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Archives of Microbiology (v.190, #5)

novE and novG act as positive regulators of novobiocin biosynthesis by Volker Dangel; Alessandra S. Eustáquio; Bertolt Gust; Lutz Heide (pp. 509-519).

The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin contains two putative regulatory genes, i.e., novE and novG. Functional proof for the role of NovG as a positive regulator of novobiocin biosynthesis had been provided previously, and we now investigated the role of novE. Heterologous expression experiments with the novobiocin biosynthetic gene cluster showed that the entire putative promoter region of novE is required to achieve optimal novobiocin production. Overexpression of novE, using a replicative vector, resulted in an increase of novobiocin formation. In contrast, inactivation of novE by in frame deletion resulted in a strong reduction of novobiocin biosynthesis. Novobiocin production could be restored by an intact copy of novE, but also by the regulatory gene novG. These observations suggest that novE is a positive regulator of novobiocin biosynthesis. NovE was expressed in E. coli and purified. However, in contrast to parallel experiments with NovG, no DNA-binding properties could be shown for NovE. RT-PCR experiments showed that expression of novG was detectable in the absence of NovE, and also that expression of novE occurred in absence of NovG.

Keywords: Streptomyces ; Novobiocin; Antibiotic; Regulation

Novel lipoarabinomannan-like lipoglycan (CdiLAM) contributes to the adherence of Corynebacterium diphtheriae to epithelial cells by L. O. Moreira; A. L. Mattos-Guaraldi; A. F. B. Andrade (pp. 521-530).

The genus Corynebacterium is part of the phylogenetic group nocardioform actinomycetes. Members of this group have a characteristic cell envelope structure composed primarily of branched long-chain lipids, termed mycolic acids, and a rich number of lipoglycans such as lipoarabinomanans (LAM) and lipomannans. In this study, we identified a novel LAM variant isolated from Corynebacterium diphtheriae named CdiLAM. The key structural features of CdiLAM are a linear α-1→6-mannan with side chains containing 2-linked α-D-Manp and 4-linked α-D-Araf residues. The polysaccharide backbone is linked to a phosphatidylinositol anchor. In contrast to the LAMs of other members of actinomycetales, CdiLAM presents an unusual substitution at position 4 of α-1→6-mannan backbone by α-D-Araf. Unlike the non-fimbrial adhesin 62–72p, CdiLAM did not function as a hemagglutinin to human red blood cells. Experimental evidences pointed to CdiLAM as an adhesin of C. diphtheriae to human respiratory epithelial cells, thereby, contributing to the pathogenesis of diphtheria.

Keywords: Lipoarabinomannan; Corynebacterium diphtheriae ; Adherence

The Cgl1281-encoding putative transporter of the cation diffusion facilitator family is responsible for alkali-tolerance in Corynebacterium glutamicum by Seiki Takeno; Mio Nakamura; Rie Fukai; Junko Ohnishi; Masato Ikeda (pp. 531-538).

Mutants of Corynebacterium glutamicum that were unable to grow under mild alkaline pH conditions were isolated by mutagenesis. Strain AL-43 exhibiting the highest sensitivity to alkaline pH among the mutants was selected and used to clone a DNA fragment that could complement the phenotype. Sequencing and subcloning of the cloned 4.0-kb EcoRI DNA fragment showed that the Cgl1281 gene was responsible for the complementation. The deduced amino acid sequence of Cgl1281 was found to show significant sequence similarity with CzcD, a Me²⁺/H⁺(K⁺) antiporter, from Bacillus subtilis and also possess the features of the cation diffusion facilitator (CDF) family: the presence of 6 putative transmembrane segments and a signature sequence, indicating that the gene product is a member of the CDF family. Chromosomal disruption of the Cgl1281 rendered C. glutamicum cells sensitive to alkaline pH as well as cobalt, while expression of the gene from a plasmid restored alkali-tolerance to the wild-type level and also led to increased cobalt resistance. These results demonstrated that the putative transporter of the CDF family mediates resistance to cobalt and also plays a physiological role in alkaline pH tolerance in C. glutamicum.

Keywords: Alkaline pH tolerance; Cation diffusion facilitator family; Cobalt resistance; Corynebacterium glutamicum

The response to a specific germinant by Bacillus anthracis spores in primary mouse macrophages is modulated by a protein encoded on the pXO1 plasmid by Arthur I. Aronson; Haijing Hu (pp. 539-546).

A Bacillus anthracis Sterne pXO1 plasmid-encoded protein designated Cot43 was found in coat extracts of purified spores. Cot43 is a tetratricopeptide repeat domain protein related to those which function as phosphatases in the sporulation phosphorelay and as regulators of competence and pathogenic factors. The synthesis of Cot43 began in the late exponential phase downstream from a sigmaA promoter (as mapped by RACE) and it was present at least until the formation of phase white endospores. There was specificity in the association of Cot43 with B. anthracis spores since Bacillus cereus producing Cot43 from a cloned gene had very little of this protein in spore coat extracts. In addition, Cot43 was synthesized by B. anthracis cells to the same extent in glucose-yeast extract and nutrient sporulation media, but was essentially absent from spores formed in the former. l-histidine is an important germinant for B. anthracis spores in macrophages, Spores produced by a mutant with a disruption of cot43 germinated in response to l-histidine both in vitro and within primary mouse macrophages earlier and more extensively than Sterne strain spores. The germination delay due to the presence of Cot43 would enhance spore survival and thus increase the chances for a successful infection.

Keywords: Spores; Germination; Bacillus anthracis ; Plasmid; Macrophage

An N-acyl homolog of mycothiol is produced in marine actinomycetes by Gerald L. Newton; Paul R. Jensen; John B. MacMillan; William Fenical; Robert C. Fahey (pp. 547-557).

Marine actinomycetes have generated much recent interest as a potentially valuable source of novel antibiotics. Like terrestrial actinomycetes the marine actinomycetes are shown here to produce mycothiol as their protective thiol. However, a novel thiol, U25, was produced by MAR2 strain CNQ703 upon progression into stationary phase when secondary metabolite production occurred and became the dominant thiol. MSH and U25 were maintained in a reduced state during early stationary phase, but become significantly oxidized after 10 days in culture. Isolation and structural analysis of the monobromobimane derivative identified U25 as a homolog of mycothiol in which the acetyl group attached to the nitrogen of cysteine is replaced by a propionyl residue. This N-propionyl-desacetyl-mycothiol was present in 13 of the 17 strains of marine actinomycetes examined, including five strains of Salinispora and representatives of the MAR2, MAR3, MAR4 and MAR6 groups. Mycothiol and its precursor, the pseudodisaccharide 1-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol, were found in all strains. High levels of mycothiol S-conjugate amidase activity, a key enzyme in mycothiol-dependent detoxification, were found in most strains. The results demonstrate that major thiol/disulfide changes accompany secondary metabolite production and suggest that mycothiol-dependent detoxification is important at this developmental stage.

Keywords: Mycothiol; N-propionyl-desacetyl-mycothiol; Marine actinomycetes; Salinispora arencola ; Mca

Effect of cra gene knockout together with edd and iclR genes knockout on the metabolism in Escherichia coli by Dayanidhi Sarkar; Khandaker Al Zaid Siddiquee; Marcos J. Araúzo-Bravo; Takahiro Oba; Kazuyuki Shimizu (pp. 559-571).

To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved.

Keywords: Escherichia coli ; cra mutant; cra.edd mutant; cra.edd.iclR mutant; DNA microarray

Adhesion of the probiotic strains Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 cells under conditions simulating the intestinal tract, and in the presence of antibiotics and anti-inflammatory medicaments by Marelize Botes; Ben Loos; Carol A. van Reenen; Leon M. T. Dicks (pp. 573-584).

Adhesion of Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 (human carcinoma epithelial) cells was visualized by fluorescent staining. Both strains showed good adhesion compared to L. casei MB1, L. casei Shirota, L. johnsonii La1 and L. rhamnosus GG. No correlation was found between hydrophobicity, aggregation and adhesion to Caco-2 cells. Presence of antibiotics and anti-inflammatory medicaments reduced adhesion of bacterial strains to Caco-2 cells. Proteins sensitive to pepsin, trypsin and pronase are involved in the adhesion of E. mundtii ST4SA and L. plantarum 423 to Caco-2 cells. Adhesion of Listeria monocytogenes ScottA to Caco-2 cells was not prevented by E. mundtii ST4SA and L. plantarum 423. Cell-free culture supernatants of strains ST4SA and 423, containing the antimicrobial peptides plantaricin 423 and peptide ST4SA, prevented the invasion of L. monocytogenes ScottA into Caco-2 cells.

Keywords: Adhesion of Enterococcus mundtii ; Lactobacillus plantarum to Caco-2 cells

Role of RpoS in stress survival, synthesis of extracellular autoinducer 2, and virulence in Vibrio alginolyticus by Yang Tian; Qiyao Wang; Qin Liu; Yue Ma; Xiaodan Cao; Yuanxing Zhang (pp. 585-594).

Vibrio alginolyticus, a marine bacterium, is an opportunistic pathogen capable of causing vibriosis with high mortality to fishes in the South China Sea. Stress resistance is very important for its survival in the natural environment and upon infection of the host. RpoS, an alternative sigma factor, is considered as an important regulator involved in stress response and virulence in many pathogens. In this study, the rpoS gene was cloned and characterized to evaluate the role of RpoS in V. alginolyticus. The predicted protein showed high identity with other reported rpoS gene products. The in-frame deleted mutation of rpoS in V. alginolyticus led to sensitivity of the strain to ethanol, hyperosmolarity, heat, and hydrogen peroxide challenges. Further studies showed that extracellular autoinducer 2 level, four of seven detected protease activities, and cytotoxicity of extracellular products were markedly decreased in the rpoS mutant compared with that in the wild-type strain. The results indicated that the global regulator RpoS was part of the regulatory networks of virulence and LuxS quorum sensing system.

Keywords: RpoS; Vibrio alginolyticus ; Stress; AI-2 synthesis; ECP

Characterization of two TonB systems in marine fish pathogen Vibrio alginolyticus: their roles in iron utilization and virulence by Qiyao Wang; Qin Liu; Xiaodan Cao; Minjun Yang; Yuanxing Zhang (pp. 595-603).

In Gram-negative bacteria, the TonB/ExbB/ExbD complex is required to energize the specific high-affinity receptors mediating iron uptake processes. In fish pathogen Vibrio alginolyticus MVP01, which exhibited capacities to scavenge iron sources with various specific iron uptake systems, we identified and characterized two sets of TonB systems. In V. alginolyticus, TonB1 and TonB2 systems were arrayed as TonB1–ExbB1–ExbD1 and ExbB2–ExbD2–TonB2, respectively. The transcription of the TonB systems was regulated by the iron concentration in culture medium. While TonB1 system specifically contributed to the hemin and hemoglobin utilization in V. alginolyticus MVP01, both of the TonB systems showed abilities to support the utilization of iron sources from ferrichrome and vibrioferrin, the endogenous siderophore of V. alginolyticus. In addition, when mutants of the TonB systems were inoculated intraperitoneally into the fish Brachydanio rerio, marked attenuation in virulence of V. alginolyticus was observed, indicating that both of the TonB systems are essential for the virulence of V. alginolyticus.

Keywords: Iron transportation; TonB system; Vibrio alginolyticus ; Virulence

Antibacterial activity of cyclodextrins against Bacillus strains by Hui-Min Zhang; Zhijun Li; Katsuyuki Uematsu; Tohru Kobayashi; Koki Horikoshi (pp. 605-609).

Growth of alkaliphilic Bacillus halodurans C-125 both on agar plates and in liquid culture was inhibited by methyl-β-cyclodextrin (CD). Furthermore, resting cells of the strain were lysed by contact with methyl-β-CD higher than 10 mM. α-CD also showed lysis activity against Bacillus and related strains. The activity was not observed with Gram-negative and Gram-positive bacteria except for Bacillus strains. Fluorescence staining and scanning electron microscopy of cells revealed that methyl-β-CD disrupted cell membranes, and consequently, the cells were lysed. This is a novel physiological property of CDs.

Keywords: Methyl-β-cyclodextrin; α-cyclodextrin; Antibacterial agent; Bacillus ; Lysis

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Archives of Microbiology (v.190, #5)