Genetic and physical analyses of group E exo- mutants of Rhizobium meliloti

Mutants of Rhizobium meliloti which are deficient in exopolysaccharide synthesis have been classified into six different genetic groups (A through F) (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). Using physical and genetic techniques, we have demonstrated that the group E Exo- mutants carry deletions in the exoA-exoB region of the megaplasmid pRmeSU47b. We have constructed strains carrying defined deletions which remove up to 200 kilobases of pRmeSU47b, including the exoA-exoB region. These derivatives have the same phenotypes as do the group E mutants.     

Symbiotic loci of Rhizobium meliloti identified by random TnphoA mutagenesis.

We have developed a system for using TnphoA (TnphoA is Tn5 IS50L::phoA), which generates fusions to alkaline phosphatase (C. Manoil and J. Beckwith, Proc. Natl. Acad. Sci. USA 82:8129-8133, 1985), in Rhizobium meliloti. Active fusions expressing alkaline phosphatase can arise only when this transposon inserts in genes encoding secreted or membrane-spanning proteins. By confining our screening to 1,250 TnphoA-generated mutants of R. meliloti that expressed alkaline phosphatase, we efficiently identified 25 symbiotically defective mutants, all of which formed ineffective (Fix-) nodules on alfalfa. Thirteen of the mutants were unable to synthesize an acidic exopolysaccharide (exo::TnphoA) that is required for nodule invasion. Twelve of the mutations created blocked at later stages of nodule development (fix::TnphoA) and were assigned to nine symbiotic loci. One of these appeared to be a previously undescribed locus located on the pRmeSU47a megaplasmid and to encode a membrane protein. Two others were located on the pRmeSU47b megaplasmid: one was a new locus which was induced by luteolin and encoded a membrane protein, and the other was dctA, the structural gene for dicarboxylic acid transport. The remaining six loci were located on the R. meliloti chromosome. One of these was inducible by luteolin and encoded a membrane protein which determined lipopolysaccharide structure. Three additional chromosomal loci also appeared to encode membrane proteins necessary for symbiosis. The remaining two chromosomal loci encoded periplasmic proteins required for symbiosis.     

Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes.

Using physical and genetic data, we have demonstrated that Rhizobium meliloti SU47 has a symbiotic megaplasmid, pRmeSU47b, in addition to the previously described nod-nif megaplasmid pRmeSU47a. This plasmid includes four loci involved in exopolysaccharide (exo) synthesis as well as two loci involved in thiamine biosynthesis. Mutations at the exo loci have previously been shown to result in the formation of nodules which lack infection threads (Inf-) and fail to fix nitrogen (Fix-). Thus, both megaplasmids contain genes involved in the formation of nitrogen-fixing root nodules. Mutations at two other exo loci were not located on either megaplasmid. To mobilize the megaplasmids, the oriT of plasmid RK2 was inserted into them. On alfalfa, Agrobacterium tumefaciens strains containing pRmeSU47a induced marked root hair curling with no infection threads and Fix- nodules, as reported by others. This plant phenotype was not observed to change with A. tumefaciens strains containing both pRmeSU47a and pRmeSU47b megaplasmids, and strains containing pRmeSU47b alone failed to curl root hairs or form nodules.     

Genetic map of Rhizobium meliloti megaplasmid pRmeSU47b.

A circular linkage map of the Rhizobium meliloti megaplasmid pRmeSU47b was constructed. The map consists of transposon insertions carrying alternating antibiotic resistance markers linked by phi M12 transduction. Data from conjugation experiments utilizing donor strains carrying Tn5-oriT insertions in the megaplasmid supported the proposed genetic map. In addition, the positions of previously identified Fix, exopolysaccharide synthetic, thiamine synthetic, and C4-dicarboxylate transport loci on the megaplasmid map were determined. By converting cotransduction frequencies to physical distance, we calculated the replicon to be 1,600 kilobases in size, which compares favorably with previous physical estimates.     

ndvF, a novel locus located on megaplasmid pRmeSU47b (pEXO) of Rhizobium meliloti, is required for normal nodule development.

Rhizobium meliloti strains carrying either of two overlapping deletions (delta 5408 and delta F114) of the megaplasmid pRmeSU47b form nodules on alfalfa which fail to fix N2 (Fix-). Strains carrying these deletions also fail to fluoresce on media containing calcofluor, indicating a defect in synthesis of the acidic exopolysaccharide (Exo-) of R. meliloti. We have isolated cosmid clones (pTH21 and pTH22) which complement the Fix- but not the Exo- phenotype of the strains carrying the delta 5408 and delta F114 deletions. In addition, cosmid clones which complement the Exo- phenotype fail to complement the Fix- phenotype of these deletions; thus, the Exo- phenotype is not related to the Fix- phenotype. A 5-kb region within a 7.3-kb BamHI restriction fragment was found to be required for complementation of the Fix- phenotype of the delta 5408 and delta F114 deletion strains. Tn5 insertions in the 5-kb region generated a Fix- phenotype when recombined into the wild-type genome. We have designated this locus ndvF, for nodule development. TnphoA mutagenesis of this region generated active alkaline-phosphatase gene fusions, indicating that ndvF encodes extracytoplasmic protein(s). Induction of nodules by the ndvF mutants was delayed by 2 to 3 days compared with induction by the wild-type strain. Light microscopy of nodules elicited by strains carrying the large 150-kb delta F114 deletion, a 12-kb deletion removing ndvF, or an individual ndvF::Tn5 insertion mutation demonstrated that many nodules contained few infected cortical cells, indicating that nodule development was blocked early in the infection process, before the release of bacteria from the infection threads.     

Rhizobium meliloti exoG and exoJ mutations affect the exoX-exoY system for modulation of exopolysaccharide production.

R. meliloti Rm1021 normally produces an acidic Calcofluor-binding exopolysaccharide, called succinoglycan or EPS I, which is required for successful nodulation of alfalfa by this strain. At least 13 loci affecting production of EPS I were previously mapped to a cluster on the second of two symbiotic megaplasmids in Rm1021, pRmeSU47b. A putative regulatory region was originally defined by the exoG and exoJ mutations. exoG and exoJ mutants produced less exopolysaccharide than wild-type strains and induced nitrogen-fixing nodules on alfalfa with reduced efficiency compared with the wild type. These mutants appeared to produce only a low-molecular-weight form of EPS I. Mutations called exoX cause an increase in exopolysaccharide production and map in the same region as the exoG and exoJ mutations. The DNA sequence of this region reveals that it contains two open reading frames, called exoX and exoY, which have homologs in other Rhizobium species. Interestingly, the exoG insertion mutations fall in an intergenic region and may affect the expression of exoX or exoY. The exoJ mutation falls in the 3' portion of the exoX open reading frame and is probably an allele of exoX that results in altered function. exoG and exoJ mutations limit EPS I production in the presence of exoR95 or exoS96 mutations, which cause overproduction of EPS I. Gene regulation studies suggest that ExoX and ExoY constitute a system that modulates exopolysaccharide synthesis at a posttranslational level. The deduced sequence of ExoY is homologous to a protein required for an early step in xanthan gum biosynthesis, further suggesting that the modulatory system may affect the exopolysaccharide biosynthetic apparatus.     

Mutants of Rhizobium meliloti defective in succinate metabolism.

We characterized mutants of Rhizobium meliloti SU47 that were unable to grow on succinate as the carbon source. The mutants fell into five groups based on complementation of the succinate mutations by individual recombinant plasmids isolated from a R. meliloti clone bank. Enzyme analysis showed that mutants in the following groups lacked the indicated common enzyme activities: group II, enolase (Eno); group III, phosphoenolpyruvate carboxykinase (Pck); group IV, glyceraldehyde-3-phosphate dehydrogenase (Gap), and 3-phosphoglycerate kinase (Pgk). Mutants in groups I and V lacked C4-dicarboxylate transport (Dct-) activity. Wild-type cells grown on succinate as the carbon source had high Pck activity, whereas no Pck activity was detected in cells that were grown on glucose as the carbon source. It was found that in free-living cells, Pck is required for the synthesis of phosphoenolpyruvate during gluconeogenesis. In addition, the enzymes of the lower half of the Embden-Meyerhoff-Parnas pathway were absolutely required for gluconeogenesis. Eno, Gap, Pck, and one of the Dct loci (ntrA) mapped to different regions of the chromosome; the other Dct locus was tightly linked to a previously mapped thi locus, which was located on the megaplasmid pRmeSU47b.     

Spontaneous deletion of citrate-utilizing ability promoted by insertion sequences.

The citrate utilization (Cit+) transposon Tn3411 was shown to be flanked by directly repeated sequences (IS3411L and IS3411R) by restriction enzyme analysis and electron microscope observation. Cit- deletion mutants were frequently found to be generated in pBR322::Tn3411 by intramolecular recombination between the two copies of IS3411. The flanking IS3411 elements of Tn3411 were shown to be functional insertion sequences by Tn3411-mediated direct and inverse transposition. Tn3411-mediated inverse transposition from pBR322::Tn3411 to the F-plasmid derivative pED100 occurred more efficiently than that of direct transposition of the Cit+ determinant. This was thought to be due to the differential transposability of IS3411L and IS3411R in the transposition process. The frequency of transposition of IS3411 marked with a chloramphenicol resistance determinant was much higher than IS3411-mediated cointegrate formation, suggesting that replicon fusions are not essential intermediates in the transposition process of Tn3411 or IS3411. Spontaneous deletions occurred with high frequency in recA hosts. The spontaneous deletion promoted by homologous recombination between two IS3411 elements in Tn3411 was examined with deletion mutants.     

紫云英根瘤菌菌株107exo基因簇的遗传互补分析

紫云英根瘤菌菌株１０７经Ｔｎ５插入诱变,得到１２株胞外多糖缺陷型变种,以质粒ｐＭＮ２为载体,从其中７株ＥＰＳ－变种内分别构建了７个Ｒ－Ｐｒｉｍｅ质粒（ｅｘｏＲ＇）,大部分变种的ＥＰＳ－表型可被ｅｘｏＲ＇互补,恢复野生型表型（ＥＰＳ＋）。互补表明,１２株ＥＰＳ－变种可分为６个不同的互补群,其中５个在遗传上连锁。ｅｘｏＲ＇酶切分析,除ｅｘｏＲ＇－０２,ｅｘｏＲ＇－０４外,其余５只的外源片段均整合于ＰＭＮ２的两同向重复序列ＩＳ２１之间。     

Evolution of rhizobial symbiosis islands through insertion sequence-mediated deletion and duplication

Symbiosis between organisms influences their evolution via adaptive changes in genome architectures. Immunity of soybean carrying the Rj2 allele is triggered by NopP (type III secretion system [T3SS]-dependent effector), encoded by symbiosis island A (SymA) in B. diazoefficiens USDA122. This immunity was overcome by many mutants with large SymA deletions that encompassed T3SS (rhc) and N₂ fixation (nif) genes and were bounded by insertion sequence (IS) copies in direct orientation, indicating homologous recombination between ISs. Similar deletion events were observed in B. diazoefficiens USDA110 and B. japonicum J5. When we cultured a USDA122 strain with a marker gene sacB inserted into the rhc gene cluster, most sucrose-resistant mutants had deletions in nif/rhc gene clusters, similar to the mutants above. Some deletion mutants were unique to the sacB system and showed lower competitive nodulation capability, indicating that IS-mediated deletions occurred during free-living growth and the host plants selected the mutants. Among 63 natural bradyrhizobial isolates, 2 possessed long duplications (261–357 kb) harboring nif/rhc gene clusters between IS copies in direct orientation via homologous recombination. Therefore, the structures of symbiosis islands are in a state of flux via IS-mediated duplications and deletions during rhizobial saprophytic growth, and host plants select mutualistic variants from the resultant pools of rhizobial populations. Our results demonstrate that homologous recombination between direct IS copies provides a natural mechanism generating deletions and duplications on symbiosis islands.Subject terms: Soil microbiology, Molecular evolution     

Genetic analyses of Rhizobium meliloti exopolysaccharides

We have recently obtained strong genetic evidence that the acidic Calcofluor-binding exopolysaccharide (EPS I) of Rhizobium meliloti Rm1021 is required for nodule invasion and possibly for later events in nodule development. Thirteen loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I. Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules. exoH mutants fail to succinylate their EPS I and form empty Fix- nodules. We have identified two unlinked regulatory loci, exoR and exoS, whose products play negative roles in the regulation of expression of the exo genes. We have recently discovered that R. meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts. Possible roles for Rhizobium exopolysaccharides in nodulation are discussed.     

Genomic deletions of the Drosophila melanogaster Hsp70 genes

Homologous recombination can produce directed mutations in the genomes of a number of model organisms, including Drosophila melanogaster. One of the most useful applications has been to delete target genes to generate null alleles. In Drosophila, specific gene deletions have not yet been produced by this method. To test whether such deletions could be produced by homologous recombination in D. melanogaster we set out to delete the Hsp70 genes. Six nearly identical copies of this gene, encoding the major heat-shock protein in Drosophila, are found at two separate but closely linked loci. This arrangement has thwarted standard genetic approaches to generate an Hsp70-null fly, making this an ideal test of gene targeting. In this study, ends-out targeting was used to generate specific deletions of all Hsp70 genes, including one deletion that spanned approximately 47 kb. The Hsp70-null flies are viable and fertile. The results show that genomic deletions of varied sizes can be readily generated by homologous recombination in Drosophila.     

Molecular Genetics of the Brown (B)-Locus Region of Mouse Chromosome 4. II. Complementation Analyses of Lethal Brown Deletions

Numerous new mutations at the brown (b) locus in mouse chromosome 4 have been recovered over the years in germ-cell mutagenesis experiments performed at the Oak Ridge National Laboratory. A large series of radiation- and chemical-induced b mutations known to be chromosomal deletions, and also known to be prenatally lethal when homozygous, were analyzed by pairwise complementation crosses as well as by pseudodominance tests involving flanking loci defined by externally visible phenotypes. These crosses were designed to determine the extent of each deletion on the genetic and phenotype map of the chromosomal region surrounding the b locus; the crosses also provided basic data that assigned deletions to complementation groups and defined four new loci associated with aberrancies in normal development. Specifically, the pseudodominance tests identified deletions that include the proximally mapping whirler (wi) and the distally mapping depilated (dep) genes, thereby bracketing these loci defined by visible developmental abnormalities with landmarks (deletion breakpoints) that are easily identified on the physical map. Furthermore, the complementation crosses, which were supplemented with additional crosses that allowed determination of the gross time of lethality of selected deletions, defined four new loci required for normal development. Homozygous deletion of one of these loci (b-associated fitness, baf) results in a runting syndrome evident during postnatal development; deletion of one locus [l(4)2Rn] causes death in the late gestation/neonatal period; and deletion of either of two loci [l(4)1Rn or l(4)3Rn] results in embryonic death, most likely in pre-, peri- or postimplantation stages. The placement of these new functionally defined loci on the evolving molecular map of the b region should be useful for continuing the analysis of the roles played in development by genes in this segment of chromosome 4.     

A Deletion Map of cyc1 Mutants and Its Correspondence to Mutationally Altered Iso-1-Cytochromes c of Yeast

Mutants arising spontaneously from sporulated cultures of certain strains of yeast, Saccharomyces cerevisiae, contained deletions of the CYC1 gene which controls the primary structure of iso-1-cytochrome c. At least 60 different kinds of deletions were uncovered among the 104 deletions examined and these ranged in length from those encompassing only two adjacent point mutants to those encompassing at least the entire CYC1 gene. X-ray-induced recombination rates of crosses involving these deletions and cyc1 point mutants resulted in the assignment of 211 point mutants to 47 mutational sites and made it possible to unambiguously order 40 of these 47 sites. Except for one mutant, cyc1-15, there was a strict colinear relationship between the deletion map and the positions of 13 sites that were previously determined by amino acid alterations in iso-1-cytochromes c from intragenic revertants.     

Evidence for non-homologous end joining and non-allelic homologous recombination in atypical NF1 microdeletions

NF1 microdeletion syndrome is caused by haploinsufficiency of the NF1 gene and of gene(s) located in adjacent flanking regions. Most of the NF1 deletions originate by non-allelic homologous recombination between repeated sequences (REP-P and -M) mapped to 17q11.2, while the remaining deletions show unusual breakpoints. We performed high-resolution FISH analysis of 18 NF1 microdeleted patients with the aims of mapping non-recurrent deletion breakpoints and verifying the presence of additional recombination-prone architectural motifs. This approach allowed us to obtain the sequence of the first junction fragment of an atypical deletion. By conventional FISH, we identified 16 patients with REP-mediated common deletions, and two patients carrying atypical deletions of 1.3 Mb and 3 Mb. Following fibre-FISH, we identified breakpoint regions of 100 kb, which led to the generation of several locus-specific probes restricting the atypical deletion endpoint intervals to a few kilobases. Sequence analysis provided evidence of small blocks of REPs, clustered around the 1.3-Mb deletion breakpoints, probably involved in intrachromatid non-allelic homologous recombination (NAHR), while isolation and sequencing of the 3-Mb deletion junction fragment indicated that a non-homologous end joining (NHEJ) mechanism is implicated.M. Venturin and C. Gervasini contributed equally to the study     

Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum

The classic strategy to achieve gene deletion variants is based on double-crossover integration of nonreplicating vectors into the genome. In addition, recombination systems such as Cre-lox have been used extensively, mainly for eukaryotic organisms. This study presents the construction of a Cre-lox-based system for multiple gene deletions in Lactobacillus plantarum that could be adapted for use on gram-positive bacteria. First, an effective mutagenesis vector (pNZ5319) was constructed that allows direct cloning of blunt-end PCR products representing homologous recombination target regions. Using this mutagenesis vector, double-crossover gene replacement mutants could be readily selected based on their antibiotic resistance phenotype. In the resulting mutants, the target gene is replaced by a lox66-P(32)-cat-lox71 cassette, where lox66 and lox71 are mutant variants of loxP and P(32)-cat is a chloramphenicol resistance cassette. The lox sites serve as recognition sites for the Cre enzyme, a protein that belongs to the integrase family of site-specific recombinases. Thus, transient Cre recombinase expression in double-crossover mutants leads to recombination of the lox66-P(32)-cat-lox71 cassette into a double-mutant loxP site, called lox72, which displays strongly reduced recognition by Cre. The effectiveness of the Cre-lox-based strategy for multiple gene deletions was demonstrated by construction of both single and double gene deletions at the melA and bsh1 loci on the chromosome of the gram-positive model organism Lactobacillus plantarum WCFS1. Furthermore, the efficiency of the Cre-lox-based system in multiple gene replacements was determined by successive mutagenesis of the genetically closely linked loci melA and lacS2 in L. plantarum WCFS1. The fact that 99.4% of the clones that were analyzed had undergone correct Cre-lox resolution emphasizes the suitability of the system described here for multiple gene replacement and deletion strategies in a single genetic background.     

IS6110-mediated deletion polymorphism in the direct repeat region of clinical isolates of Mycobacterium tuberculosis

This study investigates the phenomenon of IS6110-mediated deletion polymorphism in the direct repeat (DR) region of the genome of Mycobacterium tuberculosis. Clinical isolates and their putative predecessors were compared using a combination of DR region restriction fragment length polymorphism, IS6110 DNA fingerprinting, spoligotyping, and DNA sequencing, which allowed the mapping of chromosome structure and deletion junctions. The data suggest that adjacently situated IS6110 elements mediate genome deletion. However, in contrast to previous reports, deletions appear to be mediated by inversely oriented IS6110 elements. This suggests that these events may occur via mechanisms other than RecA-mediated homologous recombination. The results underscore the important role of IS6110-associated deletion hypervariability in driving M. tuberculosis genome evolution.     

Determination of the endpoints of partial deletion mutants of the attachment site of bacteriophage lambda by DNA sequencing.

The deletion mutants b508 and b522 of bacteriophage lambda both end within the attachment site. The formation of such deletions is dependent upon the presence of intact integrase, and thus the deletion endpoints may be related to the normal crossover site in site-specific recombination. We have determined the DNA sequences of the attachment site regions of these deletions. Comparison of the sequences with lambda wildtype shows that both the deletions end within the central common homology region but at different positions. The consequences of these findings for current models of site-specific recombination are discussed.