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Archives of Microbiology (v.185, #3)
Effect of glucose limitation and specific mutations in the module 5 enoyl reductase domains in the nystatin and amphotericin polyketide synthases on polyene macrolide biosynthesis by Sven E.F. Borgos; Håvard Sletta; Espen Fjærvik; Trygve Brautaset; Trond E. Ellingsen; Ole-Martin Gulliksen; Sergey B. Zotchev (pp. 165-171).
Enoyl reductase (ER) domains in module 5 of nystatin and amphotericin polyketide synthase (PKS) are responsible for reduction of the C28–C29 unsaturated bond on the nascent polyketide chain during biosynthesis of both macrolides, resulting in production of tetraenes nystatin A1 and amphotericin A, respectively. Data obtained in fermentations under glucose limitation conditions demonstrated that the efficiency of the ER5 domain can be influenced by carbon source availability in the amphotericin producer Streptomyces nodosus, but not in the nystatin producer Streptomyces noursei. Two S. noursei ER5 domain mutants were constructed, GG5073SP and S5016N, both producing the heptaene nystatin analogue S44HP with unsaturated C28–C29 bond. While the GG5073SP mutant, with altered ER5 NADPH binding site, produced S44HP exclusively, the S5016N mutant synthesized a mixture of nystatin and S44HP. Comparative studies on the S5016N S. noursei mutant and S. nodosus, both producing mixtures of tetraenes and heptaenes, revealed that the ratio between these two types of metabolites was significantly more affected by glucose limitation in S. nodosus. These data suggest that mutation S5016N in NysC “locks” the ER5 domain in a state of intermediate activity which, in contrast to the ER5 domain in the amphotericin PKS, is not significantly influenced by physiological conditions.
Keywords: Nystatin polyketide synthase; Enoyl reductase domain; Site-specific mutagenesis
Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov.: two anaerobic bacteria that degrade phthalate isomers in syntrophic association with hydrogenotrophic methanogens by Yan-Ling Qiu; Yuji Sekiguchi; Satoshi Hanada; Hiroyuki Imachi; I-Cheng Tseng; Sheng-Shung Cheng; Akiyoshi Ohashi; Hideki Harada; Yoichi Kamagata (pp. 172-182).
An anaerobic phthalate isomer-degrading strain (JTT) that we previously isolated was characterized. In addition, a strictly anaerobic, mesophilic, syntrophic phthalate isomer-degrading bacterium, designated strain JIT, was isolated and characterized in this study. Both were non-motile rods that formed spores. In both strains, the optimal growth was observed at temperatures around 37°C and neutral pH. In syntrophic co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei, both strains could utilize two or three phthalate isomers for growth, and produce acetate and methane as end products. Strain JTT was able to grow on isophthalate, terephthalate, and a number of low-molecular weight aromatic compounds, such as benzoate, hydroquinone, 2-hydroxybenzoate, 3-hydroxybenzoate, 2,5-dihydroxybenzoate, 3-phenylpropionate in co-culture with M. hungatei. It could also grow on crotonate, hydroquinone and 2,5-dihydroxybenzoate in pure culture. Strain JIT utilized all of the three phthalate isomers as well as benzoate and 3-hydroxybenzoate for growth in co-culture with M. hungatei. No substrates were, however, found to support the axenic growth of strain JIT. Neither strain JTT nor strain JIT could utilize sulfate, sulfite, thiosulfate, nitrate, fumarate, Fe (III) or 4-hydroxybenzoate as electron acceptor. Phylogenetically, strains JTT and JIT were relatively close to the members of the genera Pelotomaculum and Cryptanaerobacter in ‘Desulfotomaculum lineage I’. Physiological and chemotaxonomic characteristics indicated that the two isolates should be classified into the genus Pelotomaculum, creating two novel species for them. Here, we propose Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov. for strain JTT and strain JIT, respectively. The type strains are strains JTT (= DSM 16121T = JCM 11824T = NBRC 100523T) and JIT (= JCM 12282T = BAA-1053T) for P. terephthalicum and P. isophthalicum, respectively.
Keywords: Anaerobic phthalate-oxidation; Pelotomaculum terephthalicum sp. nov.; Pelotomaculum isophthalicum sp. nov
Extracellular metalloendopeptidase of Streptomyces rimosus by Ljubinka Vitale; Bojana Vukelić; Igor Križaj (pp. 183-191).
Metalloendopeptidase was isolated from Streptomyces rimosus culture filtrates in a homogeneous form. It was determined to be a 15 kDa basic protein, most active around pH 7.5, and susceptible to inhibition by chelating agents, N-bromosuccinimide, thiorphan, and 10−4 M zinc. The enzyme was highly specific for phenylalanine at the N-side of endopeptide bonds. Determination of amino acid sequence of the enzyme’s NH2-part allowed the recognition of its structure homology with isolated and predicted metallopeptidases from several Streptomyces species. The data contribute to the definition of M7 family of metalloendopeptidases in streptomycetes.
Keywords: Metalloendopeptidase; Specificity; Amino acid sequence; Streptomyces rimosus
Degradation of and sensitivity to cholate in Pseudomonas sp. strain Chol1 by Bodo Philipp; Henrike Erdbrink; Marc J. -F. Suter; Bernhard Schink (pp. 192-201).
A facultative anaerobic bacterium, Pseudomonas sp. strain Chol1, degrading cholate and other bile acids was isolated from soil. We investigated how strain Chol1 grew with cholate and whether growth was affected by the toxicity of this compound. Under anoxic conditions with nitrate as electron acceptor, strain Chol1 grew by transformation of cholate to 7,12-dihydroxy-1,4-androstadiene-3,17-dione (DHADD) as end product. Under oxic conditions, strain Chol1 grew by transformation of cholate to 3,7,12-trihydroxy-9,10-seco-1,3,5(10)-androstatriene-9,17-dione (THSATD), which accumulated in the culture supernatant before its further oxidation to CO2. Strain Chol1 converted DHADD into THSATD by an oxygenase-dependent reaction. Addition of cholate (≥10 mM) to cell suspensions of strain Chol1 caused a decrease of optical density and viable counts but aerobic growth with these toxic cholate concentrations was possible. Addition of CCCP or EDTA strongly increased the sensitivity of the cells to 10 mM cholate. EDTA also increased the sensitivity of the cells to DHADD and THSATD (≤1.7 mM). The toxicity of cholate and its degradation intermediates with a steroid structure indicates that strain Chol1 requires a strategy to minimize these toxic effects during growth with cholate. Apparently, the proton motive force and the outer membrane are necessary for protection against these toxic effects.
Keywords: Pseudomonas ; Bile acids; Cholate; Degradation; Steroids; Androstandienedione; Sensitivity
Characterization of a mercury-reducing Bacillus cereus strain isolated from the Pulicat Lake sediments, south east coast of India by Seralathan Kamala Kannan; Subramony Mahadevan; Ramaswamy Krishnamoorthy (pp. 202-211).
Pulicat Lake sediments are often severely polluted with the toxic heavy metal mercury. Several mercury-resistant strains of Bacillus species were isolated from the sediments and all the isolates exhibited broad spectrum resistance (resistance to both organic and inorganic mercuric compounds). Plasmid curing assay showed that all the isolated Bacillus strains carry chromosomally borne mercury resistance. Polymerase chain reaction and southern hybridization analyses using merA and merB3 gene primers/probes showed that five of the isolated Bacillus strains carry sequences similar to known merA and merB3 genes. Results of multiple sequence alignment revealed 99% similarity with merA and merB3 of TnMERI1 (class II transposons). Other mercury resistant Bacillus species lacking homology to these genes were not able to volatilize mercuric chloride, indicating the presence of other modes of resistance to mercuric compounds.
Keywords: Mercury reduction; Bacillus cereus ; Pulicat Lake; Heavy metals; Horizontal gene exchange
Isolation and characterization of Acidicaldus organivorus, gen. nov., sp. nov.: a novel sulfur-oxidizing, ferric iron-reducing thermo-acidophilic heterotrophic Proteobacterium by D. Barrie Johnson; Bethan Stallwood; Sakurako Kimura; Kevin B. Hallberg (pp. 212-221).
Thermo-acidophilic prokaryotes isolated from geothermal sites in Yellowstone National Park were identified as novel α-Proteobacteria, distantly related (~93% 16S rRNA gene identity) to the mesophilic acidophile Acidisphaera rubrifaciens. One of these isolates (Y008) was shown to be more thermophilic than all previously characterized acidophilic proteobacteria, with a temperature optimum for growth between 50 and 55°C and a temperature maximum of 65°C. Growth was observed in media maintained at pH between 1.75 and 3.0 and was fastest at pH between 2.5 and 3.0. The G + C content of Y008 was 71.8±0.9 mol%. The acidophile was able to grow heterotrophically on a range of organic substrates, including various monosaccharides, alcohols and amino acids and phenol, though growth on single organic compounds required the provision of one or more growth factors. The isolate oxidized sulfur to sulfuric acid in media containing yeast extract, but was not capable of autotrophic growth with sulfur as energy source. Growth occurred under aerobic conditions and also in the absence of oxygen via anaerobic respiration using ferric iron as terminal electron acceptor. Based on these genotypic and phenotypic traits, it is proposed that Y008 represents the type species of Acidicaldus organivorus, gen. nov., sp. nov.
Keywords: Acidophile; Iron reduction; Phenol; Sulfur oxidation; Thermophile
Anaerobic cells of Bacillus cereus F4430/73 respond to low oxidoreduction potential by metabolic readjustments and activation of enterotoxin expression by A. Zigha; E. Rosenfeld; P. Schmitt; C. Duport (pp. 222-233).
In the present study, a food-borne pathogen strain of Bacillus cereus (F4430/73) was anaerobically grown in controlled-batch conditions under low initial oxidoreduction potential (ORP=–148 mV) using hydrogen gas as reducing agent. Its physiological characteristics, including growth, glucose fermentation capacity and enterotoxin production, were compared with anaerobic conditions generated by nitrogen gas (ORP=+ 45 mV). The results showed that low ORP affected growth mainly during the early stages. Maximal specific rates of growth and glucose consumption were reduced, and drastic changes in time profiles of fermentation product concentration were observed. Production of lactate was promoted at the expense of acetate. Nevertheless, low ORP did not affect final biomass yield. Under both ORP conditions, Non-haemolytic enterotoxin (Nhe) was produced early during the exponential growth phase as a first enterotoxin and Haemolysin BL (Hbl) later during the early stationary growth phase as a second enterotoxin. The major effect of low ORP was the strong stimulation of Hbl production and, to a lesser extent, Nhe production. This control was complex, involving different levels of regulation. We discussed the regulation of enterotoxin expression and the involvement of the pleiotropic regulator PlcR.
Keywords: Bacillus cereus ; Enterotoxin; Fermentation; Growth; Oxidoreduction potential; RT-PCR
The effect of IHF on σ S selectivity of the phoA and pst promoters of Escherichia coli by Natalia Pasternak Taschner; Ezra Yagil; Beny Spira (pp. 234-237).
The pst operon, a member of the PHO regulon of Escherichia coli, encodes a high-affinity phosphate transport system whose expression is induced when the cells enter a phase of phosphate starvation. The expression of pst is stimulated by the integration host factor (IHF). Transcription of the PHO regulon genes is initiated by the RNA polymerase complexed with σ D (Eσ D). Owing to a cytosine residue at position −13 of the pst promoter its transcription can also be initiated by Eσ S. Here, we show that inactivation of IHF in vivo abolishes the σ S-dependent transcription initiation of the pst operon, indicating that both −13C residue and IHF are required to confer on pst the ability to be transcribed by Eσ S. Introduction of a −13C residue in the promoter region of phoA, another PHO regulon gene that is not directly affected by IHF, did not affect its exclusive transcription initiation by Eσ D.
Keywords: PHO regulon; pst operon; IHF; rpoS ; Escherichia coli
Plasma membrane of Synechocystis PCC 6803: a heterogeneous distribution of membrane proteins by Renu Srivastava; Natalia Battchikova; Birgitta Norling; Eva-Mari Aro (pp. 238-243).
Proteomic studies carried out previously on the plasma membrane of Synechocystis have identified several peripheral and integral proteins. The distribution of these proteins along the membrane still remains obscure. In this study, the distribution of proteins along the plasma membrane of Synechocystis was carried out using subfractions, the right-side-out (RSO) and inside-out (ISO) vesicles, fractionated from a pure and specific fraction of the plasma membrane. These subfractions were analyzed and quantified for several proteins by immunoblotting. It was found that the ISO fraction contained higher quantities of preD1, D1 and PsaD, the integral proteins of photosystem I and II known to be present also in the plasma membrane. Lower amounts of peripheral vesicle inducing protein Vipp1 and nitrate/nitrite binding protein NrtA were present in the ISO compared to the RSO fraction. On the contrary, the distribution of two integral transporter proteins, SbtA and PxcA, was found equal in both fractions. Our studies clearly establish that the plasma membrane of Synechocystis has a heterogeneous composition with respect to protein distribution. The accumulation of photosynthesis-associated proteins in the ISO fraction provides evidence that the discrete regions of the plasma membrane harbor sites for biogenesis of photosystems.
Keywords: Plasma membrane; Right-side-out vesicles; Inside-out vesicles; Heterogeneity; Cyanobacteria