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  • 1
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    Online Resource
    SmoXYB1C1Z of Mycobacterium sp. Strain NBB4: a Soluble Methane Monooxygenase (sMMO)-Like Enzyme, Active on C 2 to C 4 Alkanes and Alkenes
    American Society for Microbiology ; 2014
    In:  Applied and Environmental Microbiology Vol. 80, No. 18 ( 2014-09-15), p. 5801-5806
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 80, No. 18 ( 2014-09-15), p. 5801-5806
    Abstract: Monooxygenase (MO) enzymes initiate the aerobic oxidation of alkanes and alkenes in bacteria. A cluster of MO genes ( smoXYB1C1Z ) of thus-far-unknown function was found previously in the genomes of two Mycobacterium strains (NBB3 and NBB4) which grow on hydrocarbons. The predicted Smo enzymes have only moderate amino acid identity (30 to 60%) to their closest homologs, the soluble methane and butane MOs (sMMO and sBMO), and the smo gene cluster has a different organization from those of sMMO and sBMO. The smoXYB1C1Z genes of NBB4 were cloned into pMycoFos to make pSmo, which was transformed into Mycobacterium smegmatis mc 2 -155. Cells of mc 2 -155(pSmo) metabolized C 2 to C 4 alkanes, alkenes, and chlorinated hydrocarbons. The activities of mc 2 -155(pSmo) cells were 0.94, 0.57, 0.12, and 0.04 nmol/min/mg of protein with ethene, ethane, propane, and butane as substrates, respectively. The mc 2 -155(pSmo) cells made epoxides from ethene, propene, and 1-butene, confirming that Smo was an oxygenase. Epoxides were not produced from larger alkenes (1-octene and styrene). Vinyl chloride and 1,2-dichloroethane were biodegraded by cells expressing Smo, with production of inorganic chloride. This study shows that Smo is a functional oxygenase which is active against small hydrocarbons. M. smegmatis mc 2 -155(pSmo) provides a new model for studying sMMO-like monooxygenases.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.01338-14
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
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    detail.hit.zdb_id: 1478346-0
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  • 2
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    Role of IncP-1β Plasmids pWDL7:: rfp and pNB8c in Chloroaniline Catabolism as Determined by Genomic and Functional Analyses
    American Society for Microbiology ; 2012
    In:  Applied and Environmental Microbiology Vol. 78, No. 3 ( 2012-02), p. 828-838
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 78, No. 3 ( 2012-02), p. 828-838
    Abstract: Broad-host-range catabolic plasmids play an important role in bacterial degradation of man-made compounds. To gain insight into the role of these plasmids in chloroaniline degradation, we determined the first complete nucleotide sequences of an IncP-1 chloroaniline degradation plasmid, pWDL7:: rfp and its close relative pNB8c, as well as the expression pattern, function, and bioaugmentation potential of the putative 3-chloroaniline (3-CA) oxidation genes. Based on phylogenetic analysis of backbone proteins, both plasmids are members of a distinct clade within the IncP-1β subgroup. The plasmids are almost identical, but whereas pWDL7:: rfp carries a duplicate inverted catabolic transposon, Tn 6063 , containing a putative 3-CA oxidation gene cluster, dcaQTA1A2BR , pNB8c contains only a single copy of the transposon. No genes for an aromatic ring cleavage pathway were detected on either plasmid, suggesting that only the upper 3-CA degradation pathway was present. The dcaA1A2B gene products expressed from a high-copy-number vector were shown to convert 3-CA to 4-chlorocatechol in Escherichia coli . Slight differences in the dca promoter region between the plasmids and lack of induction of transcription of the pNB8c dca genes by 3-CA may explain previous findings that pNB8C does not confer 3-CA transformation. Bioaugmentation of activated sludge with pWDL7:: rfp accelerated removal of 3-CA, but only in the presence of an additional carbon source. Successful bioaugmentation requires complementation of the upper pathway genes with chlorocatechol cleavage genes in indigenous bacteria. The genome sequences of these plasmids thus help explain the molecular basis of their catabolic activities.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.07480-11
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2012
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
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  • 3
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    ExpR Coordinates the Expression of Symbiotically Important, Bundle-Forming Flp Pili with Quorum Sensing in Sinorhizobium meliloti
    American Society for Microbiology ; 2014
    In:  Applied and Environmental Microbiology Vol. 80, No. 8 ( 2014-04-15), p. 2429-2439
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 80, No. 8 ( 2014-04-15), p. 2429-2439
    Abstract: Type IVb pili in enteropathogenic bacteria function as a host colonization factor by mediating tight adherence to host cells, but their role in bacterium-plant symbiosis is currently unknown. The genome of the symbiotic soil bacterium Sinorhizobium meliloti contains two clusters encoding proteins for type IVb pili of the Flp (fimbrial low-molecular-weight protein) subfamily. To establish the role of Flp pili in the symbiotic interaction of S. meliloti and its host, Medicago sativa , we deleted pilA1 , which encodes the putative pilin subunit in the chromosomal flp-1 cluster and conducted competitive nodulation assays. The pilA1 deletion strain formed 27% fewer nodules than the wild type. Transmission electron microscopy revealed the presence of bundle-forming pili protruding from the polar and lateral region of S. meliloti wild-type cells. The putative pilus assembly ATPase CpaE1 fused to mCherry showed a predominantly unilateral localization. Transcriptional reporter gene assays demonstrated that expression of pilA1 peaks in early stationary phase and is repressed by the quorum-sensing regulator ExpR, which also controls production of exopolysaccharides and motility. Binding of acyl homoserine lactone-activated ExpR to the pilA1 promoter was confirmed with electrophoretic mobility shift assays. A 17-bp consensus sequence for ExpR binding was identified within the 28-bp protected region by DNase I footprinting analyses. Our results show that Flp pili are important for efficient symbiosis of S. meliloti with its plant host. The temporal inverse regulation of exopolysaccharides and pili by ExpR enables S. meliloti to achieve a coordinated expression of cellular processes during early stages of host interaction.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.04088-13
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
    detail.hit.zdb_id: 223011-2
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  • 4
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    Utility of the Clostridial Site-Specific Recombinase TnpX To Clone Toxic-Product-Encoding Genes and Selectively Remove Genomic DNA Fragments
    American Society for Microbiology ; 2014
    In:  Applied and Environmental Microbiology Vol. 80, No. 12 ( 2014-06-15), p. 3597-3603
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 80, No. 12 ( 2014-06-15), p. 3597-3603
    Abstract: TnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn 4451 and Tn 4453 in Clostridium perfringens and Clostridium difficile , respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids in Escherichia coli and from marked chromosomal C. perfringens mutants. This methodology enabled the construction of a C. perfringens plc virR double mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic to E. coli . We show that TnpX represses expression from its own promoter, P attCI , which can be exploited to facilitate the cloning of recalcitrant genes in E. coli for subsequent expression in the heterologous host C. perfringens . Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.04285-13
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
    detail.hit.zdb_id: 223011-2
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  • 5
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    High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage
    American Society for Microbiology ; 2014
    In:  Applied and Environmental Microbiology Vol. 80, No. 13 ( 2014-07), p. 3826-3834
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 80, No. 13 ( 2014-07), p. 3826-3834
    Abstract: Genetic modifications of bacterial chromosomes are important for both fundamental and applied research. In this study, we developed an efficient, easy-to-use system for genetic modification of the Escherichia coli chromosome, a two-plasmid method involving lambda Red (λ-Red) recombination and I-SceI cleavage. An intermediate strain is generated by integration of a resistance marker gene(s) and I-SceI recognition sites in or near the target gene locus, using λ-Red PCR targeting. The intermediate strain is transformed with a donor plasmid carrying the target gene fragment with the desired modification flanked by I-SceI recognition sites, together with a bifunctional helper plasmid for λ-Red recombination and I-SceI endonuclease. I-SceI cleavage of the chromosome and the donor plasmid allows λ-Red recombination between chromosomal breaks and linear double-stranded DNA from the donor plasmid. Genetic modifications are introduced into the chromosome, and the placement of the I-SceI sites determines the nature of the recombination and the modification. This method was successfully used for cadA knockout, gdhA knock-in, seamless deletion of pepD , site-directed mutagenesis of the essential metK gene, and replacement of metK with the Rickettsia S -adenosylmethionine transporter gene. This effective method can be used with both essential and nonessential gene modifications and will benefit basic and applied genetic research.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.00313-14
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
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  • 6
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    Revision of N 2 O-Producing Pathways in the Ammonia-Oxidizing Bacterium Nitrosomonas europaea ATCC 19718
    American Society for Microbiology ; 2014
    In:  Applied and Environmental Microbiology Vol. 80, No. 16 ( 2014-08-15), p. 4930-4935
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 80, No. 16 ( 2014-08-15), p. 4930-4935
    Abstract: Nitrite reductase (NirK) and nitric oxide reductase (NorB) have long been thought to play an essential role in nitrous oxide (N 2 O) production by ammonia-oxidizing bacteria. However, essential gaps remain in our understanding of how and when NirK and NorB are active and functional, putting into question their precise roles in N 2 O production by ammonia oxidizers. The growth phenotypes of the Nitrosomonas europaea ATCC 19718 wild-type and mutant strains deficient in expression of NirK, NorB, and both gene products were compared under atmospheric and reduced O 2 tensions. Anoxic resting-cell assays and instantaneous nitrite (NO 2 − ) reduction experiments were done to assess the ability of the wild-type and mutant N. europaea strains to produce N 2 O through the nitrifier denitrification pathway. Results confirmed the role of NirK for efficient substrate oxidation of N. europaea and showed that NorB is involved in N 2 O production during growth at both atmospheric and reduced O 2 tensions. Anoxic resting-cell assays and measurements of instantaneous NO 2 − reduction using hydrazine as an electron donor revealed that an alternate nitrite reductase to NirK is present and active. These experiments also clearly demonstrated that NorB was the sole nitric oxide reductase for nitrifier denitrification. The results of this study expand the enzymology for nitrogen metabolism and N 2 O production by N. europaea and will be useful to interpret pathways in other ammonia oxidizers that lack NirK and/or NorB genes.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.01061-14
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
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  • 7
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    Uptake and Metabolism of N -Acetylglucosamine and Glucosamine by Streptococcus mutans
    American Society for Microbiology ; 2014
    In:  Applied and Environmental Microbiology Vol. 80, No. 16 ( 2014-08-15), p. 5053-5067
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 80, No. 16 ( 2014-08-15), p. 5053-5067
    Abstract: Glucosamine and N -acetylglucosamine are among the most abundant sugars on the planet, and their introduction into the oral cavity via the diet and host secretions, and through bacterial biosynthesis, provides oral biofilm bacteria with a source of carbon, nitrogen, and energy. In this study, we demonstrated that the dental caries pathogen Streptococcus mutans possesses an inducible system for the metabolism of N -acetylglucosamine and glucosamine. These amino sugars are transported by the phosphoenolpyruvate:sugar phosphotransferase system (PTS), with the glucose/mannose enzyme II permease encoded by manLMN playing a dominant role. Additionally, a previously uncharacterized gene product encoded downstream of the manLMN operon, ManO, was shown to influence the efficiency of uptake and growth on N -acetylglucosamine and, to a lesser extent, glucosamine. A transcriptional regulator, designated NagR, was able to bind the promoter regions in vitro , and repress the expression in vivo , of the nagA and nagB genes, encoding N -acetylglucosamine-6-phosphate deacetylase and glucosamine-6-phosphate deaminase, respectively. The binding activity of NagR could be inhibited by glucosamine-6-phosphate in vitro . Importantly, in contrast to the case with certain other Firmicutes , the gene for de novo synthesis of glucosamine-6-phosphate in S. mutans , glmS , was also shown to be regulated by NagR, and NagR could bind the glmS promoter region in vitro . Finally, metabolism of these amino sugars by S. mutans resulted in the production of significant quantities of ammonia, which can neutralize cytoplasmic pH and increase acid tolerance, thus contributing to enhanced persistence and pathogenic potential.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.00820-14
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
    detail.hit.zdb_id: 223011-2
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  • 8
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    Surface Expression of ω-Transaminase in Escherichia coli
    American Society for Microbiology ; 2014
    In:  Applied and Environmental Microbiology Vol. 80, No. 7 ( 2014-04), p. 2293-2298
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 80, No. 7 ( 2014-04), p. 2293-2298
    Abstract: Chiral amines are important for the chemical and pharmaceutical industries, and there is rapidly growing interest to use transaminases for their synthesis. Since the cost of the enzyme is an important factor for process economy, the use of whole-cell biocatalysts is attractive, since expensive purification and immobilization steps can be avoided. Display of the protein on the cell surface provides a possible way to reduce the mass transfer limitations of such biocatalysts. However, transaminases need to dimerize in order to become active, and furthermore, they require the cofactor pyridoxal phosphate; consequently, successful transaminase surface expression has not been reported thus far. In this work, we produced an Arthrobacter citreus ω-transaminase in Escherichia coli using a surface display vector based on the autotransporter a dhesin i nvolved in d iffuse a dherence (AIDA-I), which has previously been used for display of dimeric proteins. The correct localization of the transaminase in the E. coli outer membrane and its orientation toward the cell exterior were verified. Furthermore, transaminase activity was detected exclusively in the outer membrane protein fraction, showing that successful dimerization had occurred. The transaminase was found to be present in both full-length and proteolytically degraded forms. The removal of this proteolysis is considered to be the main obstacle to achieving sufficient whole-cell transaminase activity.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.03678-13
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
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  • 9
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    HrcT Is a Key Component of the Type III Secretion System in Xanthomonas spp. and Also Regulates the Expression of the Key hrp Transcriptional Activator HrpX
    American Society for Microbiology ; 2014
    In:  Applied and Environmental Microbiology Vol. 80, No. 13 ( 2014-07), p. 3908-3919
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 80, No. 13 ( 2014-07), p. 3908-3919
    Abstract: The type III secretion system (T3SS), encoded by hrp (hypersensitive response and pathogenicity) genes in Gram-negative phytopathogenic bacteria, delivers repertoires of T3SS effectors (T3SEs) into plant cells to trigger the hypersensitive response (HR) in nonhost or resistant-host plants and promote pathogenicity in susceptible plants. The expression of hrp genes in Xanthomonas is regulated by two key regulatory proteins, HrpG and HrpX. However, the interactions between hrp gene products in directing T3SE secretion are largely unknown. Here we demonstrated that HrcT of X. oryzae pv. oryzicola functions as a T3SS component and positively regulates the expression of hrpX . Transcription of hrcT occurs via two distinct promoters; one (T1) is with the hrpB operon and the second (T3) within hrpB7 Via either promoter T1 or T3, the defect in Hrp phenotype by hrcT deletion was corrected in the presence of hrcT only from Xanthomonas species but not from other phytopathogenic bacteria. An N-terminally truncated HrcT was able to bind the hrpX promoter and activate the expression of hrpX , supporting that HrcT is a positive regulator of hrpX . A revised model showing the regulatory interactions between HrcT, HrpX, and HrpG is proposed.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.00308-14
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
    detail.hit.zdb_id: 223011-2
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  • 10
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    Involvement of the Global Regulator GlxR in 3-Hydroxybenzoate and Gentisate Utilization by Corynebacterium glutamicum
    American Society for Microbiology ; 2014
    In:  Applied and Environmental Microbiology Vol. 80, No. 14 ( 2014-07-15), p. 4215-4225
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 80, No. 14 ( 2014-07-15), p. 4215-4225
    Abstract: Corynebacterium glutamicum is an industrially important producer of amino acids and organic acids, as well as an emerging model system for aromatic assimilation. An IclR-type regulator GenR has been characterized to activate the transcription of genDFM and genKH operons for 3-hydroxybenzoate and gentisate catabolism and represses its own expression. On the other hand, GlxR, a global regulator of the cyclic AMP (cAMP) receptor protein-fumarate nitrate reductase regulator (CRP-FNR) type, was also predicted to be involved in this pathway. In this study, electrophoretic mobility shift assays and footprinting analyses demonstrated that GlxR bound to three sites in the promoter regions of three gen operons. A combination of site-directed mutagenesis of the biding sites, promoter activity assay, and GlxR overexpression demonstrated that GlxR repressed their expression by binding these sites. One GlxR binding site (DFMx) was found to be located −13 to +8 bp upstream of the genDFM promoter, which was involved in negative regulation of genDFM transcription. The GlxR binding site R-KHx01 (located between positions −11 to +5) was upstream of the genKH promoter sequence and involved in negative regulation of its transcription. The binding site R-KHx02, at which GlxR binds to genR promoter to repress its expression, was found within a footprint extending from positions −71 to −91 bp. These results reveal that GlxR represses the transcription of all three gen operons and then contributes to the synchronization of their expression for 3-hydroxybenzoate and gentisate catabolism in collaboration with the specific regulator GenR.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.00290-14
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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