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1.
J Biotechnol ; 152(3): 77-86, 2011 Mar 20.
Article in English | MEDLINE | ID: mdl-21320555

ABSTRACT

The alcohol dehydrogenase gene adhA in Corynebacterium glutamicum is subject to a complex carbon source-dependent regulation mediated by RamA, RamB and GlxR. In this study we identified SucR as the fourth transcriptional regulator involved in expression control of the adhA gene. SucR specifically binds to the adhA promoter and acts as transcriptional repressor independent of the carbon source used. Furthermore, we found that SucR negatively controls the expression of its own gene. This negative autoregulation is mediated by binding of SucR to at least one of four identified binding sites located in the promoter region of sucR. EMSA experiments and subsequent sequence analysis led to the identification of the SucR consensus binding sequence YYAACAWMAW. This binding motif is different from the binding site (ACTCTAGGGG) recently described for SucR in the promoter region of the sucCD operon. However, we were not able to detect a specific interaction of purified SucR protein with this motif present in the sucCD promoter region.


Subject(s)
Alcohol Dehydrogenase/genetics , Bacterial Proteins/genetics , Corynebacterium glutamicum/enzymology , Corynebacterium glutamicum/genetics , Gene Expression Regulation, Bacterial , Alcohol Dehydrogenase/metabolism , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Base Sequence , Binding Sites , DNA, Bacterial/genetics , Gene Silencing , Genes, Bacterial/genetics , Genetic Loci/genetics , Molecular Sequence Data , Plasmids/genetics , Promoter Regions, Genetic/genetics , Protein Binding , Transcription, Genetic
2.
Microbiology (Reading) ; 155(Pt 1): 150-164, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19118356

ABSTRACT

The gene products of the rbsRACBD (rbs) operon of C. glutamicum (cg1410-cg1414) encode a ribose-specific ATP-binding cassette (ABC) transport system and its corresponding regulatory protein (RbsR). Deletion of the structural genes rbsACBD prohibited ribose uptake. Deletion of the regulatory gene rbsR resulted in an increased mRNA level of the whole operon. Analysis of the promoter region of the rbs operon by electrophoretic mobility shift assays identified a catabolite-responsive element (cre)-like sequence as the RbsR-binding site. Additional RbsR-binding sites were identified in front of the recently characterized uriR operon (uriR-rbsK1-uriT-uriH) and the ribokinase gene rbsK2. In vitro, the repressor RbsR bound to its targets in the absence of an effector. A probable negative effector of RbsR in vivo is ribose 5-phosphate or a derivative thereof, since in a ribokinase (rbsK1 rbsK2) double mutant, no derepression of the rbs operon in the presence of ribose was observed. Analysis of the ribose stimulon in the C. glutamicum wild-type revealed transcriptional induction of the uriR and rbs operons as well as of the rbsK2 gene. The inconsistency between the existence of functional RbsR-binding sites upstream of the ribokinase genes, their transcriptional induction during growth on ribose, and the missing induction in the rbsR mutant suggested the involvement of a second transcriptional regulator. Simultaneous deletion of the regulatory genes rbsR and uriR finally demonstrated a transcriptional co-control of the rbs and uriR operons and the rbsK2 gene by both regulators, RbsR and UriR, which were furthermore shown to recognize the same cognate DNA sequences in the operators of their target genes.


Subject(s)
Bacterial Proteins/metabolism , Corynebacterium glutamicum/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Bacterial , Repressor Proteins/metabolism , Ribose/metabolism , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Binding Sites , Biological Transport , Corynebacterium glutamicum/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Gene Deletion , Molecular Sequence Data , Operon , Repressor Proteins/chemistry , Repressor Proteins/genetics
3.
BMC Genomics ; 9: 449, 2008 Sep 30.
Article in English | MEDLINE | ID: mdl-18826580

ABSTRACT

BACKGROUND: Bordetella petrii is the only environmental species hitherto found among the otherwise host-restricted and pathogenic members of the genus Bordetella. Phylogenetically, it connects the pathogenic Bordetellae and environmental bacteria of the genera Achromobacter and Alcaligenes, which are opportunistic pathogens. B. petrii strains have been isolated from very different environmental niches, including river sediment, polluted soil, marine sponges and a grass root. Recently, clinical isolates associated with bone degenerative disease or cystic fibrosis have also been described. RESULTS: In this manuscript we present the results of the analysis of the completely annotated genome sequence of the B. petrii strain DSMZ12804. B. petrii has a mosaic genome of 5,287,950 bp harboring numerous mobile genetic elements, including seven large genomic islands. Four of them are highly related to the clc element of Pseudomonas knackmussii B13, which encodes genes involved in the degradation of aromatics. Though being an environmental isolate, the sequenced B. petrii strain also encodes proteins related to virulence factors of the pathogenic Bordetellae, including the filamentous hemagglutinin, which is a major colonization factor of B. pertussis, and the master virulence regulator BvgAS. However, it lacks all known toxins of the pathogenic Bordetellae. CONCLUSION: The genomic analysis suggests that B. petrii represents an evolutionary link between free-living environmental bacteria and the host-restricted obligate pathogenic Bordetellae. Its remarkable metabolic versatility may enable B. petrii to thrive in very different ecological niches.


Subject(s)
Bordetella/genetics , Bordetella/metabolism , Bordetella/pathogenicity , Genome, Bacterial , Bacterial Proteins/genetics , Base Composition , Biological Evolution , Bordetella bronchiseptica/genetics , Bordetella parapertussis/genetics , Bordetella pertussis/genetics , Chromosomes, Bacterial , Genes, Bacterial , Genomic Library , Interspersed Repetitive Sequences , Molecular Sequence Data , Synteny , Virulence/genetics , Virulence Factors, Bordetella/genetics
4.
J Bacteriol ; 190(6): 2138-49, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18192381

ABSTRACT

Clavibacter michiganensis subsp. michiganensis is a plant-pathogenic actinomycete that causes bacterial wilt and canker of tomato. The nucleotide sequence of the genome of strain NCPPB382 was determined. The chromosome is circular, consists of 3.298 Mb, and has a high G+C content (72.6%). Annotation revealed 3,080 putative protein-encoding sequences; only 26 pseudogenes were detected. Two rrn operons, 45 tRNAs, and three small stable RNA genes were found. The two circular plasmids, pCM1 (27.4 kbp) and pCM2 (70.0 kbp), which carry pathogenicity genes and thus are essential for virulence, have lower G+C contents (66.5 and 67.6%, respectively). In contrast to the genome of the closely related organism Clavibacter michiganensis subsp. sepedonicus, the genome of C. michiganensis subsp. michiganensis lacks complete insertion elements and transposons. The 129-kb chp/tomA region with a low G+C content near the chromosomal origin of replication was shown to be necessary for pathogenicity. This region contains numerous genes encoding proteins involved in uptake and metabolism of sugars and several serine proteases. There is evidence that single genes located in this region, especially genes encoding serine proteases, are required for efficient colonization of the host. Although C. michiganensis subsp. michiganensis grows mainly in the xylem of tomato plants, no evidence for pronounced genome reduction was found. C. michiganensis subsp. michiganensis seems to have as many transporters and regulators as typical soil-inhabiting bacteria. However, the apparent lack of a sulfate reduction pathway, which makes C. michiganensis subsp. michiganensis dependent on reduced sulfur compounds for growth, is probably the reason for the poor survival of C. michiganensis subsp. michiganensis in soil.


Subject(s)
Actinobacteria/genetics , DNA, Bacterial/genetics , Genome, Bacterial , Solanum lycopersicum/microbiology , Actinobacteria/pathogenicity , Base Composition/genetics , DNA, Bacterial/chemistry , Electrophoresis, Gel, Pulsed-Field , Genes, Bacterial/genetics , Genomic Islands/genetics , Models, Genetic , Molecular Sequence Data , Operon/genetics , Plasmids/genetics , Sequence Analysis, DNA , Serine Endopeptidases/genetics
5.
BMC Mol Biol ; 8: 104, 2007 Nov 15.
Article in English | MEDLINE | ID: mdl-18005413

ABSTRACT

BACKGROUND: The major uptake system responsible for the transport of fructose, glucose, and sucrose in Corynebacterium glutamicum ATCC 13032 is the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The genes encoding PTS components, namely ptsI, ptsH, and ptsF belong to the fructose-PTS gene cluster, whereas ptsG and ptsS are located in two separate regions of the C. glutamicum genome. Due to the localization within and adjacent to the fructose-PTS gene cluster, two genes coding for DeoR-type transcriptional regulators, cg2118 and sugR, are putative candidates involved in the transcriptional regulation of the fructose-PTS cluster genes. RESULTS: Four transcripts of the extended fructose-PTS gene cluster that comprise the genes sugR-cg2116, ptsI, cg2118-fruK-ptsF, and ptsH, respectively, were characterized. In addition, it was shown that transcription of the fructose-PTS gene cluster is enhanced during growth on glucose or fructose when compared to acetate. Subsequently, the two genes sugR and cg2118 encoding for DeoR-type regulators were mutated and PTS gene transcription was found to be strongly enhanced in the presence of acetate only in the sugR deletion mutant. The SugR regulon was further characterized by microarray hybridizations using the sugR mutant and its parental strain, revealing that also the PTS genes ptsG and ptsS belong to this regulon. Binding of purified SugR repressor protein to a 21 bp sequence identified the SugR binding site as an AC-rich motif. The two experimentally identified SugR binding sites in the fructose-PTS gene cluster are located within or downstream of the mapped promoters, typical for transcriptional repressors. Effector studies using electrophoretic mobility shift assays (EMSA) revealed the fructose PTS-specific metabolite fructose-1-phosphate (F-1-P) as a highly efficient, negative effector of the SugR repressor, acting in the micromolar range. Beside F-1-P, other sugar-phosphates like fructose-1,6-bisphosphate (F-1,6-P) and glucose-6-phosphate (G-6-P) also negatively affect SugR-binding, but in millimolar concentrations. CONCLUSION: In C. glutamicum ATCC 13032 the DeoR-type regulator SugR acts as a pleiotropic transcriptional repressor of all described PTS genes. Thus, in contrast to most DeoR-type repressors described, SugR is able to act also on the transcription of the distantly located genes ptsG and ptsS of C. glutamicum. Transcriptional repression of the fructose-PTS gene cluster is observed during growth on acetate and transcription is derepressed in the presence of the PTS sugars glucose and fructose. This derepression of the fructose-PTS gene cluster is mainly modulated by the negative effector F-1-P, but reduced sensitivity to the other effectors, F-1,6-P or G-6-P might cause differential transcriptional regulation of genes of the general part of the PTS (ptsI, ptsH) and associated genes encoding sugar-specific functions (ptsF, ptsG, ptsS).


Subject(s)
Bacterial Proteins/metabolism , Corynebacterium glutamicum/genetics , Gene Expression Regulation, Bacterial/physiology , Gene Expression Regulation, Enzymologic/physiology , Genes, Bacterial/physiology , Multigene Family/physiology , Transcription Factors/genetics , Bacterial Proteins/genetics , Biological Transport/physiology , Corynebacterium glutamicum/enzymology , Corynebacterium glutamicum/growth & development , Fructose/genetics , Fructose/metabolism , Glucose/genetics , Glucose/metabolism , Phosphoenolpyruvate Sugar Phosphotransferase System , Transcription Factors/metabolism , Transcription, Genetic/physiology
6.
Nat Biotechnol ; 25(11): 1281-9, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17965706

ABSTRACT

The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strain's complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase-like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.


Subject(s)
Genome, Bacterial/genetics , Myxococcales/genetics , Myxococcales/metabolism , Base Sequence , Biotechnology , Molecular Sequence Data , Myxococcales/classification , Phylogeny , Sequence Analysis, DNA
7.
BMC Genomics ; 7: 205, 2006 Aug 10.
Article in English | MEDLINE | ID: mdl-16901339

ABSTRACT

BACKGROUND: Corynebacterium glutamicum, a Gram-positive bacterium of the class Actinobacteria, is an industrially relevant producer of amino acids. Several methods for the targeted genetic manipulation of this organism and rational strain improvement have been developed. An efficient transposon mutagenesis system for the completely sequenced type strain ATCC 13032 would significantly advance functional genome analysis in this bacterium. RESULTS: A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, C. glutamicum RES167, with an IS6100-containing non-replicative plasmid. Transposon mutants had stable cointegrates between the transposon vector and the chromosome. Altogether 172 transposon integration sites have been determined by sequencing of the chromosomal inserts, revealing that each integration occurred at a different locus. Statistical target site analyses revealed an apparent absence of a target site preference. From the library, auxotrophic mutants were obtained with a frequency of 2.9%. By auxanography analyses nearly two thirds of the auxotrophs were further characterized, including mutants with single, double and alternative nutritional requirements. In most cases the nutritional requirement observed could be correlated to the annotation of the mutated gene involved in the biosynthesis of an amino acid, a nucleotide or a vitamin. One notable exception was a clone mutagenized by transposition into the gene cg0910, which exhibited an auxotrophy for histidine. The protein sequence deduced from cg0910 showed high sequence similarities to inositol-1(or 4)-monophosphatases (EC 3.1.3.25). Subsequent genetic deletion of cg0910 delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown essential L-histidinol-phosphate phosphatase (EC 3.1.3.15) in C. glutamicum. The cg0910 gene, renamed hisN, and its encoded enzyme have putative orthologs in almost all Actinobacteria, including mycobacteria and streptomycetes. CONCLUSION: The absence of regional and sequence preferences of IS6100-transposition demonstrate that the established system is suitable for efficient genome-scale random mutagenesis in the sequenced type strain C.glutamicum ATCC 13032. The identification of the hisN gene encoding histidinol-phosphate phosphatase in C. glutamicum closed the last gap in histidine synthesis in the Actinobacteria. The system might be a valuable genetic tool also in other bacteria due to the broad host-spectrum of IS6100.


Subject(s)
Bacterial Proteins/genetics , Corynebacterium glutamicum/genetics , DNA Transposable Elements/genetics , DNA, Bacterial/genetics , Genes, Bacterial , Histidine/biosynthesis , Histidinol-Phosphatase/genetics , Mutagenesis, Insertional , Actinobacteria/classification , Actinobacteria/genetics , Corynebacterium glutamicum/classification , Corynebacterium glutamicum/enzymology , Gene Deletion , Gene Library , Genetic Complementation Test , Genetics, Microbial/methods , Phenotype , Phosphoric Monoester Hydrolases/classification , Phosphoric Monoester Hydrolases/genetics , Phylogeny , Sequence Homology , Species Specificity
8.
J Bacteriol ; 187(21): 7254-66, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16237009

ABSTRACT

The gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is the causative agent of bacterial spot disease in pepper and tomato plants, which leads to economically important yield losses. This pathosystem has become a well-established model for studying bacterial infection strategies. Here, we present the whole-genome sequence of the pepper-pathogenic Xanthomonas campestris pv. vesicatoria strain 85-10, which comprises a 5.17-Mb circular chromosome and four plasmids. The genome has a high G+C content (64.75%) and signatures of extensive genome plasticity. Whole-genome comparisons revealed a gene order similar to both Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and a structure completely different from Xanthomonas oryzae pv. oryzae. A total of 548 coding sequences (12.2%) are unique to X. campestris pv. vesicatoria. In addition to a type III secretion system, which is essential for pathogenicity, the genome of strain 85-10 encodes all other types of protein secretion systems described so far in gram-negative bacteria. Remarkably, one of the putative type IV secretion systems encoded on the largest plasmid is similar to the Icm/Dot systems of the human pathogens Legionella pneumophila and Coxiella burnetii. Comparisons with other completely sequenced plant pathogens predicted six novel type III effector proteins and several other virulence factors, including adhesins, cell wall-degrading enzymes, and extracellular polysaccharides.


Subject(s)
DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Genome, Bacterial , Sequence Analysis, DNA , Xanthomonas campestris/genetics , Adhesins, Bacterial/genetics , Base Composition , Chromosomes, Bacterial/genetics , Coxiella burnetii/genetics , Gene Order , Legionella pneumophila/genetics , Molecular Sequence Data , Plasmids/genetics , Polysaccharides, Bacterial/genetics , Protein Transport/genetics , Synteny , Virulence/genetics , Virulence Factors/genetics , Xanthomonas campestris/physiology
9.
J Biotechnol ; 104(1-3): 5-25, 2003 Sep 04.
Article in English | MEDLINE | ID: mdl-12948626

ABSTRACT

The complete genomic sequence of Corynebacterium glutamicum ATCC 13032, well-known in industry for the production of amino acids, e.g. of L-glutamate and L-lysine was determined. The C. glutamicum genome was found to consist of a single circular chromosome comprising 3282708 base pairs. Several DNA regions of unusual composition were identified that were potentially acquired by horizontal gene transfer, e.g. a segment of DNA from C. diphtheriae and a prophage-containing region. After automated and manual annotation, 3002 protein-coding genes have been identified, and to 2489 of these, functions were assigned by homologies to known proteins. These analyses confirm the taxonomic position of C. glutamicum as related to Mycobacteria and show a broad metabolic diversity as expected for a bacterium living in the soil. As an example for biotechnological application the complete genome sequence was used to reconstruct the metabolic flow of carbon into a number of industrially important products derived from the amino acid L-aspartate.


Subject(s)
Amino Acids/biosynthesis , Aspartic Acid/metabolism , Corynebacterium/genetics , Corynebacterium/metabolism , Genome, Bacterial , Proteome/genetics , Proteome/metabolism , Vitamins/biosynthesis , Amino Acid Sequence , Amino Acids/genetics , Aspartic Acid/genetics , Base Sequence , Corynebacterium/classification , Databases, Genetic , Gene Expression Profiling , Molecular Sequence Data , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Recombinant Proteins/biosynthesis , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Vitamins/genetics
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