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Archives of Microbiology (v.187, #2)
A cryptic type I polyketide synthase (cpk) gene cluster in Streptomyces coelicolor A3(2) by Krzysztof Pawlik; Magdalena Kotowska; Keith F. Chater; Katarzyna Kuczek; Eriko Takano (pp. 87-99).
The chromosome of Streptomyces coelicolor A3(2), a model organism for the genus Streptomyces, contains a cryptic type I polyketide synthase (PKS) gene cluster which was revealed when the genome was sequenced. The ca. 54-kb cluster contains three large genes, cpkA, cpkB and cpkC, encoding the PKS subunits. In silico analysis showed that the synthase consists of a loading module, five extension modules and a unique reductase as a terminal domain instead of a typical thioesterase. All acyltransferase domains are specific for a malonyl extender, and have a B-type ketoreductase. Tailoring and regulatory genes were also identified within the gene cluster. Surprisingly, some genes show high similarity to primary metabolite genes not commonly identified in any antibiotic biosynthesis cluster. Using western blot analysis with a PKS subunit (CpkC) antibody, CpkC was shown to be expressed in S. coelicolor at transition phase. Disruption of cpkC gave no obvious phenotype.
Keywords: Streptomyces ; Polyketide biosynthesis; Post-polyketide modifications; Antibiotic biosynthesis
Mutation in cyaA in Enterobacter cloacae decreases cucumber root colonization by Daniel P. Roberts; Laurie F. McKenna; Xiaojia Hu; Scott M. Lohrke; Hye Suk Kong; Jorge T. de Souza; C. Jacyn Baker; John Lydon (pp. 101-115).
Strains of Enterobacter cloacae show promise as biological control agents for Pythium ultimum-induced damping-off on cucumber and other crops. Enterobacter cloacae M59 is a mini-Tn5 Km transposon mutant of strain 501R3. Populations of M59 were significantly lower on cucumber roots and decreased much more rapidly than those of strain 501R3 with increasing distance from the soil line. Strain M59 was decreased or deficient in growth and chemotaxis on most individual compounds detected in cucumber root exudate and on a synthetic cucumber root exudate medium. Strain M59 was also slightly less acid resistant than strain 501R3. Molecular characterization of strain M59 demonstrated that mini-Tn5 Km was inserted in cyaA, which encodes adenylate cyclase. Adenylate cyclase catalyzes the formation of cAMP and cAMP levels in cell lysates from strain M59 were approximately 2% those of strain 501R3. Addition of exogenous, nonphysiological concentrations of cAMP to strain M59 restored growth (1 mM) and chemotaxis (5 mM) on synthetic cucumber root exudate and increased cucumber seedling colonization (5 mM) by this strain without serving as a source of reduced carbon, nitrogen, or phosphorous. These results demonstrate a role for cyaA in colonization of cucumber roots by Enterobacter cloacae.
Keywords: Biological control; Colonization; Cyclic AMP; cyaA ; Rhizosphere
Bacteriocin-like inhibitor substances produced by Mexican strains of Bacillus thuringiensis by J. Eleazar Barboza-Corona; Herminia Vázquez-Acosta; Dennis K. Bideshi; Rubén Salcedo-Hernández (pp. 117-126).
Bacteriocins are antimicrobial peptides synthesized and secreted by bacteria and could potentially be used as natural food preservatives. Here, we report the production of bacteriocin-like inhibitor substances (Bt-BLIS) by five Mexican strains of Bacillus thuringiensis. Bacillus thuringiensis subsp. morrisoni (LBIT 269), B. thuringiensis subsp. kurstaki (LBIT 287), B. thuringiensis subsp kenyae (LBIT 404), B. thuringiensis subsp. entomocidus (LBIT 420) and B. thuringiensis subsp. tolworthi (LBIT 524) produced proteinaceous Bt-BLIS with high levels of activity against Bacillus cereus and other gram-positive bacteria. Although none was active against the gram-negative bacteria, Escherichia coli, Shigella species and Pseudomonas aeruginosa, the five Bt-BLIS demonstrated antimicrobial activity against Vibrio cholerae, the etiologic agent of cholera. Biochemical and biophysical studies demonstrated that the five Bt-BLIS could be categorized into two groups, those produced by LBIT 269 and 287 (Group A) and LBIT 404, 420, 524 (Group B), based on relative time of peptide synthesis, distinctive bacterial target specificity and stability in a wide range of temperatures and pH. Because of their stability and bactericidal activities against B. cereus and V. cholerae agents of emetic, diarrheal and lethal syndromes in humans, these Bt-BLIS could potentially be used as biodegradable preservatives in the food industry.
Keywords: Bacillus thuringiensis ; Bacteriocins; Mexican strains
Evidence for a tellurite-dependent generation of reactive oxygen species and absence of a tellurite-mediated adaptive response to oxidative stress in cells of Pseudomonas pseudoalcaligenes KF707 by Valentina Tremaroli; Stefano Fedi; Davide Zannoni (pp. 127-135).
Tellurite (TeO 3 2− ) is the most toxic and soluble oxyanion among tellurium (Te) compounds. The effects of the metalloid anion on the oxidative stress response of the obligate aerobe Pseudomonas pseudoalcaligenes KF707 were investigated. Cells treated with sub-lethal concentrations of TeO 3 2− showed neither adaptation to it nor cross-protection against oxidants such as 1,1′-4,4′-bipyridinium dichloride (paraquat, PQ2+), diazenedicarboxylic acid bis-N,N-dimethylamide (diamide), tert-buthyl hydroperoxide (tBH) and hydrogen peroxide (H2O2). Notably, TeO 3 2− exerted a synergic effect on the toxicity of these latter oxidants. Tellurite was shown to decrease the cellular content of reduced thiols (RSH) with a consequent increase in the production of reactive oxygen species (ROS) and stimulation of the superoxide dismutase (SOD) activity. However, since the time course of ROS production by TeO 3 2 (t 1/2 > 30 min) was much slower than that with PQ2+ and/or diamide (t 1/2 ≤ 10 min), in the former case the SOD activity was poorly activated. We conclude that in P. pseudoalcaligenes KF707 cells: (1) the TeO 3 2− acts as a pro-oxidant by stimulating ROS production; (2) the release of superoxide oxyanions is directly linked to the mechanism of toxicity; (3) TeO 3 2− is unable to induce an adaptive response to oxidative stress.
Keywords: Adaptive response; Oxidative stress; Tellurite; Pseudomonas pseudoalcaligenes KF707
Substitutions of amino acids in α-helix-4 of gyrase A confer fluoroquinolone resistance on Clostridium perfringens by Fatemeh Rafii; Miseon Park (pp. 137-144).
DNA gyrase, an essential enzyme that regulates DNA topology in bacteria, is the target of fluoroquinolones. Three fluoroquinolone-resistant mutants derived from one strain of Clostridium perfringens had amino acid substitutions of glycine 81 to cysteine, aspartic acid 87 to tyrosine, or both, in α-helix-4 of gyrase A. The gyrase mutations affected the growth kinetics of mutants differently when the mutants were exposed to increasing concentrations of gatifloxacin and ciprofloxacin. Fluoroquinolone concentration-dependent effects observed during growth in the exponential and stationary phases depended on the presence of particular gyrA mutations. Introduction of a wild-type gyrA gene into the mutants enhanced their susceptibility to fluoroquinolones and decreased their growth rates proportional to increases in fluoroquinolone concentrations. Amino acid substitutions in α-helix-4 of gyrase A protected C. perfringens from fluoroquinolones, and a strain with two substitutions was the most resistant.
Keywords: Fluoroquinolones; Gyrase A; Mutations; Clostridium ; Resistance
Induction of α-l-arabinofuranosidase activity by monomeric carbohydrates in Bifidobacterium longum and ubiquity of encoding genes by Miguel Gueimonde; Luis Noriega; Abelardo Margolles; Clara G. de los Reyes-Gavilán (pp. 145-153).
Bifidobacterium longum can be isolated from human faeces, some strains being considered probiotics. B. longum NIZO B667 produces an exo-acting α-l-arabinofuranosidase, AbfB, previously purified by us, that releases l-arabinose from arabinan and arabinoxylan. This activity was subjected to two–seven-fold induction by l-arabinose, d-xylose, l-arabitol and xylitol and to repression by glucose. Maximum activity was obtained at 48 h incubation except for d-xylose that was at 24 h. High concentrations (200 mM) of l-arabitol also caused repression of the arabinofuranosidase. A unique band of activity showing the same migration pattern as the purified AbfB was found in zymograms of cell free extracts, indicating that the activity was likely due to this sole enzyme. The assessment of the influence of inducers and repressors on the activity of AbfB and on the expression of the abfB gene by real time PCR indicated that regulation was transcriptional. DNA amplifications using a pair of degenerated primers flanking an internal fragment within α-l-arabinofuranosidase genes of the family 51 of glycoside hydrolases evidenced that these enzymes are widespread in Bifidobacterium. The aminoacidic sequences of bifidobacteria included a fragment of four to six residues in the position 136–141 that was absent in other microorganisms
Keywords: Bifidobacterium ; Arabinofuranosidase; Induction; Repression
Production of phytoestrogen S-equol from daidzein in mixed culture of two anaerobic bacteria by Xiu-Ling Wang; Ho-Jin Kim; Su-Il Kang; Su-Il Kim; Hor-Gil Hur (pp. 155-160).
An anaerobic incubation mixture of two bacterial strains Eggerthella sp. Julong 732 and Lactobacillus sp. Niu-O16, which have been known to transform dihydrodaidzein to S-equol and daidzein to dihydrodaidzein respectively, produced S-equol from daidzein through dihydrodaidzein. The biotransformation kinetics of daidzein by the mixed cultures showed that the production of S-equol from daidzein was significantly enhanced, as compared to the production of S-equol from dihydrodaidzein by Eggerthella sp. Julong 732 alone. The substrate daidzein in the mixed culture was almost completely converted to S-equol in 24 h of anaerobic incubation. The increased production of S-equol from daidzein by the mixed culture is likely related to the increased bacterial numbers of Eggerthella sp. Julong 732. In the mixture cultures, the growth of Eggerthella sp. Julong 732 was significantly increased while the growth of Lactobacillus sp. Niu-O16 was suppressed as compared to either the single culture of Eggerthella sp. Julong 732 or Lactobacillus sp. Niu-O16. This is the first report in which two metabolic pathways to produce S-equol from daidzein by a mixed culture of bacteria isolated from human and bovine intestinal environments were successfully linked under anaerobic conditions.
Keywords: Isoflavone; Daidzein; S-equol; Intestinal; Anaerobic bacteria
Characterization of a novel ferredoxin with N-terminal extension from Clostridium acetobutylicum ATCC 824 by Razia Kutty; George N. Bennett (pp. 161-169).
A gene (CAC2657) encoding a ferredoxin (EFR1) from the strictly anaerobic soil bacterium Clostridium acetobutylicum was cloned and expressed in Escherichia coli. The ferredoxin gene encodes a polypeptide of 27 kDa that incorporates 2[4Fe–4S] clusters. An extended N-terminal region of 187 amino acid (aa) residues precedes ferredoxin domain. The EFR1 expressed in E. coli is a trimeric protein. The iron and sulfur content of the reconstituted protein agrees with that expected of a trimeric form of the protein. The ferredoxin domain of EFR1 is closely related to ferredoxin of C. pasteurianum; and can be fitted to the X-ray crystal structure with a root mean square deviation of 0.62 As for the Cα atoms of the generated 3D simulation model. In cultures of C. acetobutylicum the efr1 gene shows higher relative expression on induction with Trinitrotoluene (TNT) compared to that from uninduced control cultures.
Keywords: Ferredoxin; Polyferredoxin; Clostridium acetobutylicum ; Electron acceptors; Trinitrotoluene biotransformation; Hydrogenase; Iron storage proteins; Iron–sulfur clusters