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Archives of Microbiology (v.194, #7)

Phylogeny and photoheterotrophy in the acidophilic phototrophic purple bacterium Rhodoblastus acidophilus by Megan L. Kempher; Michael T. Madigan (pp. 567-574).

Norbert Pfennig isolated the first acidophilic purple bacterium over 40 years ago and named the organism Rhodopseudomonas acidophila (now Rhodoblastus acidophilus). Since the original work of Pfennig, no systematic study has been conducted on the phylogeny and carbon nutrition of a collection of strains of Rbl. acidophilus. We have isolated six new strains of Rbl. acidophilus from a Canadian peat bog. These strains, three of the original Pfennig strains and two additional putative R. acidophilus strains isolated several years ago in this laboratory, were characterized as to their pigments, phylogeny, and carbon sources supporting photoheterotrophic growth. Phototrophic cultures were either purple or orange in color, and the color of a particular strain was linked to phylogeny. As for the Pfennig strains of Rbl. acidophilus, all new strains grew photoheterotrophically at pH 5 on a variety of organic and fatty acids. However, in addition to methanol and ethanol, the new strains as well as the Pfennig strains grew on several other primary alcohols, results not reported in the original species description. Our work shows that some phylogenetic and physiological diversity exists within the species Rbl. acidophilus and supports the observation that few species of acidophilic purple bacteria appear to exist in nature.

Keywords: Purple nonsulfur bacteria; Acidophiles; Rhodoblastus acidophilus

Isolation and identification of a bacteriocin with antibacterial and antibiofilm activity from Citrobacter freundii by Robert M. Q. Shanks; Aliza Dashiff; Jason S. Alster; Daniel E. Kadouri (pp. 575-587).

Multi- and pan-antibiotic-resistant bacteria are a major health challenge in hospital settings. Furthermore, when susceptible bacteria establish surface-attached biofilm populations, they become recalcitrant to antimicrobial therapy. Therefore, there is a need for novel antimicrobials that are effective against multi-drug-resistant and surface-attached bacteria. A screen to identify prokaryote-derived antimicrobials from a panel of over 100 bacterial strains was performed. One compound isolated from Citrobacter freundii exhibited antimicrobial activity against a wide range of Gram-negative bacteria and was effective against biofilms. Random transposon mutagenesis was performed to find mutants unable to produce the antimicrobial compound. Transposons mapped to a bacteriocin gene located on a small plasmid capable of replication in Escherichia coli. The plasmid was sequenced and found to be highly similar to a previously described colicinogenic plasmid. Expression of the predicted bacteriocin immunity gene conferred bacteriocin immunity to E. coli. The predicted bacteriocin gene, colA-43864, expressed in E. coli was sufficient to generate anti-microbial activity, and purified recombinant ColA-43864 was highly effective in killing E. coli, Citrobacter species, and Klebsiella pneumoniae cells in a planktonic and biofilm state. This study suggests that bacteriocins can be an effective way to control surface-attached pathogenic bacteria.

Keywords: Citrobacter freundii ; Bacteriocin; Antimicrobial; Biofilm

Mutagenesis and biochemical studies on AuaA confirmed the importance of the two conserved aspartate-rich motifs and suggested difference in the amino acids for substrate binding in membrane-bound prenyltransferases by Edyta Stec; Shu-Ming Li (pp. 589-595).

AuaA is a membrane-bound farnesyltransferase from the myxobacterium Stigmatella aurantiaca involved in the biosynthesis of aurachins. Like other known membrane-bound aromatic prenyltransferases, AuaA contains two conserved aspartate-rich motifs. Several amino acids in the first motif NXxxDxxxD were proposed to be responsible for prenyl diphosphate binding via metal ions like Mg²⁺. Site-directed mutagenesis experiments demonstrated in this study that asparagine, but not the arginine residue in NRxxDxxxD, is important for the enzyme activity of AuaA, differing from the importance of NQ or ND residues in the NQxxDxxxD or NDxxDxxxD motifs observed in some membrane-bound prenyltransferases. The second motif of known membrane-bound prenyltransferases was proposed to be involved in the binding of their aromatic substrates. KDIxDxEGD, also found in AuaA, had been previously speculated to be characteristic for binding of flavonoids or homogenisate. Site-directed mutagenesis experiments with AuaA showed that KDIxDxEGD was critical for the enzyme activity. However, this motif is very likely not specific for flavonoid or homogenisate prenyltransferases, because none of the tested flavonoids was accepted by AuaA or its mutant R53A in the presence of farnesyl, geranyl or dimethylallyl diphosphate.

Keywords: Aurachin biosynthesis; Farnesyltransferase; Membrane-bound prenyltransferase; Myxobacteria; Mutagenesis

Variable composition of heme oxygenases with different regiospecificities in Pseudomonas species by Bjoern Gisk; Jessica Wiethaus; Marco Aras; Nicole Frankenberg-Dinkel (pp. 597-606).

Heme oxygenases (HO) degrade heme yielding iron, carbon monoxide and one of four possible biliverdin (BV) isomers. Pseudomonas aeruginosa PAO1 is thus far the only organism to contain two HOs with different regiospecificities: BphO and PigA. While BphO cleaves heme to exclusively yield BV IXα, PigA produces the BV isomers IXβ and IXδ. We bioinformatically identified putative HOs in diverse Pseudomonas strains, tested their enzymatic functionality and determined their regiospecificity. Surprisingly, even high amino acid sequence identities to the P. aeruginosa HOs were not sufficient to correctly predict the HO regiospecificity in all cases. Based on our results, Pseudomonas strains differ in their HO composition containing either BphO or PigA or both HO types. Concomitantly with the existence of bphO is the occurrence of at least one gene encoding a bacterial phytochrome implying that only BV IXα is the sufficient phytochrome chromophore. In contrast, pigA genes are organized in gene clusters associated with iron utilization implying a role of PigA in iron acquisition. However, at least in strains containing no PigA this function maybe fulfilled by BphO. Only a combination of homology searches and analyses of genetic environments is appropriate for a reliable prediction of the regiospecificity of Pseudomonas HOs.

Keywords: Biliverdin; Heme oxygenase; Pseudomonas ; Phytochrome; Iron acquisition

Characteristics and distribution of plasmids in a clonally diverse set of methicillin-resistant Staphylococcus aureus strains by Lucie Kuntová; Roman Pantůček; Jana Rájová; Vladislava Růžičková; Petr Petráš; Ivana Mašlaňová; Jiří Doškař (pp. 607-614).

The aim of this study was to compare the plasmid contents of methicillin-resistant Staphylococcus aureus (MRSA) strains classified into different clonal clusters (CCs). The isolates were collected from 15 Czech hospitals in 2000–2008. Plasmid DNA was detected in 65 (89%) strains, and 33 of them harbored more than one plasmid type. Altogether 24 different types of plasmids were identified, ranging in size from 1.3 to 55 kb. Restriction endonuclease analysis, plasmid elimination, DNA hybridization, and sequencing were used for their further characterization. It has been found that the conjugative, erythromycin resistance and enterotoxin D encoding plasmids are harbored by strains from different CCs. On the other hand, chloramphenicol and tetracycline resistance plasmids, and most of the penicillinase and cryptic plasmids were only detected in certain CCs. Especially, the pUSA300-like plasmids were found exclusively in the USA300 clone strains. The high diversity in plasmid content detected in the study strains implies that plasmids play a major role in evolution of MRSA clonal lineages.

Keywords: Methicillin-resistant Staphylococcus aureus ; Plasmids; Genetic diversity; Antibiotic resistance; Molecular typing

Identification of N-acyl homoserine lactones produced by Gluconacetobacter diazotrophicus PAL5 cultured in complex and synthetic media by Carlos G. Nieto-Peñalver; Elisa V. Bertini; Lucía I. C. de Figueroa (pp. 615-622).

The endophytic diazotrophic Gluconacetobacter diazotrophicus PAL5 was originally isolated from sugarcane (Saccharum officinarum). The biological nitrogen fixation, phytohormones secretion, solubilization of mineral nutrients and phytopathogen antagonism allow its classification as a plant growth-promoting bacterium. The recent genomic sequence of PAL5 unveiled the presence of a quorum sensing (QS) system. QS are regulatory mechanisms that, through the production of signal molecules or autoinducers, permit a microbial population the regulation of the physiology in a coordinated manner. The most studied autoinducers in gram-negative bacteria are the N-acyl homoserine lactones (AHLs). The usage of biosensor strains evidenced the presence of AHL-like molecules in cultures of G. diazotrophicus PAL5 grown in complex and synthetic media. Analysis of AHLs performed by LC-APCI-MS permitted the identification of eight different signal molecules, including C6-, C8-, C10-, C12- and C14-HSL. Mass spectra confirmed that this diazotrophic strain also synthesizes autoinducers with carbonyl substitutions in the acyl chain. No differences in the profile of AHLs could be determined under both culture conditions. However, although the level of short-chain AHLs was not affected, a decrease of 30% in the production of long-chain AHLs could be measured in synthetic medium.

Keywords: Gluconacetobacter diazotrophicus ; Quorum sensing; N-Acyl homoserine lactone; Autoinducer; Diazotrophic

New clusters of arsenite oxidase and unusual bacterial groups in enrichments from arsenic-contaminated soil by Munawar Sultana; Susann Vogler; Kamrun Zargar; Anne-Christine Schmidt; Chad Saltikov; Jana Seifert; Michael Schlömann (pp. 623-635).

In the present study cultivation-dependent and molecular methods were applied in combination to investigate the arsenite-oxidizing communities in enrichment cultures from arsenic and lead smelter-impacted soils with respect to both 16S rRNA and arsenite oxidase gene diversity. Enrichments with arsenite as the only electron donor resulted in completely different communities than enrichments with yeast extract and the simultaneous presence of arsenite. The lithoautotrophic community appeared to be dominated by Ferrimicrobium-related Actinobacteria, unusual Acidobacteria, Myxobacteria, and α-Proteobacteria but the heterotrophic community comprised many Dokdonella-related γ-Proteobacteria. Gene sequences of clones encoding arsenite oxidase from the enrichment for lithoautotrophs belonged to three major clusters with sequences from non-cultivated microorganisms. So, primers used to detect arsenite oxidase genes could amplify the genes from many α-, β- and γ-Proteobacteria, but not from various strains of the other phyla present in the enrichment for lithotrophs. This was also observed for the isolates where arsenite oxidase genes from new proteobacterial isolates of the genera Burkholderia, Bosea, Alcaligenes, Bradyrhizobium and Methylobacterium could be amplified but the genes of the new Rhodococcus isolate S43 could not. The results indicate that the ability to oxidize arsenite is widespread in various unusual taxa, and molecular methods for their detection require further improvement.

Keywords: Smelter-impacted soil; Arsenite-oxidizing bacteria; Rhodococcus ; Arsenite oxidase gene diversity; Microbial community

The inner membrane protein, YhiM, is necessary for Escherichia coli (E. coli) survival in acidic conditions by Tuan M. Nguyen; Rebecca L. Sparks-Thissen (pp. 637-641).

Escherichia coli must be able to survive extreme acidic conditions. We were interested in determining the role of the inner membrane protein YhiM in survival in acidic conditions. Previous data demonstrated that the yhiM gene was upregulated in acidic conditions (Tucker et al. in J Bacteriol. 184:6551–6558, 2002). We therefore tested tn10 insertions into the yhiM gene for their ability to survive at low pH (pH 2.5). We show that YhiM was required for survival at pH 2.5. We also tested the YhiM dependence of the different acid resistance pathways. YhiM was required for the RpoS, glutamine and lysine–dependent acid resistance pathways. In contrast, YhiM was not required for the arginine-dependent acid resistance pathway.

Keywords: E. coli ; Acid resistance; Inner membrane protein; Acid stress

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Archives of Microbiology (v.194, #7)