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Archives of Microbiology (v.181, #4)
Incidence and function of sigma factors in Ralstonia metallidurans and other bacteria by Dietrich H. Nies (pp. 255-268).
Bacterial sigma factors are essential for directing the bacterial RNA polymerase to promoter regions during transcription initiation. Genomic sequencing of the highly heavy-metal-resistant β-proteobacterium Ralstonia metallidurans strain CH34 revealed 17 candidate genes for sigma factors. This review compares the sigma factor machinery of R. metallidurans to that of other bacteria. The sigma factors of 105 bacterial genomes were assigned to sigma factor clusters and families formed around the factors from Escherichia coli, Bacillus subtilis, and R. metallidurans. Genes for between 1 and 65 sigma-factor-related proteins were found in these genomes. Although prediction of sigma factor function from sequence comparisons can be misleading, organization of the R. metallidurans sigma factors into clusters and protein families, together with a discussion of the physiological function of members of these clusters, might yield insight into the cellular roles of bacterial sigma factors and the genes that depend on them for their expression.
Keywords: Sigma factors; Extracytoplasmic function; Alcaligenes eutrophus ; Ralstonia metallidurans
Thermobaculum terrenum gen. nov., sp. nov.: a non-phototrophic gram-positive thermophile representing an environmental clone group related to the Chloroflexi (green non-sulfur bacteria) and Thermomicrobia by Lina M. Botero; Kathy B. Brown; Sue Brumefield; Mark Burr; Richard W. Castenholz; Mark Young; Timothy R. McDermott (pp. 269-277).
A novel bacterium was cultivated from an extreme thermal soil in Yellowstone National Park, Wyoming, USA, that at the time of sampling had a pH of 3.9 and a temperature range of 65–92 °C. This organism was found to be an obligate aerobic, non-spore-forming rod, and formed pink-colored colonies. Phylogenetic analysis of the 16S rRNA gene sequence placed this organism in a clade composed entirely of environmental clones most closely related to the phyla Chloroflexi and Thermomicrobia. This bacterium stained gram-positive, contained a novel fatty-acid profile, had cell wall muramic acid content similar to that of Bacillus subtilis (significantly greater than Escherichia coli), and failed to display a lipopolysaccharide profile in SDS-polyacrylamide gels that would be indicative of a gram-negative cell wall structure. Ultrastructure examinations with transmission electron microscopy showed a thick cell wall (approximately 34 nm wide) external to a cytoplasmic membrane. The organism was not motile under the culture conditions used, and electron microscopic examination showed no evidence of flagella. Genomic G+C content was 56.4 mol%, and growth was optimal at 67 °C and at a pH of 7.0. This organism was able to grow heterotrophically on various carbon compounds, would use only oxygen as an electron acceptor, and its growth was not affected by light. A new species of a novel genus is proposed, with YNP1T (T=type strain) being Thermobaculum terrenum gen. nov., sp. nov. (16S rDNA gene GenBank accession AF391972). This bacterium has been deposited in the American Type Culture Collection (ATCC BAA-798) and the University of Oregon Culture Collection of Microorganisms from Extreme Environments (CCMEE 7001).
Keywords: Thermophile; Green non-sulfur; Chloroflexi; Gram-positive; Yellowstone National Park; Thermal soil
Differential expression of sialylglycoconjugates and sialidase activity in distinct morphological stages of Fonsecaea pedrosoi by Daniela S. Alviano; Marcio L. Rodrigues; Catia A. Almeida; André L. S. Santos; José N. S. S. Couceiro; Rosangela M. A. Soares; Luiz R. Travassos; Celuta S. Alviano (pp. 278-286).
The expression of sialoglycoconjugates in Fonsecaea pedrosoi conidia, mycelia, and sclerotic cells was analyzed using influenza A and C virus strains, sialidase treatment, and lectin binding. Conidium and mycelium whole cells were recognized by Limax flavus (LFA), Maackia amurensis (MAA), and Sambucus nigra (SNA) lectins, denoting the presence of surface sialoglycoconjugates containing α2,3- and α2,6-sialylgalactosyl sequences. Sialidase-treated conidia reacted more intensively with peanut agglutinin (PNA), confirming the occurrence of sialyl-galactosyl linkages. Conidial cells agglutinated in the presence of influenza A and C virus strains, which confirmed the results obtained from lectin-binding experiments and revealed the presence of sialoglycoconjugates bearing 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) surface structures. Western blotting analysis with peroxidase-labeled LFA demonstrated the occurrence of sialylglycoproteins in protein extracts from conidia and mycelia, with molecular masses corresponding to 56 and 40 kDa. An additional band of 77 kDa was detected in conidial extracts, suggesting an association between sialic acid expression and morphogenesis. Synthesis of sialic acids was correlated with sialidase expression, since both conidial and mycelial morphological stages presented secreted and cell-associated enzyme activity. Sialoglycoconjugates were not detected in F. pedrosoi sclerotic cells from in vitro and in vivo sources, which also do not express sialidase activity. The surface sialyl residues in F. pedrosoi are apparently involved in the fungal interaction with immune effector cells, since sialidase-treated conidia were less resistant to phagocytosis by human neutrophils from healthy individuals. These findings suggest that sialic acid expression in F. pedrosoi varies according to the morphological transition and may protect infecting propagules against immune destruction by host cells.
Keywords: Fonsecaea pedrosoi ; Sialic acid; Sialidase; Lectins; Phagocytosis
Cloning, identification and expression of an entE homologue angE from Vibrio anguillarum serotype O1 by Qin Liu; Yue Ma; Haizhen Wu; Mingfei Shao; Haifeng Liu; Yuanxing Zhang (pp. 287-293).
Anguibactin, an important virulent factor in Vibrio anguillarum serotype O1, is synthesized by a nonribosomal peptide synthetases (NRPS) system encoded on a 65-kb virulence plasmid pJM1. angE, as one of the NRPS genes, is responsible for selecting and activating 2,3-dihydroxybenzoic acid (2,3-DHBA), an important precursor in anguibactin synthesis, into 2,3-DHBA-AMP by adenylylation in the presence of ATP. In this work, an entE homologue, angE, was identified on pEIB1 (a pJM1-like plasmid) from virulent V. anguillarum serotype O1 strain MVM425. A recombinant clone carrying the complete angE was able to complement an Escherichia coli entE mutant. The angE-encoded protein was overexpressed in E. coli and purified by a three-step procedure. Purified AngE was then used to establish an in vitro enzymatic reaction in which its enzymatic activity of 1-(5′-monophosphate adenyl) 2,3-dihydroxybenzoic acid ligase (2,3-DHBA-AMP ligase) was proved using HPLC to detect AMP formation in the reaction mixture. Moreover, evidence at the level of both transcription and translation confirmed that angE was actively expressed in vivo in V. anguillarum MVM425, and interestingly, unlike many other iron-uptake-system-related genes, its expression is not induced by a low iron concentration in the surrounding environment.
Keywords: Vibrio anguillarrum ; pJM1-like plasmid; Iron uptake system; angE ; 2,3-DHBA-AMP ligase; Anguibactin biosynthesis; Gene expression
Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Kitasatospora setae, a bafilomycin B1 producer by Sun-Uk Choi; Chang-Kwon Lee; Yong-Il Hwang; Hiroshi Kinoshita; Takuya Nihira (pp. 294-298).
An effective transformation procedure for Kitasatospora setae was established based on transconjugation from Escherichia coli ET12567 (pUZ8002) using a ϕC31-derived integration vector, pSET152, containing oriT and attP fragments. While no transconjugation was observed under the standard transconjugation conditions for Streptomyces species, sufficient transconjugation (>1×10-6) was achieved on ISP4 medium containing 30 mM MgCl2 using a 25- to 125-fold excess of E. coli donor cells. In addition, the sequence and location of the chromosomal integration site attB of K. setae was identified for the first time in genera of non-Streptomyces actinomycetes. K. setae contains a single ϕC31 attB site. Similar to the case of Streptomyces species, the attB site of K. setae is present within an ORF encoding a pirin-homolog, but the K. setae-attB sequence deviates slightly from the consensus sequence of Streptomyces attB sequences.
Keywords: Conjugal transfer; Integration site; Kitasatospora setae ; Bafilomycin B1
‘Candidatus Hepatincola porcellionum’ gen. nov., sp. nov., a new, stalk-forming lineage of Rickettsiales colonizing the midgut glands of a terrestrial isopod by Yongjie Wang; Ulrich Stingl; Friederike Anton-Erxleben; Martin Zimmer; Andreas Brune (pp. 299-304).
The midgut glands (hepatopancreas) of terrestrial isopods are densely colonized by hitherto uncultivated bacteria. In the case of the Common Woodlouse, Porcellio scaber (Crustacea: Isopoda), the symbionts represent a novel lineage in the α-subdivision of Proteobacteria. Based on comparative sequence analysis of their 16S rRNA genes, their closest (albeit distant) relatives were among the Rickettsiales, which are intracellular symbionts or pathogens of many animals. Transmission electron microscopy and in situ hybridization with fluorescently labeled oligonucleotide probes revealed a homogeneous population of symbionts intimately associated with the endothelium of the hepatopancreas, which apparently interact with the microvilli of the brush border by means of a stalk-like cytoplasmic appendage. Based on isolated phylogenetic position and unique cytological properties, the provisional name ‘Candidatus Hepatincola porcellionum’ is proposed to classify this new taxon of Rickettsiales colonizing the hepatopancreas of P. scaber.
Keywords: Crustacea; Isopoda; Hepatopancreas; Symbionts; α-Proteobacteria; Rickettsiales; Prosthecate bacteria
Lagging strand replication of rolling-circle plasmids in Streptomyces lividans: an RNA polymerase-independent primer synthesis by Ichiro Suzuki; Masakazu Kataoka; Toshiomi Yoshida; Tatsuji Seki (pp. 305-313).
The rolling circle (RC) mechanism of DNA replication generating single-stranded DNA (ssDNA) intermediates is common in various high-copy circular plasmids in Streptomyces, and the ssDNA released after leading strand synthesis is converted to its double-stranded form (dsDNA) by the host proteins. The in vivo and in vitro lagging strand syntheses from ssDNA replicative intermediates of RC plasmid pSN22 in Streptomyces lividans was characterized. The presence or absence of the single-strand origin (sso), the replication initiation site of lagging strand synthesis, did not significantly affect the copy numbers of pSN22 derivatives. In vivo lagging strand synthesis was not affected by the rifampicin inhibition of S. lividans RNA polymerase. Likewise, in vitro lagging strand synthesis using cell-free extracts revealedsso-independent, rifampicin-resistant lagging strand synthesis in S. lividans. Although all four dNTPs are usually required for the initiation of such synthesis, the presence of only one NTP was sufficient to carry outlagging strand synthesis in vitro. Interestingly, the cell-free extract of exponential-phase cells required less ATP than that of stationary-phase cells. These results reveal a predominant RNA polymerase-independent priming system in S. lividans that may be a result of the stabilization of RC plasmids lacking sso in S. lividans.
Keywords: Replication; Streptomyces; Priming; Lagging strand
Bacillus amyloliquefaciens strains isolated from moisture-damaged buildings produced surfactin and a substance toxic to mammalian cells by Raimo Mikkola; Maria A. Andersson; Pavel Grigoriev; Vera V. Teplova; Nils-Erik L. Saris; Frederick A. Rainey; Mirja S. Salkinoja-Salonen (pp. 314-323).
Fungicidic Bacillus amyloliquefaciens strains isolated from the indoor environment of moisture-damaged buildings contained heat-stable, methanol-soluble substances that inhibited motility of boar spermatozoa within 15 min of exposure and killed feline lung cells in high dilution in 1 day. Boar sperm cells lost motility, cellular ATP, and NADH upon contact to the bacterial extract (0.2 μg dry wt/ml). Two bioactive substances were purified from biomass of the fungicidal isolates. One partially characterized substance, 1,197 Da, was moderately hydrophobic and contained leucine, proline, serine, aspartic acid, glutamic acid and tyrosine, in addition to chromophore(s) absorbing at 365 nm. In boar sperm and human neural cells (Paju), the compound depolarized the transmembrane potentials of mitochondria (ΔΨ m) and the plasma membrane (ΔΨ p) after a 20-min exposure and formed cation-selective channels in lipid membranes, with a selectivity K+:Na+:Ca2+ of 26:15:3.5. The other substance was identified as a plasma-membrane-damaging lipopeptide surfactin. Plate-grown biomass of indoor Bacillus amyloliquefaciens contained ca. 7% of dry weight of the two substances, 1,197 Da and surfactin, in a ratio of 1:6 (w:w). The in vitro observed simultaneous collapse of both cytosolic and mitochondrial ATP in the affected mammalian cell, induced by the 1,197-Da cation channel, suggests potential health risks for occupants of buildings contaminated with such toxins.
Keywords: Bacillus amyloliquefaciens ; Black lipid membrane; Cation channel; Ionophore; Membrane-damage; Mitochondria; Moisture-damaged building; Plasma membrane; Surfactin; Toxin
Purification and characterization of an H2O-forming NADH oxidase from Clostridium aminovalericum: existence of an oxygen-detoxifying enzyme in an obligate anaerobic bacteria by Shinji Kawasaki; Jun Ishikura; Daisuke Chiba; Tomoko Nishino; Youichi Niimura (pp. 324-330).
Clostridium aminovalericum, an obligate anaerobe, is unable to form colonies on PYD agar plates in the presence of 1% O2. When grown anaerobically in PYD liquid medium, the strain can continue normal growth after the shift from anoxic (sparged with O2-free N2 carrier-gas) to microoxic (sparged with 3% O2/97% N2 mixed carrier-gas) growth conditions in the mid exponential phase (OD660=1.0). When the strain grew under 3% O2/97% N2, the medium remains anoxic. Thirty minutes after beginning aeration with 3% O2, the activity of NADH oxidase in cell-free extracts increased more than five-fold from the level before aeration. We purified NADH oxidase to determine the characteristics of this enzyme in an obligate anaerobe. The purified NADH oxidase dominated the NADH oxidase activity detected in cell-free extracts. The enzyme is a homotetramer composed of a subunit with a molecular mass of 45 kDa. The enzyme shows a spectrum typical of a flavoprotein, and flavin adenine dinucleotide (FAD) was identified as a cofactor. The final product of NADH oxidation was H2O, and the estimated K m for oxygen was 61.9 μM. These data demonstrate that an O2-response enzyme that is capable of detoxifying oxygen to water exists in C. aminovalericum.
Keywords: NADH oxidase; Clostridium ; Obligate anaerobe; Oxidative stress
Screening for highly active β-lactam antibiotics against Agrobacterium tumefaciens by Yoichi Ogawa; Masahiro Mii (pp. 331-336).
Quantitative in vitro antibacterial activities, i.e., minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs), of 12 β-lactam antibiotics against Agrobacterium tumefaciens strains LBA4404 and EHA101 were examined, in order to identify antibiotics effective in eliminating the bacteria in Agrobacterium-mediated plant genetic transformation. The antibacterial activities of β-lactams tested against strain EHA101 were equal to or less than those tested against strain LBA4404. Cefotaxime, cefbuperazone, and meropenem had high activities against strain LBA4404 (MBC <1 mg l−1). Against strain EHA101, however, only meropenem showed activity comparable to that against strain LBA4404. The production of β-lactamase was observed only in strain EHA101.
Keywords: Agrobacterium tumefaciens ; Bactericidal activity; Bacteristatic activity; β-Lactam antibiotics; β-Lactamase