A directional, high-frequency chromosomal mobilization system for genetic mapping of Rhizobium meliloti

Klein, Shoshana; Lohman, Karin N.; Clover, Ralph; Walker, Graham C.; Signer, Ethan R.

doi:10.1128/jb.174.1.324-326.1992

Cited by 16 publications

(13 citation statements)

References 18 publications

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“…In this work we have characterized a 5.5-kb SstI DNA fragment which contains the S. meliloti lpsB chromosomal gene (15,23,33). In addition to lpsB, five other ORFs were identified, including one corresponding to the previously reported lpsC gene (15) and two others with sequence similarities to the regions coding for the transcription factors GreA and Lrp, respectively, found in several other gram-negative bacteria (9,37).…”

Section: Discussionmentioning

confidence: 99%

Genetic Characterization of a Sinorhizobium meliloti Chromosomal Region Involved in Lipopolysaccharide Biosynthesis

et al. 2001

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The genetic characterization of a 5.5-kb chromosomal region of Sinorhizobium meliloti 2011 that contains lpsB, a gene required for the normal development of symbiosis with Medicago spp., is presented. The nucleotide sequence of this DNA fragment revealed the presence of six genes: greA and lpsB, transcribed in the forward direction; and lpsE, lpsD, lpsC, and lrp, transcribed in the reverse direction. Except for lpsB, none of the lps genes were relevant for nodulation and nitrogen fixation. Analysis of the transcriptional organization of lpsB showed that greA and lpsB are part of separate transcriptional units, which is in agreement with the finding of a DNA stretch homologous to a "nonnitrogen" promoter consensus sequence between greA and lpsB. The opposite orientation of lpsB with respect to its first downstream coding sequence, lpsE, indicated that the altered LPS and the defective symbiosis of lpsB mutants are both consequences of a primary nonpolar defect in a single gene. Global sequence comparisons revealed that the greA-lpsB and lrp genes of S. meliloti have a genetic organization similar to that of their homologous loci in R. leguminosarum bv. viciae. In particular, high sequence similarity was found between the translation product of lpsB and a core-related biosynthetic mannosyltransferase of R. leguminosarum bv. viciae encoded by the lpcC gene. The functional relationship between these two genes was demonstrated in genetic complementation experiments in which the S. meliloti lpsB gene restored the wild-type LPS phenotype when introduced into lpcC mutants of R. leguminosarum. These results support the view that S. meliloti lpsB also encodes a mannosyltransferase that participates in the biosynthesis of the LPS core. Evidence is provided for the presence of other lpsB-homologous sequences in several members of the family Rhizobiaceae.The infection of legume roots by soil bacteria of the genera Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium results in the development of specialized root organs, the nitrogen-fixing nodules (53). The establishment of functional root nodules is the result of a complex process that involves an active signal exchange between the host roots and the infecting rhizobia (7). One of the earliest events in the symbiotic dialog involves the secretion of flavonoid compounds by the host plant and the subsequent production of nodulation (Nod) factors by the rhizobia (38). Although the Nod factors are the best-characterized signal molecules of rhizobia, independent evidence supports the view that some of the bacterial surface polysaccharides are also active in signaling the plant (2,16,19,22,39,40,57,60). It has thus been shown that the pretreatment of alfalfa roots with specific fractions of Sinorhizobium meliloti exopolysaccharides (EPS) conferred on symbiosis-deficient EPS mutants the ability to develop nitrogen-fixing nodules at a significant rate (2,22,57,60). This and similar results for other plant-rhizobium associations (19) indicate that the EPSs induc...

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Section: Discussionmentioning

confidence: 99%

Genetic Characterization of a Sinorhizobium meliloti Chromosomal Region Involved in Lipopolysaccharide Biosynthesis

et al. 2001

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“…Cold Spring Harbor Laboratory Press on April 29, 2019 -Published by genesdev.cshlp.org Downloaded from tance did not cotransfer with either pSym (Finan et al 1986), and subsequently by conjugation analysis (Glazebrook et al 1992;Klein et al 1992), finding that the B4 mutation lies between fix-382 and ura-501 (Fig. 1A).…”

Section: Genes and Developmentmentioning

confidence: 99%

“…Mapping of the Tn5-Tn233 in B4Sp2 was done using approaches described by Klein et al (1992) and Glazebrook et al (1992). The B4Sp2 marker transferred with the highest efficienties from TnS-Mob fl611 and f~615 about equally (Ogawa 1993), indicating that the Tn5 insertion in mutant B4 lies between trp-33 and pyr-49 on the R. meliloti chromosome.…”

Section: Genetic Techniquesmentioning

confidence: 99%

The Rhizobium meliloti groELc locus is required for regulation of early nod genes by the transcription activator NodD.

Ogawa¹,

Long²

1995

Genes Dev.

107

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The molecular chaperones related to GroEL (hsp60, cpn60) interact with partially folded proteins and appear to assist them to attain active and correctly folded conformation. They are required for cell viability but are probably more important for some processes than for others. Through a random genetic search to find loci that are required for expression of the Rhizobium meliloti nod (nodulation) genes, we isolated a mutant (B4) defective in luteolin-dependent activation of nod gene expression, and found it carries a Tn5 insertion within a chromosomal groEL gene (groELc) located just downstream of a groESc gene. The groELc mutation affected activity of three related LysR-type activator proteins NodD1, NodD3, and SyrM; on plants, the mutants formed nodules late, and the nodules were Fix-. Hybridization and protein expression analysis show that a similar groESL locus (groESLa) maps to the Rml021 megaplasmid pSyma. Southern blot analysis revealed additional, but less closely related sequences hybridizing to groELc and groESc probes elsewhere in the R. meliloti genome. Clones of groESLc and groESLa can each restore robust phage ~ growth on an Escherichia coli groE mutant. Likewise each clone can complement all of the phenotypes observed for B4 mutants; thus, the two appear to be functionally equivalent if expression is controlled. We determined that groELc is required for normal DNA binding of the NodD target sequence in R. meliloti. GroEL coimmunopurifies with NodD1 from R. meliloti, which suggests a direct physical association between these proteins. GroEL is thus probably involved in the folding or assembly of transcriptionally active NodD.

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“…To map the ftsZ genes roughly with respect to other chromosomal markers, we used conjugational mapping with several oriT::TnS insertions in the R. meliloti chromosome (19). First, Spr markers linked to ftsZl and ftsZ2 were transduced into the oriT::TnS strains Rm6727, Rm6743, and 6761.…”

Section: Methodsmentioning

confidence: 99%

Rhizobium meliloti contains a novel second homolog of the cell division gene ftsZ

Margolin

Long

1994

J Bacteriol

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We have identified a second homolog of the cell division gene,ftsZ, in the endosymbiont Rhizobium meliloti. The ftsZ2 gene was cloned by screening a genomic A library with a probe derived from PCR amplification of a highly conserved domain. It encodes a 36-kDa protein which shares a high level of sequence similarity with the FtsZ proteins of Escherichia coli and BaciUus subtilis and FtsZl (Zl) of R. meliloti but lacks the carboxy-terminal region conserved in other FtsZ proteins. The identity of theftsZ2 gene product was confirmed both by in vitro transcription-translation in an R. meliloti S-30 extract and by overproduction in R. meliloti cells. As with Zl, the overproduction of FtsZ2 in E. coli inhibited cell division and induced filamentation, although to a lesser extent than with Zl. However, the expression offtsZ2 in E. coli under certain conditions caused some cells to coil dramatically, a phenotype not observed during Zl overproduction. Although several Tn3-GUS (glucuronidase) insertions in a plasmid-borneftsZ2 gene failed to cross into the chromosome, one interruption in the chromosomal ftsZ2 gene was isolated, suggesting that ftsZ2 is nonessential for viability. The two ftsZ genes were genetically mapped to the R. meliloti main chromosome, approximately 100 kb apart.The earliest known step in Escherichia coli cell division is the formation of a ring consisting of the FtsZ protein at the division site (5). FtsZ is also necessary for the formation of both vegetative and asymmetric sporulation septa in Bacillus subtilis (2). Cells containing very high levels of FtsZ or a temperature-sensitive FtsZ under nonpermissive conditions will form long, nonseptate filaments, presumably because the division ring cannot be formed (3, 39). It is not known how FtsZ, which is uniformly distributed during most of the cell cycle, assembles at the correct time in the center of the predivisional cell. However, the recent demonstrations that E. coli FtsZ binds GTP and has GTPase activity (10,26,29) suggest that GTP binding and hydrolysis by FtsZ could catalyze the assembly of the ring structure, in a manner analogous to filament formation by tubulins (34). The three previously sequenced FtsZ proteins, from E. coli, B. subtilis, and Rhizobium meliloti (1,21,40), all share a completely conserved 13-amino-acid domain that is very similar to the proposed GTP binding domain of tubulins (10) MATERIALS AND METHODSBacterial strains and plasmids. All strains and plasmids used in this study are listed in Table 1.Bacterial growth and induction conditions. All E. coli strains were grown in Luria broth (LB) (30) plus 10 ,ug of tetracycline and 50 ,ug of ampicillin per ml for plasmids in host strain XL1-Blue. Isopropyl-,B-D-thiogalactopyranoside (IPTG) inductions of E. coli cells were done by streaking colonies on agar plates containing LB, antibiotics, and 1 mM IPTG. R. meliloti cells were induced for protein overproduction by the addition of 1 mM IPTG to cells growing in LB plus 10 ,ug of tetracycline per ml in late exponential phase.Pre...

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A directional, high-frequency chromosomal mobilization system for genetic mapping of Rhizobium meliloti

Abstract: A system for mapping of the Rhizobium meliloti chromosome that utilizes transposon TnS-Mob, which carries the mobilization site of IncP plasmid RP4 (R. Simon, Mol. Gen. Genet. 196:413-420, 1984)

Cited by 16 publications

References 18 publications

Genetic Characterization of a Sinorhizobium meliloti Chromosomal Region Involved in Lipopolysaccharide Biosynthesis

Genetic Characterization of a Sinorhizobium meliloti Chromosomal Region Involved in Lipopolysaccharide Biosynthesis

The Rhizobium meliloti groELc locus is required for regulation of early nod genes by the transcription activator NodD.

Rhizobium meliloti contains a novel second homolog of the cell division gene ftsZ

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