Genetic analysis of carbamoylphosphate synthesis in Rhizobium meliloti 104A14

We have previously isolated ineffective (Fix-) mutants of Rhizobium meliloti 104A14 requiring both arginine and uracil, and thus probably defective in carbamoylphosphate synthetase. We describe here the molecular and genetic analysis of the R. meliloti genes coding for carbamoylphosphate synthetase. Plasmids that complement the mutations were isolated from a R. meliloti gene bank. Restriction analysis of these plasmids indicated that complementation involved two unlinked regions of the R. meliloti chromosome, carA and carB. Genetic complementation between the plasmids and mutants demonstrated a single complementation group for carA, but two overlapping complementation groups for carB. The cloned R. meliloti genes hybridize to the corresponding E. coli carA and carB genes which encode the two subunits of carbamoylphosphate synthetase. Transposon Tn5 mutagenesis was used to localize the carA and carB genes on the cloned R. meliloti DNA. The cloned R. meliloti carA and carB genes were unable to complement E. coli carA or carB mutants alone or in combination. We speculate on the mechanism of the unusual pattern of genetic complementation at the R. meliloti carB locus.     

Cloning, characterization, and complementation of lesions causing acid sensitivity in Tn5-induced mutants of Rhizobium meliloti WSM419.

Four Tn5-induced mutants of Rhizobium meliloti WSM419 were unable to grow or maintain intracellular pH at an external pH of 5.6. Restriction analysis of DNA fragments carrying Tn5 and flanking sequences cloned from these mutants indicated that all four cloned mutations are unique and that the two strains (TG1-6 and TG1-11) carry Tn5 insertions which are within 4.4 kilobases of each other on a single EcoRI fragment. Southern analysis of total mutant DNA indicated a single copy of Tn5 in each mutant. A limited cosmid gene bank of wild-type WSM419 DNA was probed for homology to mutant DNA cloned from the acid-sensitive mutants. Dot hybridization experiments identified one cosmid element within this bank carrying wild-type DNA sequences corresponding to DNA implicated in acid tolerance. This cosmid was able to complement defects in growth and intracellular pH maintenance in TG1-11 but not TG1-6.     

Isolation and characterization of Azospirillum brasilense loci that correct Rhizobium meliloti exoB and exoC mutations.

The occurrence in Azospirillum brasilense of genes that code for exopolysaccharide (EPS) synthesis was investigated through complementation studies of Rhizobium meliloti Exo- mutants. These mutants are deficient in the synthesis of the major acidic EPS of Rhizobium species and form empty, non-nitrogen-fixing root nodules on alfalfa (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We demonstrated that the exoC mutation of R. meliloti could be corrected for EPS production by several cosmid clones of a clone bank of A. brasilense ATCC 29145. However, the EPS produced differed in structure from the wild-type R. meliloti EPS, and the symbiotic deficiency of the exoC mutation was not reversed by any of these cosmid clones. The exoB mutation could be corrected not only for EPS production but also for the ability to form nitrogen-fixing nodules on alfalfa by one particular cosmid clone of A. brasilense. Tn5 insertions in the cloned DNA were isolated and used to construct Azospirillum mutants with mutations in the corresponding loci by marker exchange. It was found that these mutants failed to produce the wild-type high-molecular-weight EPS, but instead produced EPSs of lower molecular weight.     

Isolation and characterization of the recA gene of Rhizobium meliloti.

Interspecific complementation of an Escherichia coli recA mutant with plasmids containing a gene bank of Rhizobium meliloti DNA was used to identify a clone which contains the recA gene of R. meliloti. The R. meliloti recA protein can function in recombination and in response to DNA damage when expressed in an E. coli recA host, and hybridization studies have shown that DNA sequence homology exists between the recA gene of E. coli and that of R. meliloti. The isolated R. meliloti recA DNA was used to construct a recA R. meliloti, and this bacterium was not deficient in its ability to carry out symbiotic nitrogen fixation.     

Isolation and characterization of an R-prime plasmid from Rhizobium meliloti.

Using a simple enrichment procedure, we isolated an R-prime derivative of plasmid R68.45 carrying a 17.8-megadalton segment of the Rhizobium meliloti 41 chromosome. The chromosomal segment carried on this plasmid (pGY1) includes the markers cys-24+, cys-46+, and att16-3. Plasmid pGY1 mobilized the chromosome in a polarized way starting from the region of homology, but cannot promote chromosome transfer from other sites. The att16-3 site on pGY1 allowed the integration of phage 16-3 into pGY1, and a composite plasmid of 91.8 megadaltons was formed. This vector (pGY2) is suitable for the introduction of Rhizobium bacteriophage 16-3 into other gram-negative bacteria.     

Characterization of polysaccharides of Rhizobium meliloti exo mutants that form ineffective nodules.

Mutants of Rhizobium meliloti SU47 with defects in the production of the Calcofluor-binding expolysaccharide succinoglycan failed to gain entry into alfalfa root nodules. In order to define better the polysaccharide phenotypes of these exo mutants, we analyzed the periplasmic oligosaccharide cyclic (1-2)-beta-D-glucan and lipopolysaccharide (LPS) in representative mutants. The exoC mutant lacked the glucan and had abnormal LPS which appeared to lack a substantial portion of the O side chain. The exoB mutant had a spectrum of LPS species which differed from those of both the wild-type parental strain and the exoC mutant. The presence of the glucan and normal LPS in the exoA, exoD, exoF, and exoH mutants eliminated defects in these carbohydrates as explanations for the nodule entry defects of these mutants. We also assayed for high- and low-molecular-weight succinoglycans. All of the exo mutants except exoD and exoH completely lacked both forms. For the Calcofluor-dim exoD mutant, the distribution of high- and low-molecular-weight forms depended on the growth medium. The haloless exoH mutant produced high-molecular-weight and only a trace of low-molecular-weight succinoglycan; the succinyl modification was missing, as was expected from the results of previous studies. The implications of these observations with regard to nodule entry are discussed.     

Symbiotic pseudorevertants of Rhizobium meliloti ndv mutants.

Nodule development (ndv) mutants of Rhizobium meliloti cannot invade alfalfa to establish a nitrogen-fixing symbiosis and instead induce the formation of small, white, unoccupied nodules on alfalfa roots. Such mutants also fail to produce the unusual cyclic oligosaccharide beta-(1----2)-glucan and show defects in several aspects of vegetative growth and function. Here we show that ndv mutants are severely reduced, although not totally deficient, in the ability to attach to and initiate infection threads on alfalfa seedlings, and we demonstrate that the symbiotic deficiency can be separated from the rest of the mutant phenotype by isolating second-site pseudorevertants. Pseudorevertants selected for restoration of motility, a vegetative property, regained a substantial amount of attachment capability but only slight infection thread initiation and symbiotic ability. Such strains also regained partial tolerance to growth at low osmolarity, even though they did not recover the ability to synthesize periplasmic beta-(1----2)-glucan. Pseudorevertants selected on alfalfa for restoration of symbiosis were unrestored for beta-(1----2)-glucan production or any other vegetative property and regained little or no attachment or infection thread initiation capability. We take these data to indicate that wild-type R. meliloti normally has considerable excess capability for both attachment and infection thread initiation and that the symbiotic block in ndv mutants lies further along the developmental pathway than either of these processes, probably at the level of infection thread extension. Further, the fact that neither type of pseudorevertant recovered the ability to produce periplasmic beta-(1----2)-glucan raises the possibility that this oligosaccharide is not directly required for nodule development.     

Symbiotic mutants of Rhizobium meliloti that uncouple plant from bacterial differentiation

Spontaneous mutants at a new symbiotic locus in Rhizobium meliloti SU47 are resistant to several phages and are conditionally insensitive to a monoclonal antibody to the bacterial surface, apparently because they are deficient in a wild-type exopolysaccharide. On alfalfa, the mutants do not curl root hairs, but penetrate the epidermis directly, forming nodules that contain no visible infection threads or "bacteroids," have a few bacteria in superficial intercellular spaces only and not within the nodule cells, and fail to fix nitrogen (Fix-). Evidently, infection threads are not essential for cell proliferation and nodule formation, which are here induced by a bacterial signal at a distance and uncoupled from the bacterial differentiation that normally goes on as well.     

Isolation of Klebsiella aerogenes mutants cis-dominant for glutamine synthetase expression

We have isolated three strains of Klebsiella aerogenes that failed to show repression of glutamine synthetase even when grown under the most repressing conditions for the wild-type strain. These mutant strains were selected as glutamine-independent derivatives of a strain that is merodiploid for the glnA region and contains a mutated glnF allele. The mutation responsible for the Gln+ phenotype in each strain was tightly linked to glnA, the structural gene for glutamine synthetase, and was dominant to the wild-type allele. These mutations are probably lesions in the control region of the glnA gene, since each mutation was cis-dominant for constitutive expression of the enzyme in hybrid merodiploid strains. Strains harboring this class of mutations were unable to produce a high level of glutamine synthetase unless they also contained an intact glnF gene, and unless cells were grown in derepressing medium. This study supports the idea that the glnA gene is regulated both positively and negatively, and that the deoxyribonucleic acid sites critical for positive control and negative control are functionally distinct.     

Isolation and characterization of the constitutive acyl carrier protein from Rhizobium meliloti.

Rhizobium species produce an inducible acyl carrier protein (ACP), encoded by the nodF gene, that somehow functions in an exchange of cell signals between bacteria and specific plant hosts, leading to nodulation of plant roots and symbiotic nitrogen fixation, as well as a constitutive ACP needed for the synthesis of essential cell lipids. The periplasmic cyclic glucans of Rhizobium spp. are also involved in specific rhizobium-plant interaction. These glucans are strongly similar to the periplasmic membrane-derived oligosaccharides (MDO) of Escherichia coli. E. coli ACP is an essential component of a membrane-bound transglucosylase needed for the biosynthesis of MDO, raising the possibility that either or both of the rhizobial ACPs might have a similar function. We have now isolated the constitutive ACP of R. meliloti and determined its primary structure. We have also examined its function, together with those of ACPs from E. coli, Rhodobacter sphaeroides, and spinach, in the MDO transglucosylase system and as substrate for the E. coli ACP acylase enzyme. All four ACPs act as acceptors of acyl residues, but only the E. coli ACP functions in the transglucosylase system.     

Cloning and characterization of a second acid phosphatase from Sinorhizobium meliloti strain 104A14

Sinorhizobium meliloti has two nonspecific periplasmic acid phosphatases. The NapD enzyme has been previously described, and a second acid phosphatase, NapE, is described in this report. NapE was partially purified from an S. meliloti napD mutant and characterized with respect to molecular mass and substrate range. As predicted from SDS-PAGE analysis, the subunit molecular mass of NapE is approximately 35.8 kDa and gel filtration experiments estimated the native molecular mass to be approximately 70 kDa, indicating that the active enzyme is a homodimer. NapE demonstrated significant activity with p-nitrophenyl phosphate, phenyl phosphate, and alpha-naphthyl-phosphate. The pH optimum was between 4.5 and 5.0. The gene encoding NapE was also sequenced and the inferred amino acid sequence from the predicted ORF was found to be 60% identical and 75% similar to that encoded by napD. An S. meliloti napE mutant was constructed and assessed for symbiotic competence. This mutant did not differ from the wild-type parent strain in nodulation and symbiotic efficiency.     

Isolation and characterization of glutamine synthetase from the marine diatom Skeletonema costatum.

Two peaks of glutamine synthetase (GS) activity were resolved by anion-exchange chromatography from the marine diatom Skeletonema costatum Grev. The second peak of activity accounted for greater than 93% of total enzyme activity, and this isoform was purified over 200-fold. Results from denaturing gel electrophoresis and gel-filtration chromatography suggest that six 70-kD subunits constitute the 400-kD native enzyme. The structure of the diatom GS, therefore, appears more similar to that of a type found in bacteria than to the type common among other eukaryotes. Apparent Michaelis constant values were 0.7 mM for NH4(+), 5.7 mM for glutamic acid, and 0.5 mM for ATP. Enzyme activity was inhibited by serine, alanine, glycine, phosphinothricin, and methionine sulfoximine. Polyclonal antiserum raised against the purified enzyme localized a single polypeptide on western blots of S. costatum cell lysates and recognized the denatured, native enzyme. Western analysis of the two peak fractions derived from anion-exchange chromatography demonstrated that the 70-kD protein was present only in the later eluting peak of enzyme activity. This form of GS does not appear to be unique to S. costatum, since the antiserum recognized a similar-sized protein in cell lysates of other chromophytic algae.     

Isolation and characterization of CHO cell mutants with altered asparagine synthetase.

Two asparagine auxotrophic mutants (N3, N4) were isolated from the Gat- strain of Chinese hamster ovary cells, using a selection procedure modified from that of Goldfarb et al. (1). The defect in these mutants is due to a deficiency in asparagine synthetase activity. N3, in particular, had no measurable enzyme activity. Complementation analysis by PEG-mediated cell fusion showed that the auxotrophic phenotype behaved as a recessive trait; complementation was obtained between N3 or N4 and the pseudoauxotroph, Asn3, which has a temperature-sensitive asparagyl-tRNA synthetase activity. Revertants obtained by plating N3 or N4 in asparagine-free medium had about normal levels of asparagine synthetase activity and were produced with a probability of about 10(-6) per cell per generation. Three particular revertants of N3 and one revertant of N4 were shown to have asparagine synthetase activities that were different in thermolability from that of the wild type. This observation is consistent with the suggestion that N3 and N4 have defective structural genes rather than defective regulatory genes for asparagine synthetase.     

Isolation and characterization of Rhizobium etli mutants altered in degradation of asparagine.

Rhizobium etli mutants unable to grow on asparagine as the nitrogen and carbon source were isolated. Two kinds of mutants were obtained: AHZ1, with very low levels of aspartase activity, and AHZ7, with low levels of asparaginase and very low levels of aspartase compared to the wild-type strain. R. etli had two asparaginases differentiated by their thermostabilities, electrophoretic mobilities, and modes of regulation. The AHZ mutants nodulated as did the wild-type strain and had nitrogenase levels similar to that of the wild-type strain.     

Isolation and characterization of thymidylate synthetase mutants of Xanthomonas maltophilia

Thymidylate synthetase mutants of Xanthomonas maltophilia ATCC 13270 were isolated on a solid minimal medium containing 50 mg/l thymidine and a high concentration of trimethoprim (500 mg/l). It was found that a high concentration of trimethoprim was required to prevent background growth of the wild-type strain. The isolated mutants could grow on thymidine or dTMP at a concentration of 50 mg/l while they were unable to grow on 1000 mg/l thymine or 50 mg/l deoxyridine. Thymidylate synthetase activity was assayed in the wild-type cells and in the mutant cells but only the wild-type cells contained measurable enzyme activity.