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Archives of Microbiology (v.191, #12)
Anaerobic metabolism of phenol in proteobacteria and further studies of phenylphosphate carboxylase by Sirko Schmeling; Georg Fuchs (pp. 869-878).
Anaerobic phenol metabolism was studied in three facultative aerobic denitrifying bacteria, Thauera aromatica, “Aromatoleum aromaticum” strain EbN1 (Betaproteobacteria), and Magnetospirillum sp. (Alphaproteobacterium). All species formed phenylphosphate and contained phenylphosphate carboxylase but not phenol carboxylase activity. This is in contrast to direct phenol carboxylation by fermenting bacteria. Antisera raised against subunits of the Thauera phenylphosphate synthase and phenylphosphate carboxylase partly cross-reacted with the corresponding proteins in the other species. Some unsolved features of phenylphosphate carboxylase were addressed in T. aromatica. The core sub-complex of this enzyme consists of three different subunits and catalyzes the exchange of 14CO2 with the carboxyl group of 4-hydroxybenzoate, but not phenylphosphate carboxylation. It was inactivated by oxygen or by the oxidizing agent thionin and fully reactivated under reducing conditions. The purified recombinant phosphatase subunit alone had only low phenylphosphate phosphatase activity in the absence of the other components. However, activity was strongly enhanced in the presence of the core enzyme resulting in phenylphosphate carboxylation. Hence, a tight interaction of the carboxylase subunits is required for dephosphorylation of phenylphosphate, which is coupled to the concomitant carboxylation of the produced phenolate to 4-hydroxybenzoate, thus preventing a futile cycle.
Keywords: Anaerobic metabolism of phenol; Phenylphosphate synthase; Phenylphosphate carboxylase; Thauera aromatica ; Aromatoleum aromaticum strain EbN1; Magnetospirillum sp. strain CC-26
Interaction of sortase A and lipase 2 in the inhibition of Staphylococcus aureus biofilm formation by Ning Xiong; Chunyan Hu; Yong Zhang; Shiyun Chen (pp. 879-884).
Recombinant sortase A (SrtA) was used to immune rabbit, and the inhibitory activity of anti-SrtA serum on Staphylococcus aureus biofilm formation was tested. Biofilm formation was inhibited by anti-SrtA rabbit serum in S. aureus ATCC25923 and two clinical isolated strains. The antiserum was separated into two fractions, and the main component with the inhibitory activity was demonstrated to be the IgG fraction. Two proteins interact with the IgG fraction were identified by using an in vitro pull-down assay and were confirmed to be lipase 2 and γ-hemolysin by mass spectrometry. Cross-interaction between SrtA and lipase 2 was further confirmed by Western blotting. Addition of anti-lipase 2 serum in the culture medium also showed inhibitory effect against biofilm formation. Together, our study suggests anti-SrtA serum inhibits S. aureus biofilm formation and lipase 2 is one of the targets of anti-SrtA serum in this inhibition process. This is the first study to demonstrate the roles of antisera against SrtA and lipase 2 in the inhibition of biofilm formation in S. aureus.
Keywords: Staphylococcus aureus ; Sortase A; Biofilm; Lipase 2
Mixotrophic metabolism in Burkholderia kururiensis subsp. thiooxydans subsp. nov., a facultative chemolithoautotrophic thiosulfate oxidizing bacterium isolated from rhizosphere soil and proposal for classification of the type strain of Burkholderia kururiensis as Burkholderia kururiensis subsp. kururiensis subsp. nov. by Rangasamy Anandham; Pandiyan Indira Gandhi; Soon Wo Kwon; Tong Min Sa; Yong Ki Kim; Hyeong Jin Jee (pp. 885-894).
A thiosulfate-oxidizing facultative chemolithoautotrophic Burkholderia sp. strain ATSB13T was previously isolated from rhizosphere soil of tobacco plant. Strain ATSB13T was aerobic, Gram-staining-negative, rod shaped and motile by means of sub-terminal flagellum. Strain ATSB13T exhibited mixotrophic growth in a medium containing thiosulfate plus acetate. A phylogenetic study based on 16S rRNA gene sequence analysis indicated that strain ATSB13T was most closely related to Burkholderia kururiensis KP23T (98.7%), Burkholderia tuberum STM678T (96.5%) and Burkholderia phymatum STM815T (96.4%). Chemotaxonomic data [G+C 64.0 mol%, major fatty acids, C18:1 ω7c (28.22%), C16:1 ω7c/15 iso 2OH (15.15%), and C16:0 (14.91%) and Q-8 as predominant respiratory ubiquinone] supported the affiliation of the strain ATSB13T within the genus Burkholderia. Though the strain ATSB13T shared high 16S rRNA gene sequence similarity with the type strain of B. kururiensis but considerably distant from the latter in terms of several phenotypic and chemotaxonomic characteristics. DNA–DNA hybridization between strain ATSB13T and B. kururiensis KP23T was 100%, and hence, it is inferred that strain ATSB13T is a member of B. kururiensis. On the basis of data obtained from this study, we propose that B. kururiensis be subdivided into B. kururiensis subsp. kururiensis subsp. nov. (type strain KP23T = JCM 10599T = DSM 13646T) and B. kururiensis subsp. thiooxydans subsp. nov. (type strain ATSB13T = KACC 12758T).
Keywords: Burkholderia kururiensis subsp. kururiensis ; Burkholderia kururiensis subsp. thiooxydans ; Mixotrophic growth; Reduced sulfur compounds oxidation ; Thiosulfate-oxidizing bacteria
Comparison of the metabolic activities of four wild-type Clostridium perfringens strains with their gatifloxacin-selected resistant mutants by Fatemeh Rafii; Miseon Park; Gonçalo Gamboa da Costa; Luisa Camacho (pp. 895-902).
The production of short-chain fatty acids, reductive enzymes, and hydrolytic enzymes by four gatifloxacin-selected, fluoroquinolone-resistant, mutant strains of C. perfringens, with stable mutations either in DNA gyrase or in both DNA gyrase and topoisomerase IV, was compared with that produced by the wild-type parent strains to investigate the effect of mutations associated with the selection of gatifloxacin resistance on bacterial metabolic activities. The mutants differed from their respective wild-type parent strains in the enzymatic activities of azoreductase, nitroreductase, and β-glucosidase and in the ratio of butyric acid to acetic acid production. Microarray analysis of one wild type and the corresponding mutant revealed different levels of mRNA expression for the enzymes involved in short-chain fatty acid (SCFA) synthesis and for β-glucosidase and oxidoreductases. In addition to mutations in the target genes, selection of resistance to gatifloxacin resulted in strain-specific physiological changes in the resistant mutants of C. perfringens that affected their metabolic activities.
Keywords: Clostridium perfringens ; Fluoroquinolone; Resistance; Azoreductase; Nitroreductase; Gatifloxacin
Identification, cloning, and functional characterization of EmrD-3, a putative multidrug efflux pump of the major facilitator superfamily from Vibrio cholerae O395 by Kenneth P. Smith; Sanath Kumar; Manuel F. Varela (pp. 903-911).
A putative multidrug efflux pump, EmrD-3, belonging to the major facilitator superfamily (MFS) of transporters and sharing homology with the Bcr/CflA subfamily, was identified in Vibrio cholerae O395. We cloned the emrD-3 gene and evaluated its role in antimicrobial efflux in a hypersensitive Escherichia coli strain. The efflux activity of this membrane protein resulted in lowering the intracellular concentration of ethidium. The recombinant plasmid carrying emrD-3 conferred enhanced resistance to several antimicrobials. Among the antimicrobials tested, the highest relative increase in minimum inhibitory concentration (MIC) of 102-fold was observed for linezolid (MIC = 256 μg/ml), followed by an 80.1-fold increase for tetraphenylphosphonium chloride (TPCL) (156.2 μg/ml), 62.5-fold for rifampin (MIC = 50 μg/ml), >30-fold for erythromycin (MIC = 50 μg/ml) and minocycline (MIC = 2 μg/ml), 20-fold for trimethoprim (MIC = 0.12 μg/ml), and 18.7-fold for chloramphenicol (MIC = 18.7 μg/ml). Among the fluorescent DNA-binding dyes, the highest relative increase in MIC of 41.7-fold was observed for ethidium bromide (125 μg/ml) followed by a 17.2-fold increase for rhodamine 6G (100 μg/ml). Thus, we demonstrate that EmrD-3 is a multidrug efflux pump of V. cholerae, the homologues of which are present in several Vibrio spp., some members of Enterobacteriaceae family, and Gram-positive Bacillus spp.
Keywords: EmrD-3; Efflux; Antimicrobial resistance; Vibrio cholerae ; Linezolid; MIC
Roles of PprA, IrrE, and RecA in the resistance of Deinococcus radiodurans to germicidal and environmentally relevant UV radiation by Anja Bauermeister; Esma Bentchikou; Ralf Moeller; Petra Rettberg (pp. 913-918).
To study the role of different DNA repair genes in the resistance of Deinococcus radiodurans to mono- and polychromatic UV radiation, wild-type strain and knockout mutants in RecA, PprA, and IrrE of D. radiodurans were irradiated with UV-C (254 nm), UV-(A + B) (280–400 nm) and UV-A (315–400 nm) radiation, and survival was monitored. The strain deficient in recA was highly sensitive to UV-C radiation compared to the wild-type, but showed no loss of resistance against irradiation with UV-(A + B) and UV-A, while pprA and irrE-deficient strains exhibited elevated sensitivity to UV-A and UV-(A + B) radiation. These results suggest that the repair of DNA double-strand breaks is essential after treatment with highly energetic UV-C radiation, whereas protection from oxidative stress may play a greater role in resistance to environmentally relevant UV radiation.
Keywords: Deinococcus radiodurans ; 254 nm UV-C; Environmental UV radiation; DNA repair
Proteolytic processing of Escherichia coli twin-arginine signal peptides by LepB by Iris Lüke; Jennifer I. Handford; Tracy Palmer; Frank Sargent (pp. 919-925).
The twin-arginine translocation (Tat) apparatus is a protein targeting system found in the cytoplasmic membranes of many prokaryotes. Substrate proteins of the Tat pathway are synthesised with signal peptides bearing SRRxFLK ‘twin-arginine’ amino acid motifs. All Tat signal peptides have a common tripartite structure comprising a polar N-terminal region, followed by a hydrophobic region of variable length and a polar C-terminal region. In Escherichia coli, Tat signal peptides are proteolytically cleaved after translocation. The signal peptide C-terminal regions contain conserved AxA motifs, which are possible recognition sequences for leader peptidase I (LepB). In this work, the role of LepB in Tat signal peptide processing was addressed directly. Deliberate repression of lepB expression prevented processing of all Tat substrates tested, including SufI, AmiC, and a TorA-23K reporter protein. In addition, electron microscopy revealed gross defects in cell architecture and membrane integrity following depletion of cellular LepB protein levels.
Keywords: Escherichia coli ; Tat protein transport pathway; Signal peptidase I; LepB protein