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Archives of Microbiology (v.184, #5)
The three-dimensional structure of the cyanobacterium Synechocystis sp. PCC 6803 by Allison M.L. van de Meene; Martin F. Hohmann-Marriott; Wim F.J. Vermaas; Robert W. Roberson (pp. 259-270).
To advance our knowledge of the model cyanobacterium Synechocystis sp. PCC 6803 we investigated the three-dimensional organization of the cytoplasm using standard transmission electron microscopy and electron tomography. Electron tomography allows a resolution of ~5 nm in all three dimensions, superior to the resolution of most traditional electron microscopy, which is often limited in part by the thickness of the section (70 nm). The thylakoid membrane pairs formed layered sheets that followed the periphery of the cell and converged at various sites near the cytoplasmic membrane. At some of these sites, the margins of thylakoid membranes associated closely along the external surface of rod-like structures termed thylakoid centers, which sometimes traversed nearly the entire periphery of the cell. The thylakoid membranes surrounded the central cytoplasm that contained inclusions such as ribosomes and carboxysomes. Lipid bodies were dispersed throughout the peripheral cytoplasm and often juxtaposed with cytoplasmic and thylakoid membranes suggesting involvement in thylakoid maintenance or biogenesis. Ribosomes were numerous and mainly located throughout the central cytoplasm with some associated with thylakoid and cytoplasmic membranes. Some ribosomes were attached along internal unit-membrane-like sheets located in the central cytoplasm and appeared to be continuous with existing thylakoid membranes. These results present a detailed analysis of the structure of Synechocystis sp. PCC 6803 using high-resolution bioimaging techniques and will allow future evaluation and comparison with gene-deletion mutants.
Keywords: Electron tomography; Field-emission cryo-scanning electron microscopy; Serial section reconstruction; Transmission electron microscopy; Thylakoid membrane biogenesis; Cyanobacteria
Differential antibacterial activity of genistein arising from global inhibition of DNA, RNA and protein synthesis in some bacterial strains by Katarzyna Ulanowska; Aleksandra Tkaczyk; Grażyna Konopa; Grzegorz Węgrzyn (pp. 271-278).
Antibacterial activities of various flavonoids have been reported previously, but mechanism(s) of their action on bacterial cells remain(s) largely unknown. Here, we investigated effects of genistein, an isoflavone, and representatives of other flavonoids: daidzein (another isoflavone), apigenin (a flavone), naringenin (a flavanone) and kaempferol (a flavonol), on commonly used laboratory strains of model bacterial species: Escherichia coli, Vibrio harveyi and Bacillus subtilis. We found that E. coli was resistant to all tested flavonoids at concentrations up to 0.1 mM, while high sensitivity of V. harveyi to most of them (except daidzein, which exhibited significantly less pronounced effect) was observed. Effects of the flavonoids on B. subtilis were relatively intermediate to the two extremes, i.e., E. coli and V. harveyi. Action of genistein on bacterial cells was investigated in more detail to indicate changed cell morphology (formation of filamentous cells) of V. harveyi and drastic inhibition of global synthesis of DNA and RNA as shortly as 15 min after addition of this isoflavone to a bacterial culture to a final concentration of 0.1 mM. Protein synthesis inhibition was also apparent, but delayed. Both cell morphology and synthesis of nucleic acids and proteins were unaffected in E. coli cultures under analogous conditions. Studies on cell survival suggest that genistein is a bacteriostatic agent rather than a bactericidal compound.
Keywords: Genistein; Flavonoids; Antibacterial agents; Nucleic acids’ synthesis inhibition; Protein synthesis
Transformation of metals and metal ions by hydrogenases from phototrophic bacteria by Oleg A. Zadvorny; Nikolay A. Zorin; Ivan N. Gogotov (pp. 279-285).
The ability of hydrogenases isolated from Thiocapsa roseopersicina and Lamprobacter modestohalophilus to reduce metal ions and oxidize metals has been studied. Hydrogenases from both phototrophic bacteria oxidized metallic Fe, Cd, Zn and Ni into their ionic forms with simultaneous evolution of molecular hydrogen. The metal oxidation rate decreased in the series Zn>Fe>Cd>Ni and depended on the pH. The presence of methyl viologen in the reaction system accelerated this process. T. roseopersicina and L. modestohalophilus cells and their hydrogenases reduced Ni(II), Pt(IV), Pd(II) or Ru(III) to their metallic forms under H2 atmosphere. These results suggest that metals or metal ions can serve as electron donors or acceptors for hydrogenases from phototrophic bacteria.
Keywords: Hydrogenase; Phototrophic bacteria; Metal ions reduction; Metal oxidation; Metals transformation by hydrogenases
Characterization of the ectoine biosynthesis genes of haloalkalotolerant obligate methanotroph “Methylomicrobium alcaliphilum 20Z” by Alexander S. Reshetnikov; Valentina N. Khmelenina; Yuri A. Trotsenko (pp. 286-297).
The genes involved in biosynthesis of the major compatible solute ectoine (1,4,5,6-tetrahydro-2-methylpyrimidine carboxylic acid) in halotolerant obligate methanotroph “Methylomicrobium alcaliphilum 20Z” were studied. The complete nucleotide sequences of the structural genes encoding l-aspartokinase (Ask), l-2,4-diaminobutyric acid transaminase (EctB), l-2,4-diaminobutyric acid acetyltransferase (EctA), and l-ectoine synthase (EctC) were defined and shown to be transcribed as a single operon ectABCask. Phylogenetic analysis revealed high sequence identities (34–63%) of the Ect proteins to those from halophilic heterotrophs with the highest amino acid identities being to Vibrio cholerae enzymes. The chromosomal DNA fragment from “M. alcaliphilum 20Z” containing ectABC genes and putative promoter region was expressed in Escherichia coli. Recombinant cells could grow in the presence of 4% NaCl and synthesize ectoine. The data obtained suggested that despite the ectoine biosynthesis pathway being evolutionary well conserved with respect to the genes and enzymes involved, some differences in their organization and regulation could occur in various halophilic bacteria.
Keywords: “Methylomicrobium alcaliphilum”; Methanotrophs; Compatible solutes; Ectoine; EctABCask gene cluster; Osmoregulation
Regulation of the nap operon encoding the periplasmic nitrate reductase of Paracoccus pantotrophus: delineation of DNA sequences required for redox control by M.J.K. Ellington; W.L.J. Fosdike; R.G. Sawers; D.J. Richardson; S.J. Ferguson (pp. 298-304).
Expression of the nap operon, encoding the periplasmic nitrate reductase in Paracoccus pantotrophus, is maximal when cells are grown aerobically, but not anaerobically, with butyrate. Two promoters, termed P1 and P2, control operon expression and the operon-proximal P2 promoter is primarily responsible for increased nap expression in the presence of butyrate. A near-perfect palindromic sequence is centred at +7, relative to the P2 transcription start site. Mutation of this palindrome demonstrated that it is important for regulation of nap operon expression in response to both the redox and the oxidation state of the carbon substrate. A 5′ deletion analysis of the nap promoter fused to lacZ revealed that full redox control of expression was retained when the DNA sequence up to position −49 bp, relative to the operon-distal P1 transcription start site, was removed. Encroaching beyond this position resulted in an ~4-fold reduction in expression when cells were grown aerobically with butyrate. Additionally, point mutations at position −38 and −45 relative to P1 also resulted in a reduction in expression during aerobic growth with butyrate. A GC-rich region of nap promoter DNA, centred on position −41 relative to the P1 transcription start site is thus proposed as a second DNA motif that is important for efficient expression of the nap operon.
Keywords: Periplasmic nitrate reductase; Butyrate metabolism; Paracoccus pantotrophus ; Redox regulation
Isolation and characterization of a gene cluster for dibenzofuran degradation in a new dibenzofuran-utilizing bacterium, Paenibacillus sp. strain YK5 by Toshiya Iida; Kaoru Nakamura; Atsushi Izumi; Yuki Mukouzaka; Toshiaki Kudo (pp. 305-315).
Spore-forming bacterial strains capable of utilizing dibenzofuran (DF) as a sole source of carbon and energy were isolated. Characteristics of the isolates justified their classification into the genus Paenibacillus, and their closest relative was P. naphthalenovorans. Degenerate primers for aromatic hydrocarbon dioxygenase alpha subunit (AhDOa) genes and genomic DNA of the strain YK5 were used for gene isolation. The nucleotide sequences of clones of the PCR products revealed that the strain YK5 carries at least five different AhDOa genes. Northern hybridization analysis showed that one of the AhDOa genes was transcribed under DF-containing culture conditions. A gene cluster encoding the AhDOa was isolated. The genes predicted to encode extradiol dioxygenase (dbfB) and hydrolase (dbfC) were found to be an upstream of genes encoding the alpha and beta subunit of the AhDO (dbfA1 and dbfA2, respectively); the latter two gene products showed 60 and 53% identity to the amino acid sequences of DbfA1 and DbfA2 of Terrabacter sp. DBF63, respectively. Two Paenibacillus validus JCM 9077 strains transformed with the dbf gene clusters acquired the ability to convert DF to 2,2′,3-trihydroxybiphenyl (THBP) and salicylic acid (SAL). These results suggest that the enzymes encoded by the gene cluster isolated in this study are involved in DF metabolism in YK5.
Keywords: Dibenzofuran; Paenibacillus ; Aromatic hydrocarbon dioxygenase; Angular dioxygenase; Extradiol dioxygenase; Hydrolase
Control of the phytopathogen Botrytis cinerea using adipic acid monoethyl ester by Begonya Vicedo; María de la O Leyva; Víctor Flors; Ivan Finiti; Gemma del Amo; Dale Walters; Maria Dolores Real; Pilar García-Agustín; Carmen González-Bosch (pp. 316-326).
The in vitro and in vivo antifungal activity of adipic acid monoethyl ester (AAME) on the necrotrophic pathogen Botrytis cinerea has been studied. This chemical effectively controlled this important phytopathogen, inhibited spore germination and mycelium development at non-phytotoxic concentrations. The effectiveness of AAME treatment is concentration-dependent and influenced by pH. Spore germination in the presence of AAME is stopped at a very early stage, preventing germ tube development. In addition, cytological changes such as retraction of the conidial cytoplasm in the fungus are observed. AAME was also found to act on membrane integrity, affecting permeability without exhibiting lytic activity, as described previously for other antifungal compounds. Polyamine content in the mycelium of B. cinerea was also affected in response to AAME treatment, resulting in putrescine reduction and spermine accumulation similar to a number of antifungal agents. Microscopic observation of treated conidia after inoculation on tomato leaves suggested that inhibited spores are not able to attach to and penetrate the leaf. Finally, AAME completely suppressed the grey mould disease of tomato fruits under controlled inoculation conditions, providing evidence for its efficacy in a biological context and for the potential use of this chemical as an alternative fungicide treatment.
Keywords: Botrytis cinerea ; Antifungal activity; Adipic acid monoethyl ester; Lycopersicon esculentum
Isolation of constitutive variants of a subfamily 10 histidine protein kinase (SppK) from Lactobacillus using random mutagenesis by Geir Mathiesen; Gunnhild W. Axelsen; Lars Axelsson; Vincent G. H. Eijsink (pp. 327-334).
The histidine protein kinase SppK is a peptide pheromone-activated kinase that regulates the production of the bacteriocin sakacin P in Lactobacillus sakei. SppK belongs to subfamily 10 of histidine protein kinases (HPKs), which regulate important processes in Gram-positive bacteria, including virulence, competence and bacteriocin production. To obtain insight into the functional properties of this relatively unknown class of HPKs, we have subjected SppK to random mutagenesis by error-prone PCR, followed by selection for mutants displaying a constitutive phenotype. Most identified mutations were clustered in a predicted coiled coil-like region, which is an important part of the HPK dimer interface and which includes the autophosphorylated histidine. Other mutations were located in the junctions between the dimerization domain and the membrane receptor domain or the catalytic kinase domain. Interestingly, two previously identified constitutive variants of ComD, an SppK homologue involved in competence regulation in Streptococcus pneumoniae, contained single mutations in the same regions.
Keywords: Histidine kinase; Signal transduction; Lactobacillus ; Bacteriocin; Gene expression; Error-prone PCR
Gastric antibacterial efficiency is different for pepsin A and C by Sören Schreiber; Roland Bücker; Claudia Groll; Marina Azevedo-Vethacke; Peter Scheid; Sören Gatermann; Christine Josenhans; Sebastian Suerbaum (pp. 335-340).
The gastric lumen represents a bactericidal barrier, whose major components are an acidic pH and a family of isoenzymes of the gastric aspartate protease, pepsin. To evaluate whether specific pepsins are specialized in antibacterial protection, we tested their effects on the gastric pathogen Helicobacter pylori. In a recent study we found pepsin to affect the motility of the bacteria, one of its most important virulence factors. We were able to show that the antibacterial effect of pepsin occurs in two phases: rapid loss of motility and subsequent destruction. In the present study we used the rapid pepsin-induced bacterial immobilization as a marker of antibacterial efficiency. The proteolytic activity of different pepsins was normalized to values between 2 and 200 U/ml in the hemoglobin degradation test of Anson, performed at pH 2 and 5. We found that pepsin C completely inactivates H. pylori at proteolytic activities of 2 (pH 5) and 20 (pH 2) U/ml. In contrast, the activities of pepsin A and chymosin required to affect Helicobacter motility were ten times higher.
Keywords: Pepsin A; Pepsin C; Bactericidal barrier; Helicobacter motility