showed that, if at all, an insignificant amount of the apoprotein of the PCE dehalogenase was present. The results indicate that the inability of strain N to dechlorinate is due to the absence of the corrinoid cofactor of the enzyme mediating PCE dechlorination.

Keywords: Corrinoid Dehalorespiration Dehalospirillum multivorans PCE pceA pceB Strain K Strain N Tetrachloroethene Tetrachloroethene reductive dehalogenase

Localization of Mn(II)-oxidizing activity and the putative multicopper oxidase, MnxG, to the exosporium of the marine Bacillus sp. strain SG-1 by Chris A. Francis; Karen L. Casciotti; Bradley M. Tebo (pp. 450-456).

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.

Keywords: Mn(II) oxidation Bacillus spores Exosporium MnxG multicopper oxidase

Influence of nitrate on oxalate- and glyoxylate-dependent growth and acetogenesis by Moorella thermoacetica by Corinna Seifritz; Jürgen M. Fröstl; Harold L. Drake; Steven L. Daniel (pp. 457-464).

Oxalate and glyoxylate supported growth and acetate synthesis by Moorella thermoacetica in the presence of nitrate under basal (without yeast extract) culture conditions. In oxalate cultures, acetate formation occurred concomitant with growth and nitrate was reduced in the stationary phase. Growth in the presence of [¹⁴C]bicarbonate or [¹⁴C]oxalate showed that CO₂ reduction to acetate and biomass or oxalate oxidation to CO₂ was not affected by nitrate. However, cells engaged in oxalate-dependent acetogenesis in the presence of nitrate lacked a membranous b-type cytochrome, which was present in cells grown in the absence of nitrate. In glyoxylate cultures, growth was coupled to nitrate reduction and acetate was formed in the stationary phase after nitrate was totally consumed. In the absence of nitrate, glyoxylate-grown cells incorporated less CO₂ into biomass than oxalate-grown cells. CO₂ conversion to biomass by glyoxylate-grown cells decreased when cells were grown in the presence of nitrate. These results suggest that: (1) oxalate-grown cells prefer CO₂ as an electron sink and bypass the nitrate block on the acetyl-CoA pathway at the level of reductant flow and (2) glyoxylate-grown cells prefer nitrate as an electron sink and bypass the nitrate block of the acetyl-CoA pathway by assimilating carbon via an unknown process that supplements or replaces the acetyl-CoA pathway. In this regard, enzymes of known pathways for the assimilation of two-carbon compounds were not detected in glyoxylate- or oxalate-grown cells.

Keywords: Moorella thermoacetica Acetyl-CoA pathway Wood–Ljungdahl pathway Acetogenesis Oxalate Glyoxylate Nitrate dissimilation Carbon assimilation Reductant flow Cellular energetics

Evidence for MoeA-dependent formation of the molybdenum cofactor from molybdate and molybdopterin in Escherichia coli by Cristinel Sandu; Roderich Brandsch (pp. 465-470).

The function of the MoeA protein in the biosynthesis of the molybdenum cofactor (MoCo) was analyzed in vitro, using purified His₆-MoeA from Escherichia coli, molybdopterin (MPT) isolated from buttermilk xanthine oxidase and molybdate. The formation of MoCo was monitored by the reconstitution of nitrate reductase activity in extracts of the Neurospora crassa nit-1 mutant. Formation of MoCo from MPT and molybdate required MoeA and L-cysteine or glutathione. The reaction proceeded at micromolar molybdate levels and was time- and MoeA concentration-dependent. A physical interaction between MoeA and MPT was demonstrated by HPLC analysis of MoeA-bound MPT.

Keywords: MoeA Molybdate Molybdenum cofactor nit-1 Neurospora crassa Xanthine oxidase

The roles of the three gene copies encoding hydroxylamine oxidoreductase in Nitrosomonas europaea by Norman G. Hommes; Luis A. Sayavedra-Soto; Daniel J. Arp (pp. 471-476).

The nitrifying bacterium Nitrosomonas europaea contains three copies of the gene (hao) encoding hydroxylamine oxidoreductase (HAO), the second enzyme in the nitrification pathway which oxidizes NH₂OH to NO₂ ^–. The nucleotide sequences of the hao genes differ by only one nucleotide. Two of the three gene copies have identical promoter sequences, while the third promoter has a different nucleotide sequence. Mutant strains with two of the three copies of hao inactivated were created by insertional inactivation, using DNA cassettes containing kanamycin- and gentamycin-resistance genes. All three double-mutant combinations were obtained. These double mutants were phenotypically identical under the conditions tested. Two of these double mutants were similar to wild-type cells or cells having a single hao copy inactivated regarding growth rates or hydroxylamine-dependent O₂ uptake activity, but had only about 50% of the wild-type level of in vitro HAO activity and hao mRNA. The third hao double mutant had an unstable genotype, resulting in recombination of the gentamycin marker into another copy of hao. The N. europaea genomic sequence was recently completed, revealing the locations of the copies of hao and other nitrification genes. Comparison with the arrangement of hao genes in the closely related strain, Nitrosomonas sp. strain ENI-11, showed a similar organization.

Keywords: Nitrosomonas europaea Nitrifier Hydroxylamine oxidoreductase hao Nitrosomonas sp. strain ENI-11 Genomic sequence pUCGM

Different effectors of dimorphism in Yarrowia lipolytica by José Ruiz-Herrera; Rafael Sentandreu (pp. 477-483).

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.

Keywords: Dimorphic fungi Mycelial cells Yeast–mycelial cell transition cAMP

Characterisation, genome size and genetic manipulation of the myxobacterium Sorangium cellulosum So ce56 by Silke Pradella; Astrid Hans; Cathrin Spröer; Hans Reichenbach; Klaus Gerth; Stefan Beyer (pp. 484-492).

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.

Keywords: Polyangium (Sorangium) cellulosum Chivosazole Etnangien Triparental mating Genome size Pulsed-field gel electrophoresis Polyketide 16S rDNA

Intracellular pyruvate flux in the methane-producing archaeon Methanococcus maripaludis by Yu-Ling Yang; John N. Glushka; William B. Whitman (pp. 493-498).

During growth of the methanogenic archaeon Methanococcus maripaludis on alanine as the sole nitrogen source under H₂/CO₂, alanine was incorporated into amino acids derived from pyruvate including leucine, isoleucine, and valine. Thus, growth with alanine was an efficient means of labeling intracellular pools of pyruvate in this lithotroph. Cells were grown with 18% [U-¹³C]alanine, and the distribution of the isotope in the branched-chain amino acids was determined by ¹³C-NMR. Carbons derived from pyruvate contained 14.5% ¹³C, indicating that most of the cellular pyruvate was obtained from alanine. In contrast, carbons derived from acetyl-CoA contained only 3–5% ¹³C, indicating that only small amounts of acetyl-CoA were formed from pyruvate. Thus, autotrophic acetyl-CoA biosynthesis continued even in the presence of an organic carbon source. Moreover, the labeling of acetyl-CoA was lower than would be predicted if pyruvate was a C-1 donor for acetyl-CoA biosynthesis. Carbon derived from the C-1 of acetyl-CoA contained less ¹³C than carbon derived from the C-2 of acetyl-CoA, and this difference was attributed to the acetyl-CoA:CO₂ exchange activity of acetyl-CoA synthase. No enrichment was detected for the C-1 of valine, which was derived from the C-1 of pyruvate. This result was attributed to the pyruvate:CO₂ exchange activity of pyruvate oxidoreductase and may have important implications for isotope tracer studies utilizing pyruvate. Lastly, these results demonstrate that the breakdown of pyruvate by methanococci is very limited even under conditions where it is the sole nitrogen and major carbon source.

Keywords: Methanococcus maripaludis Methanogen Archaea Pyruvate oxidoreductase Acetyl-coA synthase

Tricarboxylic acid cycle aconitase activity during the life cycle of Streptomyces viridochromogenes Tü494 by Konstanze Muschko; Grit Kienzlen; Hans-Peter Fiedler; Wolfgang Wohlleben; Dirk Schwartz (pp. 499-505).

Previously, it was shown that inactivation of the tricarboxylic acid cycle aconitase gene acnA impairs the morphological and physiological differentiation of Streptomyces viridochromogenes Tü494, which produces the herbicide phosphinothricin tripeptide (PTT). In order to further characterize the role of the aconitase in the Streptomyces life cycle, aconitase activity was analyzed during growth of S. viridochromogenes in liquid culture. Two prominent maxima were measured in cell-free crude extracts. The first maximum was found at an early stage of growth, which is correlated with a decrease in pH when rapid glucose consumption is initiated. The second, lower maximum was detected at the beginning of the expression of the PTT-specific biosynthetic gene phsA, implying the onset of secondary metabolism. These results were confirmed by examining transcription of the acnA promoter in time-course experiments. The highest transcription rate was found during the early growth phases. In order to identify putative regulatory mechanisms, the transcriptional start site of the acnA transcript and subsequently the promoter were identified. Several putative, regulatory protein binding sites (e.g. regulators of oxygen stress or iron metabolism) were detected in the promoter region of acnA, which suggested complex regulation of acnA.

Keywords: Aconitase regulation Streptomyces viridochromogenes Phosphinothricin tripeptide

Genes involved in the anaerobic degradation of ethylbenzene in a denitrifying bacterium, strain EbN1 by Ralf Rabus; Michael Kube; Alfred Beck; Friedrich Widdel; Richard Reinhardt (pp. 506-516).

Abstract. Genes involved in anaerobic degradation of the petroleum hydrocarbon ethylbenzene in the denitrifying Azoarcus-like strain EbN1 were identified on a 56-kb DNA contig obtained from shotgun sequencing. Ethylbenzene is first oxidized via ethylbenzene dehydrogenase to (S)-1-phenylethanol; this is converted by (S)-1-phenylethanol dehydrogenase to acetophenone. Further degradation probably involves acetophenone carboxylase forming benzoylacetate, a ligase forming benzoylacetyl-CoA, and a thiolase forming acetyl-CoA and benzoyl-CoA. Genes of this pathway were identified via N-terminal sequences of proteins isolated from strain EbN1 and by sequence similarities to proteins from other bacteria. Ethylbenzene dehydrogenase is encoded by three genes (ebdABC), in accordance with the heterotrimeric enzyme structure. Binding domains for a molybdenum cofactor (in subunit EbdA) and iron/sulfur-clusters (in subunits EbdA and EbdB) were identified. The previously observed periplasmic location of the enzyme was corroborated by the presence of a twin-arginine leader peptide characteristic of the Tat system for protein export. A fourth gene (ebdD) was identified, the product of which may act as an enzyme-specific chaperone in the maturation of the molybdenum-containing subunit. A distinct gene (ped) coding for (S)-1-phenylethanol dehydrogenase apparently forms an operon with the ebdABCD genes. The ped gene product with its characteristic NAD(P)-binding motif in the N-terminal domain belongs to the short-chain dehydrogenase/reductase (SDR) superfamily. A further operon apparently contains five genes (apc1–5) suggested to code for subunits of acetophenone carboxylase. Four of the five gene products are similar to subunits of acetone carboxylase from Xanthobacter autotrophicus. Upstream of the apc genes, a single gene (bal) was identified which possibly codes for a benzoylacetate CoA-ligase and which is co-transcribed with the apc genes. In addition, an apparent operon containing almost all genes required for β-oxidation of fatty acids was detected; one of the gene products may be involved in thiolytic cleavage of benzoylacetyl-CoA. The DNA fragment also included genes for regulatory systems; these were two sets of two-component systems, two LysR homologs, and a TetR homolog. Some of these proteins may be involved in ethylbenzene-dependent gene expression.

Keywords: Ethylbenzene Denitrifying bacterium Anaerobic degradation Gene prediction Gene annotation Genomics

(R)-Benzylsuccinyl-CoA dehydrogenase of Thauera aromatica, an enzyme of the anaerobic toluene catabolic pathway by Christina Leutwein; Johann Heider (pp. 517-524).

The first intermediate of anaerobic toluene catabolism, (R)-benzylsuccinate, is formed by enzymic addition of the methyl group of toluene to a fumarate cosubstrate and is subsequently activated to (R)-2-benzylsuccinyl-CoA. This compound is then oxidised to benzoyl-CoA and succinyl-CoA by a specific β-oxidation pathway. The enzyme catalysing the first oxidation step of this pathway, (R)-benzylsuccinyl-CoA dehydrogenase, is encoded by the bbsG gene in Thauera aromatica. It was functionally overproduced in Escherichia coli, purified and characterised. The enzyme is a homotetramer with a subunit size of 45 kDa and contains one FAD per subunit. It is highly specific for (R)-benzylsuccinyl-CoA and is inhibited by (S)-benzylsuccinyl-CoA. An apparent K _m value of 110±10 µM was obtained for (R)-benzylsuccinyl-CoA. The reaction product of (R)-benzylsuccinyl-CoA dehydrogenase was identified as (E)-benzylidene-succinyl-CoA by comparison with the chemically synthesised compound, which was obtained via a new synthetic procedure. (R)-Benzylsuccinyl-CoA dehydrogenase was detected as a specifically substrate-induced protein in toluene- and m-xylene-grown cells of several bacterial species, using enzyme activity and immunological detection.

Keywords: Anaerobic toluene metabolism Benzylsuccinyl-CoA Benzylidene-succinyl-CoA Phenylitaconyl-CoA acyl-CoA dehydrogenase Flavin Stereochemistry β-Oxidation

Amino acid replacements in transmembrane domain 1 influence osmosensing but not K⁺ sensing by the sensor kinase KdpD of Escherichia coli by Iris Stallkamp; Karlheinz Altendorf; Kirsten Jung (pp. 525-530).

Expression of the kdpFABC operon coding for the high affinity K⁺-translocating KdpFABC complex of Escherichia coli is induced by K⁺ limitation or high osmolality. This process is controlled by the sensor kinase/response regulator system KdpD/KdpE. To study the importance of the transmembrane domains of KdpD for stimulus perception, each amino acid residue of the transmembrane domain 1 and Asp-424 of the adjacent periplasmic loop were replaced with Cys in a KdpD derivative devoid of native Cys residues. In vivo analysis of KdpD proteins with a single Cys residue revealed that 14 out of 18 amino acid replacements caused an altered response towards an osmotic upshift imposed by NaCl, whereby only four replacements also altered the response towards changes in the K⁺concentration. The in vitro activities of most of the KdpD derivatives were in the range of KdpD devoid of native Cys residues. The results reveal that the osmosensing and K⁺-sensing properties of KdpD can be dissected. Furthermore, the data support the hypothesis that osmosensing involves amino acid residues of the transmembrane domains.

Keywords: Escherichia coli K+ Sensor kinase Response regulator Stimulus perception Osmolarity

Genetic groups of the insect-pathogenic fungus Beauveria bassiana are associated with habitat and thermal growth preferences by Michael J. Bidochka; Fiona V. Menzies; Andrena M. Kamp (pp. 531-537).

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.

Keywords: Entomopathogenic fungi Habitat Population genetics Cold activity

Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe by Gae-Won Nam; Dong-Woo Lee; Han-Seoung Lee; Nam-Ju Lee; Byoung-Chan Kim; Eun-Ah Choe; Jae-Kwan Hwang; Maggy T. Suhartono; Yu-Ryang Pyun (pp. 538-547).

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.

Keywords: Native feathers Thermophilic anaerobe Fervidobacterium islandicum AW-1 Thermostable keratinolytic protease

In vivo assessment of the Tat signal peptide specificity in Escherichia coli by Bérengère Ize; Fabien Gérard; Long-Fei Wu (pp. 548-553).

Tat- and Sec-targeting signal peptides are specific for the cognate Tat or Sec pathways. Using two reporter proteins, the specificity and convertibility of a Tat signal peptide were assessed in vivo. The specific substitutions by RK, KR and KK for the RR motif of the TorA signal peptide had no effect on the exclusive Tat-dependent export of colicin V (ColV). By introducing multiple substitutions in a typical Tat signal peptide, altered signal peptides lacking the twin-arginine motif were obtained. Interestingly, some of these signal peptides preserved Tat-pathway targeting capacity, but resulted in a loss of exclusivity. In addition, further increasing the hydrophobicity of the n-region without modifying the h-region converted the Tat signal peptides to Sec signal peptides in the ColV transport. Replacement of positively charged residues in the c-region also abolished the Tat-exclusive targeting of ColV or green fluorescent protein (GFP), but the folded GFP could be transported only through the Tat pathway. These results strongly suggest that the overall hydrophobicity of the n-region is one of the determinants of Tat-targeting exclusivity.

Keywords: Tat system Sec machinery Signal peptide Twin-arginine motif Hydrophobicity

Inducible aluminum resistance of Acidiphilium cryptum and aluminum tolerance of other acidophilic bacteria by Jörg Fischer; Armin Quentmeier; Sven Gansel; Vera Sabados; Cornelius G. Friedrich (pp. 554-558).

Aluminum ions are highly soluble in acidic environments. Toxicity of aluminum ions for heterotrophic, facultatively and obligately chemolithoautotrophic acidophilic bacteria was examined. Acidiphilium cryptum grew in glucose-mineral medium, pH 3, containing 300 mM aluminum sulfate [Al₂(SO₄)₃] after a lag phase of about 120 h with a doubling time of 7.6 h, as compared to 5.2 h of growth without aluminum. Precultivation with 1 mM Al₂(SO₄)₃ and transfer to a medium with 300 mM Al₂(SO₄)₃ reduced the lag phase from 120 to 60 h, and immediate growth was observed when A. cryptum was precultivated with 50 mM Al₂(SO₄)₃, suggesting an aluminum-induced resistance. Aluminum resistance was not induced by Fe³⁺ ions and divalent cations. Upon exposure of A. cryptum to 300 mM Al₂(SO₄)₃, the protein profile changed significantly as determined by SDS-PAGE. When other acidophiles were cultivated with 50–200 mM aluminum sulfate, no lag phase was observed while the growth rates and the cellular yields were significantly reduced. This growth response was observed with Acidobacterium capsulatum, Acidiphilium acidophilum, Acidithiobacillus ferrooxidans, and Acidithiobacillus thiooxidans. Precultivation of these strains with aluminum ions did not alter the growth response caused by aluminum. The content of A. cryptum cultivated with 300 mM Al₂(SO₄)₃was 0.44 µg Al/mg cell dry weight, while that of the other strains cultivated with 50 mM Al₂(SO₄)₃ ranged from 0.30 to 3.47 µg Al/mg cell dry weight.

Keywords: Acidiphilium cryptum Acidophilic bacteria Aluminum resistance Aluminum tolerance

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Archives of Microbiology (v.178, #6)