A high-density physical map of
            <i>Sinorhizobium meliloti</i>
            1021 chromosome derived from bacterial artificial chromosome library

Capela, Delphine; Barloy-Hubler, Frédérique; Gatius, M.; Gouzy, Jérôme; Galibert, Francis

doi:10.1073/pnas.96.16.9357

Cited by 27 publications

(13 citation statements)

References 64 publications

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“…Transconjugants arose only on TY-Rf-Gm-Sp, and 50 of these transconjugant colonies were screened for Nm r ; all were found to be Nm s . This result clearly excludes a pRmeSU47a location for phbC and is consistent with our earlier chromosome mapping data (11) and recently reported genomic sequence data (9).…”

Section: Vol 182 2000 Enzymes For Growth On Phb Cycle Intermediatessupporting

confidence: 81%

Requirement for the Enzymes Acetoacetyl Coenzyme A Synthetase and Poly-3-Hydroxybutyrate (PHB) Synthase for Growth of Sinorhizobium meliloti on PHB Cycle Intermediates

2000

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We have identified two Sinorhizobium meliloti chromosomal loci affecting the poly-3-hydroxybutyrate degradation pathway. One locus was identified as the gene acsA, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase. Analysis of the acsA nucleotide sequence revealed that this gene encodes a putative protein with a molecular weight of 72,000 that shows similarity to acetyl-CoA synthetase in other organisms. Acetyl-CoA synthetase activity was not affected in cell extracts of glucose-grown acsA::Tn5 mutants; instead, acetoacetyl-CoA synthetase activity was drastically reduced. These findings suggest that acetoacetyl-CoA synthetase, rather than CoA transferase, activates acetoacetate to acetoacetyl-CoA in the S. meliloti poly-3-hydroxybutyrate cycle. The second locus was identified as phbC, encoding poly-3-hydroxybutyrate synthase, and was found to be required for synthesis of poly-3-hydroxybutyrate deposits.The catabolism of intracellular carbon stores is a strategy employed by many bacterial species to survive nutritionally suboptimal conditions. Polyhydroxyalkanoates (PHA), such as poly-3-hydroxybutyrate (PHB), accumulate in cells when growth is limited but carbon availability is not (2). This stored carbon can then be utilized during conditions of otherwise limiting carbon availability (45). PHB synthesis and degradation are collectively referred to as the PHB cycle. The extent of PHB accumulation is dependent on the relative rates of synthesis and degradation, which in turn are controlled by growth conditions (36,41).PHA have attracted substantial industrial interest for their use as high-quality, biodegradable plastics (2). This interest has driven research efforts directed at PHA biosynthesis. The genes encoding the enzymes responsible for PHA synthesis have been isolated and characterized from a number of bacterial species (33, 43), including Sinorhizobium meliloti strains Rm41 (49) and Rm1021 (51). The degradative portion of the cycle has not been subjected to similar attention, and it is less well defined both genetically and biochemically.We have recently isolated a number of S. meliloti mutants that are affected in the ability to utilize PHB cycle intermediates as sole carbon sources to support growth (11). The mutations mapped to loci on both the chromosome and the pRmeSU47b (pEXO) megaplasmid. Two loci on the megaplasmid have been identified via enzymatic and nucleotide sequence analyses. One encodes the enzyme 3-hydroxybutyrate dehydrogenase (3), and the other encodes methylmalonyl coenzyme A (methylmalonyl-CoA) mutase (10). In this paper, we further characterize and identify two of the chromosomal loci. MATERIALS AND METHODSStrains, plasmids, and culture conditions. Bacterial strains and plasmids are listed in Table 1. The construction of new strains is described in the text. Bacterial culture in Luria-Bertani (LB) and TY complex media and modified M9 minimal salts medium and antibiotic selection were carried out as previously described (12). Modified M9 minimal salts medium was supplemented...

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Section: Vol 182 2000 Enzymes For Growth On Phb Cycle Intermediatessupporting

confidence: 81%

Requirement for the Enzymes Acetoacetyl Coenzyme A Synthetase and Poly-3-Hydroxybutyrate (PHB) Synthase for Growth of Sinorhizobium meliloti on PHB Cycle Intermediates

2000

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“…About 1400 individual clones, representing an estimated 16-fold genome coverage based on the complete sequence of C. glutamicum ATCC 13032, were transferred into E. coli EPI300 and used for further experiments. The fosmid library was screened for the presence of the S-layer gene by PCR techniques, using the cspBspecific primer pair cspB_S1 and cspB_S2 and a defined combination of DNA pools of clones from microtitre plates (Capela et al, 1999). Despite the high genome coverage of the fosmid library, only four fosmids revealed a PCR fragment of the expected size of about 1 kb, representing an internal fragment of the cspB gene (data not shown).…”

Section: Resultsmentioning

confidence: 99%

The surface (S)-layer gene cspB of Corynebacterium glutamicum is transcriptionally activated by a LuxR-type regulator and located on a 6 kb genomic island absent from the type strain ATCC 13032

et al. 2006

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The surface (S)-layer gene region of the Gram-positive bacterium Corynebacterium glutamicum ATCC 14067 was identified on fosmid clones, sequenced and compared with the genome sequence of C. glutamicum ATCC 13032, whose cell surface is devoid of an ordered S-layer lattice. A 5·97 kb DNA region that is absent from the C. glutamicum ATCC 13032 chromosome was identified. This region includes cspB, the structural gene encoding the S-layer protomer PS2, and six additional coding sequences. PCR experiments demonstrated that the respective DNA region is conserved in different C. glutamicum wild-type strains capable of S-layer formation. The DNA region is flanked by a 7 bp direct repeat, suggesting that illegitimate recombination might be responsible for gene loss in C. glutamicum ATCC 13032. Transfer of the cloned cspB gene restored the PS2− phenotype of C. glutamicum ATCC 13032, as confirmed by visualization of the PS2 proteins by SDS-PAGE and imaging of ordered hexagonal S-layer lattices on living C. glutamicum cells by atomic force microscopy. Furthermore, the promoter of the cspB gene was mapped by 5′ rapid amplification of cDNA ends PCR and the corresponding DNA fragment was used in DNA affinity purification assays. A 30 kDa protein specifically binding to the promoter region of the cspB gene was purified. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and peptide mass fingerprinting of the purified protein led to the identification of the putative transcriptional regulator Cg2831, belonging to the LuxR regulatory protein family. Disruption of the cg2831 gene in C. glutamicum resulted in an almost complete loss of PS2 synthesis. These results suggested that Cg2831 is a transcriptional activator of cspB gene expression in C. glutamicum.

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“…In the case of a recent BAC library prepared from the total genome of the bacterium Mycobacterium tuberculosis (12), the authors reported particular difficulty in obtaining BAC clones with inserts of greater than 100 kb. Moreover, in the case of a recently reported BAC library of the S. meliloti chromosome, the average insert size was 80 kb (14). Brosch et al (12) suggested that the limit on insert size could be due to plasmid instability resulting from the lethal overexpression of certain M. tuberculosis genes in E. coli.…”

Section: Vol 182 2000 Cloning Of Large Fragments From Bacterial Genmentioning

confidence: 99%

oriT -Directed Cloning of Defined Large Regions from Bacterial Genomes: Identification of the Sinorhizobium meliloti pExo Megaplasmid Replicator Region

et al. 2000

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We have developed a procedure to directly clone large fragments from the genome of the soil bacterium Sinorhizobium meliloti. Specific regions to be cloned are first flanked by parallel copies of an origin of transfer (oriT) together with a plasmid replication origin capable of replicating large clones in Escherichia coli but not in the target organism. Supplying transfer genes in trans specifically transfers the oriT-flanked region, and in this process, site-specific recombination at the oriT sites results in a plasmid carrying the flanked region of interest that can replicate in E. coli from the inserted origin of replication (in this case, the F origin carried on a BAC cloning vector). We have used this procedure with the oriT of the plasmid RK2 to clone contiguous fragments of 50, 60, 115, 140, 240, and 200 kb from the S. meliloti pExo megaplasmid. Analysis of the 60-kb fragment allowed us to identify a 9-kb region capable of autonomous replication in the bacterium Agrobacterium tumefaciens. The nucleotide sequence of this fragment revealed a replicator region including homologs of the repA, repB, and repC genes from other Rhizobiaceae, which encode proteins involved in replication and segregation of plasmids in many organisms.With the rapid increase in the number of completed microbial genome sequences, we are entering an era in which interests in the manipulation and functional characterization of whole genomes are flourishing. Methods and techniques involved in the manipulation of large regions of genomes will increasingly become valuable tools (for example, in the generation of mosaic organisms with various catabolic and biosynthetic capabilities). Here we describe a new procedure to clone large (Ͼ100-kb) defined regions from the genome of the nitrogen-fixing bacterium Sinorhizobium meliloti.S. meliloti is a free-living gram-negative soil bacterium whose symbiotic interaction with alfalfa results in the formation of nitrogen-fixing root nodules. The genome of S. meliloti strain SU47 consists of three large replicons, the largest of which is 3,500 kb in size and appears to be similar to a conventional bacterial chromosome (4,28,36). The two other replicons are referred to as megaplasmids (47). One is 1,350 kb in size and, because it carries nodulation and nitrogen fixation genes required for symbiosis, is referred to as pSym (5,33,46); the other is 1,700 kb and is designated pExo since it carries two large gene clusters required for the synthesis of exopolysaccharides (alternate designations include pRmeSU47b and pSymb [4,13,22,30,33]).In previous work, we constructed a genetic map of the pExo megaplasmid which consists of sequential Tn5-derivative transposon insertions linked to each other in transduction (16). Strains carrying pExo megaplasmid deletions between defined insertions were isolated, and a phenotypic analysis of these strains allowed us to identify several loci involved in utilization of the carbon sources dulcitol, ␤-hydroxybutyrate, lactose, rhamnose, and protocatechuate (17). Other known g...

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A high-density physical map of Sinorhizobium meliloti 1021 chromosome derived from bacterial artificial chromosome library

Abstract: As

Cited by 27 publications

References 64 publications

Requirement for the Enzymes Acetoacetyl Coenzyme A Synthetase and Poly-3-Hydroxybutyrate (PHB) Synthase for Growth of Sinorhizobium meliloti on PHB Cycle Intermediates

Requirement for the Enzymes Acetoacetyl Coenzyme A Synthetase and Poly-3-Hydroxybutyrate (PHB) Synthase for Growth of Sinorhizobium meliloti on PHB Cycle Intermediates

The surface (S)-layer gene cspB of Corynebacterium glutamicum is transcriptionally activated by a LuxR-type regulator and located on a 6 kb genomic island absent from the type strain ATCC 13032

oriT -Directed Cloning of Defined Large Regions from Bacterial Genomes: Identification of the Sinorhizobium meliloti pExo Megaplasmid Replicator Region

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