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Archives of Microbiology (v.187, #3)

Arsenate detoxification in a Pseudomonad hypertolerant to arsenic by Prerna C. Patel; Florence Goulhen; Christopher Boothman; Andrew G. Gault; John M. Charnock; Kiran Kalia; Jonathan R. Lloyd (pp. 171-183).

Pseudomonas sp. strain As-1, obtained from an electroplating industrial effluent, was capable of growing aerobically in growth medium supplemented with up to 65 mM arsenate (As (V)), significantly higher concentrations than those tolerated by other reference arsenic resistant bacteria. The majority of the arsenic was detected in culture supernatants as arsenite (As (III)) and X-ray absorbance spectroscopy suggested that 30% of this cell-bound arsenic was As (V), 65% As (III) and 5% of arsenic was associated with sulphur. PCR analysis using primers designed against arsenic resistance genes of other Gram-negative bacteria confirmed the presence of an arsenic resistance operon comprising of three genes, arsR, arsB and arsC in order of predicted transcription, and consistent with a role in intracellular reduction of As (V) and efflux of As (III). In addition to this classical arsenic resistance mechanism, other biochemical responses to arsenic were implicated. Novel arsenic-binding proteins were purified from cellular fractions, while proteomic analysis of arsenic-induced cultures identified the upregulation of additional proteins not normally associated with the metabolism of arsenic. Cross-talk with a network of proteins involved in phosphate metabolism was suggested by these studies, consistent with the similarity between the phosphate and arsenate anions.

Keywords: Metal reduction; Metal detoxification; Bioremediation; Arsenic reductase

Identification of promoters recognized by RNA polymerase containing Mycobacterium tuberculosis stress-response sigma factor σ^F by Dagmar Homerova; Katarina Surdova; Katarina Mikusova; Jan Kormanec (pp. 185-197).

A previously optimized Escherichia coli two-plasmid system was used to identify Mycobacterium tuberculosis promoters recognized by RNA polymerase containing the M. tuberculosis stress response sigma factor σ^F. The method allowed the identification of five new σ^F-dependent promoters. Transcriptional start points of the promoters were determined by high-resolution S1-nuclease mapping using RNA prepared from E. coli containing the two-plasmid system. The promoters were confirmed by an in vitro transcription assay. The Mycobacterium smegmatis and Mycobacterium tuberculosis core RNA polymerases, after complementation with σ^F, were able to recognize all the five promoters. All the promoters contained sequences highly similar to the sequence of the previously identified M. tuberculosis σ^F-dependent promoter, usfXp1. Comparison of the promoters revealed a σ^F consensus sequence GtTtga-N_14–18–GGGTAT. The σ^F-dependent promoters may govern expression of genes encoding a transcription regulator homologous to the response regulators of bacterial two-component signal transduction systems and proteins with unknown function.

Keywords: Bacterial virulence; Pathogenesis; Promoter; RNA polymerase; Sigma factor; Mycobacterium tuberculosis ; Stress response

Purification and characterization of a catechol 1,2-dioxygenase from a phenol degrading Candida albicans TL3 by San-Chin Tsai; Yaw-Kuen Li (pp. 199-206).

A eukaryotic catechol 1,2-dioxygenase (1,2-CTD) was produced from a Candida albicans TL3 that possesses high tolerance for phenol and strong phenol degrading activity. The 1,2-CTD was purified via ammonium sulfate precipitation, Sephadex G-75 gel filtration, and HiTrap Q Sepharose column chromatography. The enzyme was purified to homogeneity and found to be a homodimer with a subunit molecular weight of 32,000. Each subunit contained one iron. The optimal temperature and pH were 25°C and 8.0, respectively. Substrate analysis showed that the purified enzyme was a type I catechol 1,2-dioxygenase. This is the first time that a 1,2-CTD from a eukaryote (Candida albicans) has been characterized. Peptide sequencing on fragments of 1,2-CTD by Edman degradation and MALDI-TOF/TOF mass analyses provided information of amino acid sequences for BLAST analysis, the outcome of the BLAST revealed that this eukaryotic 1,2-CTD has high identity with a hypothetical protein, CaO19_12036, from Candida albicans SC5314. We conclude that the hypothetical protein is 1,2-CTD.

Keywords: Catechol 1,2-dioxygenase; Candida albicans TL3; MALDI-TOF mass analysis

Proteomic analysis of phytopathogenic fungus Botrytis cinerea as a potential tool for identifying pathogenicity factors, therapeutic targets and for basic research by Francisco Javier Fernández-Acero; Inmaculada Jorge; Enrique Calvo; Inmaculada Vallejo; María Carbú; Emilio Camafeita; Carlos Garrido; Juan Antonio López; Jesús Jorrin; Jesús Manuel Cantoral (pp. 207-215).

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.

Keywords: Botrytis cinerea ; Fungal phytopathogen; Fungal proteomics; Virulence factor

Mechanism controlling the extended lag period associated with vinyl chloride starvation in Nocardioides sp. strain JS614 by Timothy E. Mattes; Nicholas V. Coleman; Adina S. Chuang; Andrea J. Rogers; Jim C. Spain; James M. Gossett (pp. 217-226).

The extended lag period associated with vinyl chloride (VC) starvation in VC- and ethene-assimilating Nocardioides sp. strain JS614 was examined. The extended lag periods were variable (3–7 days), only associated with growth on VC or ethene, and were observed in VC- or ethene-grown cultures following 24 h carbon starvation and mid-exponential phase cultures grown on non-alkene carbon sources (e.g. acetate). Alkene monooxygenase (AkMO) and epoxyalkane:coenzyme M transferase (EaCoMT) are the initial enzymes of VC and ethene biodegradation in strain JS614. Reverse-transcription PCR confirmed that the AkMO gene etnC was expressed in response to epoxyethane, a metabolic intermediate of ethene biodegradation. Epoxyethane (0.5 mM) eliminated the extended lag period in both starved and mid-exponential phase cultures, suggesting that epoxyethane accumulation activates AkMO expression in strain JS614. AkMO activity in ethene-grown cultures was not detected after 6.7 h of carbon starvation, while 40% of the initial EaCoMT activity remained after 24 h. Acetate eliminated the extended lag period in starved cultures but not in mid-exponential phase cultures suggesting that acetate reactivates extant AkMO in starved VC- or ethene-grown cultures. The imbalance between AkMO and EaCoMT activities during starvation likely contributes to the extended lag period by delaying epoxide accumulation and subsequent AkMO induction.

Biosynthesis of clorobiocin: investigation of the transfer and methylation of the pyrrolyl-2-carboxyl moiety by Christine Anderle; Silke Alt; Tanja Gulder; Gerhard Bringmann; Bernd Kammerer; Bertolt Gust; Lutz Heide (pp. 227-237).

Clorobiocin is an aminocoumarin antibiotic containing a 5-methylpyrrolyl-2-carboxyl moiety, attached by an ester bond to a deoxysugar. This pyrrolyl moiety is important for the binding of the antibiotic to its biological target, the B subunit of gyrase. Inactivation experiments had shown that two putative acyl carrier proteins, CloN5 and CloN1, and two putative acyl transferases, CloN2 and CloN7, are involved in the transfer of the pyrrolyl-2-carboxyl moiety to the deoxysugar. In this study, pyrrolyl-2-carboxyl-N-acetylcysteamine thioester was synthesized and fed to cloN1 −, cloN2 − and cloN7 − mutants, and secondary metabolite formation was analyzed by HPLC and HPLC-MS. Transfer of the pyrrolyl-2-carboxyl moiety was observed in the cloN1 − and cloN2 − mutants, but not in the cloN7 − mutant, suggesting that CloN7 is responsible for this reaction. The product of this transfer, novclobiocin 109, was not further methylated to the 5-methylpyrrolyl-2-carboxyl compound, i.e. clorobiocin, suggesting that methylation does not take place after the acyl transfer. Additional investigations for the presence of 5-methylpyrrolyl-2-carboxylic acid in the mutants, and inactivation experiments with the methyltransferase gene cloN6, suggested that methylation by CloN6 and acyl transfer by CloN7 take place in a concerted fashion, requiring the presence of both proteins for efficient product formation. A mechanism for the methylation/acyl transfer process in the late steps of clorobiocin biosynthesis, involving CloN1, CloN2, CloN5, CloN6 and CloN7 is suggested.

Keywords: Acyl transfer; Biosynthesis; Clorobiocin; Pyrrolyl-2-carboxylic acid; 5-methylpyrrolyl-2-carboxylic acid; Streptomyces

Na⁺ and flagella-dependent swimming of alkaliphilic Bacillus pseudofirmus OF4: a basis for poor motility at low pH and enhancement in viscous media in an “up-motile” variant by Shun Fujinami; Naoya Terahara; Sunmi Lee; Masahiro Ito (pp. 239-247).

Flagella-based motility of extremely alkaliphilic Bacillus species is completely dependent upon Na⁺. Little motility is observed at pH values < ∼8.0. Here we examine the number of flagella/cell as a function of growth pH in the facultative alkaliphile Bacillus pseudofirmus OF4 and a derivative selected for increased motility on soft agar plates. Flagella were produced by both strains during growth in a pH range from 7.5 to 10.3. The number of flagella/cell and flagellin levels of cells were not strongly dependent on growth pH over this range in either strain although both of these parameters were higher in the up-motile strain. Assays of the swimming speed indicated no motility at pH < 8 with 10 mM Na⁺, but significant motility at pH 7 at much higher Na⁺ concentrations. At pH 8–10, the swimming speed increased with the increase of Na⁺ concentration up to 230 mM, with fastest swimming at pH 10. Motility of the up-motile strain was greatly increased relative to wild-type on soft agar at alkaline pH but not in liquid except when polyvinylpyrrolidone was added to increase viscosity. The up-motile phenotype, with increased flagella/cell may support bundle formation that particularly enhances motility under a subset of conditions with specific challenges.

Keywords: Alkaliphiles; Motility; Bacillus pseudofirmus ; Sodium-motive force; Flagella; MotPS

N-acyl homoserine lactones are degraded via an amidolytic activity in Comamonas sp. strain D1 by Stéphane Uroz; Phil Oger; Siri Ram Chhabra; Miguel Cámara; Paul Williams; Yves Dessaux (pp. 249-256).

Comamonas strain D1 enzymatically inactivates quorum-sensing (QS) signal molecules of the N-acyl homoserine lactone (N-AHSL) family, and exhibits the broadest inactivation range of known bacteria. It degrades N-AHSL with acyl-side chains ranging from 4 to 16 carbons, with or without 3-oxo or 3-hydroxy substitutions. N-AHSL degradation yields HSL but not N-acyl homoserine: strain D1 therefore harbors an amidohydrolase activity. Strain D1 is the fifth bacterium species in which an N-AHSL amidohydrolase is described. Consistent with its N-AHSL degradation ability, strain D1 efficiently quenches various QS-dependent functions in other bacteria, such as violacein production by Chromobacterium violaceum and pathogenicity and antibiotic production in Pectobacterium.

Keywords: Comamonas ; Quorum sensing; Quorum quenching; N-acyl homoserine lactone; Acylase; Pectobacterium

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Archives of Microbiology (v.187, #3)