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New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
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Archives of Microbiology (v.189, #2)
The highly toxic oxyanion tellurite (TeO 3 2− ) enters the phototrophic bacterium Rhodobacter capsulatus via an as yet uncharacterized monocarboxylate transport system by Roberto Borghese; Daniele Marchetti; Davide Zannoni (pp. 93-100).
The facultative phototroph Rhodobacter capsulatus takes up the highly toxic oxyanion tellurite when grown under both photosynthetic and respiratory growth conditions. Previous works on Escherichia coli and R. capsulatus suggested that tellurite uptake occurred through a phosphate transporter. Here we present evidences indicating that tellurite enters R. capsulatus cells via a monocarboxylate transport system. Indeed, intracellular accumulation of tellurite was inhibited by the addition of monocarboxylates such as pyruvate, lactate and acetate, but not by dicarboxylates like malate or succinate. Acetate was the strongest tellurite uptake antagonist and this effect was concentration dependent, being already evident at 1 μM acetate. Conversely, tellurite at 100 μM was able to restrict the acetate entry into the cells. Both tellurite and acetate uptakes were energy dependent processes, since they were abolished by the protonophore FCCP and by the respiratory electron transport inhibitor KCN. Interestingly, cells grown on acetate, lactate or pyruvate showed a high level resistance to tellurite, whereas cells grown on malate or succinate proved to be very sensitive to the oxyanion. Taking these data together, we propose that: (a) tellurite enters R. capsulatus cells via an as yet uncharacterized monocarboxylate(s) transporter, (b) competition between acetate and tellurite results in a much higher level of tolerance against the oxyanion and (c) the toxic action of tellurite at the cytosolic level is significantly restricted by preventing tellurite uptake.
Keywords: Tellurite uptake; Monocarboxylate transport; Phototrophic bacteria; Rhodobacter capsulatus
Molecular diversity of 16S rRNA and gyrB genes in copper mines by Huaqun Yin; Linhui Cao; Guanzhou Qiu; Dianzuo Wang; Laurie Kellogg; Jizhong Zhou; Xinxing Liu; Zhimin Dai; Jiannan Ding; Xueduan Liu (pp. 101-110).
The molecular diversities of the microbial communities from four sites impacted by acid mine drainage (AMD) at Dexing Copper Mine in Jiangxi province of China were studied using 16S rRNA sequences and gyrB sequences. Of the four sampled sites, each habitat exhibited distinct geochemical characteristics and the sites were linked geographically allowing us to correlate microbial community structure to geochemical characteristics. In the present study, we examined the molecular diversity of 16S rRNA and gyrB genes from water at these sites using a PCR-based cloning approach. We found that the microbial community appears to be composed primarily of Proteobacteria, Acidobacteria, Actinobacteria, Nitrospira, Firmicutes, Chlorella and unknown phylotypes. Of clones affiliated with Nitrospira, Leptospirillum ferrooxidans, Leptospirillum ferriphilum and Leptospirillum group III were all detected. Principal-component analysis (PCA) revealed that the distribution of the microbial communities was influenced greatly by geochemical characteristics. The overall PCA profiles showed that the sites with similar geochemical characteristics had more similar microbial community structures. Moreover, our results also indicated that gyrB sequence analysis may be very useful for differentiating very closely related species in the study of microbial communities.
Keywords: Molecular diversity; 16S rRNA and gyrB gene; Geochemical characteristics; Principal-component analysis
Function of lanI in regulation of landomycin A biosynthesis in Streptomyces cyanogenus S136 and cross-complementation studies with Streptomyces antibiotic regulatory proteins encoding genes by Yuriy Rebets; Lilia Dutko; Bohdan Ostash; Andriy Luzhetskyy; Olexandr Kulachkovskyy; Toshio Yamaguchi; Tatsunosuke Nakamura; Andreas Bechthold; Victor Fedorenko (pp. 111-120).
The transcriptional regulator of landomycin A biosynthesis encoded by lanI gene has been inactivated within the chromosome of Streptomyces cyanogenus S136. The obtained mutant strain did not produce landomycin A and its known intermediates. Loss of landomycin A production caused significant changes in morphology of the lanI deficient strain. RT-PCR analysis confirmed complete cessation of transcription of certain lan genes, including lanJ (encoding putative proton dependent transporter) and lanK (presumably involved in lanJ expression regulation). Introduction of either lanI or lndI [lanI homologue controlling landomycin E biosynthesis in Streptomyces globisporus 1912, both encoding Streptomyces antibiotic regulatory proteins (SARPs)] restored landomycin A production in the mutant strain. Chimeric constructs ladI and ladR were generated by exchanging the DNA sequences corresponding to N- and C-terminal parts of LndI and LanI. None of these genes were able to activate the production of landomycins in regulatory mutants of S. cyanogenus and S. globisporus. Nevertheless, the production of novel unidentified compound was observed in the case of S. cyanogenus harboring ladI gene. Various genes encoding SARPs have been expressed in S. globisporus and S. cyanogenus regulatory mutants and the results of these complementation experiments are discussed.
Keywords: Streptomyces ; Antibiotics; Polyketide; SARP; Transcriptional regulators
Unusual location and characterization of Cu/Zn-containing superoxide dismutase from filamentous fungus Humicola lutea by Ekaterina Krumova; Alexander Dolashki; Svetlana Pashova; Pavlina Dolashka-Angelova; Stefan Stevanovic; Rumyana Hristova; Lilyana Stefanova; Wolfgang Voelter; Maria Angelova (pp. 121-130).
The present study aims to provide new information about the unusual location of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) in lower eukaryotes such as filamentous fungi. Humicola lutea, a high producer of SOD was used as a model system. Subcellular fractions [cytosol, mitochondrial matrix, and intermembrane space (IMS)] were isolated and tested for purity using activity measurements of typical marker enzymes. Evidence, based on electrophoretic mobility, sensitivity to KCN and H2O2 and immunoblot analysis supports the existence of Cu/Zn-SOD in mitochondrial IMS, and the Mn-SOD in the matrix. Enzyme activity is almost equally partitioned between both the compartments, thus suggesting that the intermembrane space could be one of the major sites of exposure to superoxide anion radicals. The mitochondrial Cu/Zn-SOD was purified and compared with the previously published cytosolic enzyme. They have identical molecular mass, cyanide- and H2O2-sensitivity, N-terminal amino acid sequence, glycosylation sites and carbohydrate composition. The H. lutea mitochondrial Cu/Zn-SOD is the first identified naturally glycosylated enzyme, isolated from IMS. These findings suggest that the same Cu/Zn-SOD exists in both the mitochondrial IMS and cytosol.
Keywords: Cu/Zn-superoxide dismutase; Intracellular location; Mitochondrial intermembrane space; Enzyme glycosylation; Protein sequencing
Expression of putative virulence factors in the potato pathogen Clavibacter michiganensis subsp. sepedonicus during infection by Ingrid Holtsmark; Gunnhild W. Takle; May Bente Brurberg (pp. 131-139).
The Gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus is the causal agent of bacterial wilt and ring rot of potato. So far, only two proteins have been shown to be essential for virulence, namely a plasmid-encoded cellulase CelA and a hypersensitive response-inducing protein. We have examined the relative expression of CelA and eight putative virulence factors during infection of potato and in liquid culture, using quantitative real-time PCR. The examined putative virulence genes were celB, a cellulase-encoding gene and genes encoding a pectate lyase, a xylanase and five homologues of the Clavibacter michiganensis subsp. michiganensis pathogenicity factor Pat-1 thought to encode a serine protease. Six of the nine assayed genes were up-regulated during infection of potato, including celA, celB, the xylanase gene, and two of the pat genes. The pectate lyase gene showed only slightly elevated expression, whereas three of the five examined pat genes were down-regulated during infection in potato. Interestingly, the two up-regulated pat genes showed a noticeable sequence difference compared to the three down-regulated pat genes. These results reveal several new proteins that are likely to be involved in Clavibacter michiganensis subsp. sepedonicus pathogenicity.
Keywords: Clavibacter ; Virulence factor; Infection; Gene expression
Transcript analysis of the Halothiobacillus neapolitanus cso operon by Fei Cai; Sabine Heinhorst; Jessup M. Shively; Gordon C. Cannon (pp. 141-150).
Carboxysomes are polyhedral microcompartments that sequester the CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase in many autotrophic bacteria. Their protein constituents are encoded by a set of tightly clustered genes that are thought to form an operon (the cso operon). This study is the first to systematically address transcriptional regulation of carboxysome protein expression. Quantification of transcript levels derived from the cso operon of Halothiobacillus neapolitanus, the sulfur oxidizer that has emerged as the model organism for carboxysome structural and functional studies, indicated that all cso genes are transcribed, albeit at different levels. Combined with comparative genomic evidence, this study supports the premise that the cso gene cluster constitutes an operon. Characterization of transcript 5′- and 3′-ends and examination of likely regulatory sequences and secondary structure elements within the operon suggested potential strategies by which the vastly different levels of individual carboxysome proteins in the microcompartment could have arisen.
Keywords: Carboxysome operon; Halothiobacillus neapolitanus ; Transcription; Real-time PCR
Alkaline phosphatase of Physarum polycephalum is insoluble by Kiyoshi Furuhashi (pp. 151-156).
The plasmodia of Physarum polycephalum grow as multinucleated cells in the presence of sufficient humidity and nutriment. Under non-illuminating conditions, stresses such as low temperature or high concentrations of salts transform the plasmodia into spherules whereas dehydration induces sclerotization. Some phosphatases including protein phosphatase and acid phosphatase have been purified from the plasmodia, but alkaline phosphatase remains to be elucidated. Phosphatase of the plasmodia, spherules and sclerotia was visualized by electrophoresis gel-staining assay using 5-bromo-4-chloro-3-indolyl phosphate. Insoluble fractions of the sclerotia were abundant in phosphatase activity. The phosphatase which was extracted by nonionic detergent was subjected to column chromatography and preparative electrophoresis. Purified phosphatase showed the highest activity at pH 8.8, indicating that this enzyme belongs to alkaline phosphatase. The apparent molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing condition was estimated to be 100 kDa whereas that under reducing was 105 kDa. An amount of 1% sodium dodecyl sulfate or 0.5 M NaCl had no effects on the activity although the phosphatase showed heat instability, Mg2+-dependency and sensitivity to 2-glycerophosphate or NaF. The extracting conditions and enzymatic properties suggest that this alkaline phosphatase which is in a membrane-bound form plays important roles in phosphate metabolism.
Keywords: Physarum polycephalum ; Myxomycetes; Slime mold; Plasmodia; Spherules; Sclerotia; Phosphatase
Characterisation of glutamine fructose-6-phosphate amidotransferase (EC 2.6.1.16) and N-acetylglucosamine metabolism in Bifidobacterium by Sophie Foley; Emilie Stolarczyk; Fadoua Mouni; Colette Brassart; Olivier Vidal; Eliane Aïssi; Stéphane Bouquelet; Frédéric Krzewinski (pp. 157-167).
Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial d-fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the d-fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism.
Keywords: Bifidobacterium ; GlmS; Glutamine fructose-6-phosphate amidotransferase; N-acetylglucosamine
High level of soluble expression in Escherichia coli and characterisation of the CyaA pore-forming fragment from a Bordetella pertussis Thai clinical isolate by Busaba Powthongchin; Chanan Angsuthanasombat (pp. 169-174).
Bordetella pertussis adenylate cyclase toxin-haemolysin (CyaA) can permeabilise erythrocytes by forming lytic pores. Here, a gene segment encoding CyaA pore-forming (CyaA-PF) domain cloned from genomic DNA of B. pertussis Thai isolate was over-expressed in Escherichia coli as a 126-kDa soluble protein which cross-reacted with anti-RTX monoclonal antibody. By co-expressing with acyltransferase CyaC, the CyaA-PF protein was found palmitoylated at Lys983. Unlike E. coli lysate with the non-acylated form, the lysate containing acylated CyaA-PF exhibited high haemolytic activity against sheep erythrocytes. This study presents that the recombinant CyaA-PF protein comprising pore-forming domain can be expressed separately as soluble native-folded precursor that conserves at least part of its functionality.
Keywords: Adenylate cyclase toxin; Acyltransferase; Bordetella pertussis ; Palmitoylation; Pore-forming toxin; Sheep erythrocyte
Colonization of broilers by Campylobacter jejuni internalized within Acanthamoeba castellanii by William J. Snelling; Norman J. Stern; Colm J. Lowery; John E. Moore; Emma Gibbons; Ciara Baker; James S. G. Dooley (pp. 175-179).
Although Campylobacter survives within amoeba in-vitro, it is unknown if intra-amoeba Campylobacter jejuni can colonize broilers. Five groups of 28 day-of-hatch chicks were placed into separate isolators. Groups (1) and (2) were challenged with page’s amoeba saline (PAS), and disinfected planktonic C. jejuni NCTC 11168, respectively. Groups (3), (4) and (5) were challenged with a C. jejuni positive control, C. jejuni in PAS, and intra-amoeba C. jejuni, respectively. After 1, 3, 7 and 14 days post challenge, seven birds from each unit were examined for C. jejuni colonization. For the first time we report that intra-amoeba C. jejuni colonized broilers.
Keywords: Campylobacter jejuni ; Broilers; Drinking water; Protozoa; Epidemiology
Lon protease promotes survival of Escherichia coli during anaerobic glucose starvation by Shen Luo; Megan McNeill; Timothy G. Myers; Robert J. Hohman; Rodney L. Levine (pp. 181-185).
In Escherichia coli, Lon is an ATP-dependent protease which degrades misfolded proteins and certain rapidly-degraded regulatory proteins. Given that oxidatively damaged proteins are generally degraded rather than repaired, we anticipated that Lon deficient cells would exhibit decreased viability during aerobic, but not anaerobic, carbon starvation. We found that the opposite actually occurs. Wild-type and Lon deficient cells survived equally well under aerobic conditions, but Lon deficient cells died more rapidly than the wild-type under anaerobiosis. Aerobic induction of the Clp family of ATP-dependent proteases could explain these results, but direct quantitation of Clp protein established that its level was not affected by Lon deficiency and overexpression of Clp did not rescue the cells under anaerobic conditions. We conclude that the Lon protease supports survival during anaerobic carbon starvation by a mechanism which does not depend on Clp.
Keywords: Lon protease; Clp protease; Anaerobiosis; Carbon starvation; Stationary phase survival