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Archives of Microbiology (v.189, #6)

Diversity, ecology, and genomics of the Roseobacter clade: a short overview by Thorsten Brinkhoff; Helge-Ansgar Giebel; Meinhard Simon (pp. 531-539).

Due to worldwide distribution, high abundance and availability of physiologically diverse isolates the Roseobacter clade is one of the most intensively studied groups of marine bacteria. Organisms of this clade have been detected in a large variety of habitats, from coastal regions to deep-sea sediments and from polar ice to tropical latitudes, and constitute up to 25% of the total bacterial community. Use of a multitude of organic compounds, sulfur oxidation, aerobic anoxygenic photosynthesis, oxidation of carbon monoxide, DMSP demethylation, and production of secondary metabolites are some of the important traits found in this clade. Physiological characteristics and the different isolation sources indicate that organisms of the Roseobacter clade occupy various ecological niches. Since the first description of Roseobacter spp. in 1991, 38 affiliated and validated genera have been described. More than half of these descriptions have been published within the last 3 years. Genome sequencing of currently 40 different strains demonstrates enormous interest in the genetic and metabolic diversity of these bacteria. Plasmids with an enormous size range are also widespread in the Roseobacter clade indicating an adaptive genomic structure. Comparisons with other highly relevant groups, like the SAR11 clade, have shown drastic differences in genome organization.

Keywords: Microbial ecology; Rhodobacterales ; Rhodobacteraceae ; Taxonomy

Genome-wide transcriptional responses of Nitrosomonas europaea to zinc by Sunhwa Park; Roger L. Ely (pp. 541-548).

Nitrosomonas europaea, a Gram-negative obligate chemolithoautotroph, participates in global nitrogen cycling by carrying out nitrification and derives energy for growth through oxidation of ammonia. In this work, the physiological, proteomic, and transcriptional responses of N. europaea to zinc stress were studied. The nitrite production rate and ammonia-dependent oxygen uptake rate of the cells exposed to 3.4 μM ZnCl₂ decreased about 61 and 69% within 30 min, respectively. Two proteins were notably up regulated in zinc treatment and the mRNA levels of their encoding genes started to increase by 1 h after the addition of zinc. A total of 27 genes were up regulated and 30 genes were down regulated. Up-regulated genes included mercury resistance genes (merTPCAD), inorganic ion transport genes, oxidative stress genes, toxin-antitoxin genes, and two-component signal transduction systems genes. merTPCAD was the highest up-regulated operon (46-fold). Down-regulated genes included the RubisCO operon (cbbO), biosynthesis (mrsA), and amino acid transporter.

Keywords: Nitrosomonas europaea ; 2-D SDS-PAGE; Zinc; cDNA microarray; Quantitative reverse transcriptase-PCR

Screening of genes regulated by cold shock in Shewanella piezotolerans WP3 and time course expression of cold-regulated genes by Shengkang Li; Xiang Xiao; Ping Sun; Fengping Wang (pp. 549-556).

The differential gene transcription of a deep-sea bacterium Shewanella piezotolerans WP3 in response to cold shock was analyzed by RNA arbitrarily primed PCR. Ninety primer sets were used to scan two different RNA pools derived from the culture of cold shock and its control (culture at its optimal grown temperature). Ninety-four putative differentially expressed fragments were identified and cloned. Six out of the 94 fragments were confirmed to be truly differentially transcribed in terms of cold shock by reverse Northern dot blot and then sequenced. Sequence blast analysis showed that the six differentially transcribed genes are putative genes for zonular occludens toxin, chaperon GroEL, efflux transporter, Sua5/YciO/YrdC/YwlC family protein, betaine-aldehyde dehydrogenase, and DEAD box RNA helicase, respectively. The time course expression profiles of these six genes from 0 to 90 min upon cold shock were quantified by real-time PCR. Deletion mutation of the highest induced gene—RNA helicase gene, had no significant impact on the growth of the strain no matter upon cold shock or under permanent low temperature. It is suggested that one or more additional DEAD box RNA helicase genes compensate for the loss of the function of the mutated gene.

Keywords: Shewanella piezotolerans WP3; RAP-PCR; Differential-expressed fragments; Time course expression; Mutation

Characterization of the codY gene and its influence on biofilm formation in Bacillus cereus by Yi-Huang Hsueh; Eileen B. Somers; Amy C. Lee Wong (pp. 557-568).

The foodborne pathogen Bacillus cereus can form biofilms on various food contact surfaces, leading to contamination of food products. To study the mechanisms of biofilm formation by B. cereus, a Tn5401 library was generated from strain UW101C. Eight thousand mutants were screened in EPS, a low nutrient medium. One mutant (M124), with a disruption in codY, developed fourfold less biofilm than the wild-type, and its defective biofilm phenotype was rescued by complementation. Addition of 0.1% casamino acids to EPS prolonged the duration of biofilms in the wild-type but not codY mutant. When decoyinine, a GTP synthesis inhibitor, was added to EPS, biofilm formation was decreased in the wild-type but not the mutant. The codY mutant produced three times higher protease activity than the wild-type. Zymogram and SDS-PAGE data showed that production of the protease (∼130 kDa) was repressed by CodY. Addition of proteinase K to EPS decreased biofilm formation by the wild-type. Using a dpp-lacZ fusion reporter system, it was shown that that the B. cereus CodY can sense amino acids and GTP levels. These data suggest that by responding to amino acids and intracellular GTP levels CodY represses production of an unknown protease and is involved in biofilm formation.

Keywords: CodY; Biofilm; GTP; Amino acids; Bacillus cereus

Functional analysis of a BarX homologue (SngA) as a pleiotropic regulator in Streptomyces natalensis by Kang-Mu Lee; Chang-Kwon Lee; Sun-Uk Choi; Hae-Ryong Park; Yong-Il Hwang (pp. 569-577).

The prior sequencing of the upstream region of the γ-butyrolactone autoregulator receptor gene (sngR) in Streptomyces natalensis revealed the presence of a 972-bp gene encoding a BarX homologue (SngA), which acts as a pleiotropic regulator controlling secondary metabolism and morphological differentiation. In this study, we investigated the in vivo function of SngA in S. natalensis, by comparing the natamycin production, morphology, and transcription of genes related to natamycin biosynthesis in a wild-type strain and a sngA-deleted mutant. The disruption of sngA resulted in a decrease in natamycin production, and in the induction of pigment production that had not been previously observed from S. natalensis. On the other hand, the insertion of the intact sngA with its own promoter, into the wild-type strain, resulted in a 1.7-fold increase in natamycin production. Spore formation decreased in comparison to that of the wild-type strain when the sngA-deleted mutant was grown on YEME agar, MS medium, and ISP4 medium. All phenotypes were restored to the original wild-type phenotypes upon complementation with the intact sngA, suggesting that SngA has pleiotropic functions in controlling both morphological differentiation and secondary metabolite production.

Keywords: Streptomyces natalensis ; Pleiotropic regulator; Morphological differentiation; Natamycin production

Glycoconjugates enhanced the intracellular killing of Bacillus spores, increasing macrophage viability and activation by Olga Tarasenko; Lee Soderberg; Kristin Hester; Michelle Park Kim; Deirdre McManus; Pierre Alusta (pp. 579-587).

Infections caused by Bacillus spores can be attenuated if the intracellular killing of the organism by macrophages can be enhanced. Glycoconjugate-bearing polymers, which selectively bind to Bacillus spores, were tested for modulation of intracellular killing when added prior to, during, and following macrophage exposure to B. cereus spores. In the absence of glycoconjugates, murine macrophages were ineffective at killing Bacillus spores. In presence of glycoconjugates, however, macrophages efficiently killed spores, whether the glycoconjugates were added to the cells prior to, during, and following spore addition. Glycoconjugates were shown to exert a protective influence on macrophages and increase their activation, as evidenced by viability and lactate dehydrogenase release assays. Increased levels of nitric oxide production by macrophages pretreated with glycoconjugates suggest that, under these conditions, glycoconjugates provide an activation signal to macrophages. These results indicate that glycoconjugates promote killing of Bacillus spores, while increasing macrophage activation level and viability. The selection of glycoconjugate ligands bearing immunomodulating properties could be exploited for vaccine and/or immunomodulator development and/or for the improvement of existing vaccines against B. cereus and B. anthracis.

Keywords: Glycoconjugate; Macrophage; Phagocytosis; Prior to, during, and following exposure to B. cereus spores; Nitric oxide

Addition of exogenous carbon and nitrogen sources to aphid exuviae modulates synthesis of proteases and chitinase by germinating conidia of Beauveria bassiana by Sohail S. Qazi; George G. Khachatourians (pp. 589-596).

Secretion of catabolic extracellular enzymes (ECE) is the hallmark of the infection of insects through the cuticle by entomopathogenic fungi (EPF). In this paper, we show that germinating conidia of Beauveria bassiana (Bb) regulate the synthesis of ECE through a multiple control mode during the initial stages of germination. We tested Bb conidial growth on aphid exuviae with or without supplementation of additional carbon and/or nitrogen (C/N) compounds. To understand the interrelation between conidial germination during growth, the synthesis of ECE activity, free amino nitrogen (FAN), glucose and fungal dry weight biomass were measured. Immediately (0.25 h) upon incubation of conidia, activity of subtilisin-like Pr1 and trypsin-like Pr2 enzymes and chitinase (NAGase) was observed in the culture filtrates. At 0.25 h, addition of exogenous C-source resulted in higher activities of Pr1 and Pr2, respectively. Conversely at 0.25 h, addition of N-sources repressed the synthesis of Pr2, but that of Pr1. C/N repression was observed only for exponentially growing mycelia. NAGase activity remained at basal level and unaffected by added C/N. We conclude that C/N repression occurs only when it is necessary for the Bb infective structures to establish a nutritional relationship with the host structures.

Keywords: Subtilisins (Pr1-like); Trypsin (Pr2-like); NAGase; Regulation; Beauveria bassiana ; Biocontrol

Escherichia coli tat mutant strains are able to transport maltose in the absence of an active malE gene by Isabelle Caldelari; Tracy Palmer; Frank Sargent (pp. 597-604).

The twin-arginine transport (Tat) system is a prokaryotic protein transport system. Escherichia coli mutants in this pathway show a defect in cell separation during cell division, resulting in destabilization and permeability of the outer membrane. Maltose uptake is catalysed by a membrane-bound transporter of the ATP binding cassette (ABC) superfamily, where MalE is the essential periplasmic binding protein component. Here, we report that tat mutants are unexpectedly able to transport maltose in the absence of malE. This observation is specific to the MalE component since co-inactivation of malF, which encodes one of the channel components of the transporter, completely abolishes maltose transport even when the Tat system is inactivated. Genetic repair of the outer membrane leaky phenotype of the tat mutant strain re-established the absolute requirement for MalE in maltose uptake. In addition, we demonstrate that phenotypic repair of the outer membrane defect of the tat strain can also be achieved chemically by the inclusion of high concentrations of calcium or magnesium in the growth medium.

Keywords: Protein transport; Tat pathway; Twin-arginine signal peptide; ABC transporter; Periplasmic binding protein; Outer membrane

Corynebacterium glutamicum possesses two secA homologous genes that are essential for viability by Michael Caspers; Roland Freudl (pp. 605-610).

SecA is a central component of the bacterial Sec preprotein translocase. Besides the housekeeping SecA (SecA1), some mostly pathogenic Gram-positive bacteria possess an accessory SecA (SecA2) that is involved in the export of a few substrates only. Here we show that neither of the two secA homologous genes present in the genome of the non-pathogenic bacterium Corynebacterium glutamicum can be deleted, unless a copy of the respective gene is provided in trans on a plasmid. This finding is in marked contrast to all other cases examined so far making C. glutamicum the first reported bacterium possessing two essential SecA proteins.

Keywords: Gram-positive bacteria; Corynebacterianeae; Protein secretion; Translocase subunit; SecA

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Archives of Microbiology (v.189, #6)