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G C Walker

Publications and source records attributed to G C Walker.

At least 109 records · Page 6Linked to original sources
PubMed1991

Acidic conditions permit effective nodulation of alfalfa by invasion-deficient Rhizobium meliloti exoD mutants.

Rhizobium meliloti exoD mutants are deficient in invasion of alfalfa nodules and, as a consequence, the nodules that exoD strains induce fail to fix nitrogen. These nodules appear to be arrested at the same stage as nodules induced by other exo mutants, which do not make an acidic exopolysaccharide called EPS I, or by ndv mutants, which do not produce a periplasmic cyclic beta(1,2) glucan. However previous genetic and biochemical evidence suggested that the nodule invasion defect of exoD mutants arose from a biochemical deficiency distinct from those of both EPS I-deficient exo mutants and ndv mutants. In this study, we characterize mutant phenotypes of exoD strains in both free-living and symbiotic states. Nodules induced by exoD mutants are generally small and empty of bacteria, and exhibit the same structural features as nodules induced by other invasion-deficient mutants. Putative incipient infection threads were visible in outer cortical cells of these nodules but not in the plant cells in the interior of the nodule. We show that exoD mutants are sensitive to alkaline conditions, ceasing to grow at elevated pH in liquid yeast extract cultures and exhibiting decreased viability in alkaline medium. Interestingly, we find that buffering the plant growth medium at slightly acidic pH (6.0-6.5) restores the ability of exoD mutants to invade alfalfa nodules. exoD mutants are thus alkali sensitive for both free-living and symbiotic phenotypes. This result implies that the nodule invasion defect of exoD mutants arises from their sensitivity to alkaline conditions and, furthermore, that alkaline conditions may obtain in the developing infection thread. The deduced amino acid sequence of ExoD is extremely hydrophobic, suggesting that the protein is membrane associated. We propose models whereby absence of a putative membrane protein might lead to sensitivity to alkaline conditions and consequent arrest of nodule invasion.

Amino Acid Sequence↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

The exoR gene of Rhizobium meliloti affects RNA levels of other exo genes but lacks homology to known transcriptional regulators.

Rhizobium meliloti strains mutant in the exoR gene overproduce an exopolysaccharide called succinoglycan or EPS I. Protein fusions to several different exo genes required for EPS I biosynthesis are expressed at a higher level in an exoR strain than in a wild-type strain, showing that the overproduction of EPS I in exoR strains results at least in part from increased gene expression. This regulation is important to nodulation, since exoR mutants fail to invade alfalfa nodules unless secondary suppressor mutations that cause a decrease in EPS I production occur. Here, we show that an exoR strain contains higher levels of mRNA for other exo genes than does the wild-type parental strain. ExoR therefore most probably exerts its regulatory effect at the level of transcription. In addition, we have localized, subcloned, and sequenced the exoR gene. A newly constructed insertion allele of exoR has the same phenotype as the original mutant. The deduced sequence of ExoR is 268 amino acids long but does not show homology to other sequenced genes.

Amino Acid Sequence↗
PubMed1991

Regulation of Rhizobium meliloti exo genes in free-living cells and in planta examined by using TnphoA fusions.

The exo loci of Rhizobium meliloti are necessary for the production of an acidic exopolysaccharide, EPS I, that is needed for alfalfa nodule invasion by strain Rm1021. We have isolated and characterized alkaline phosphatase fusions made with TnphoA in several exo loci of R. meliloti and used these fusions to examine the subcellular localization of exo gene products and the regulation of exo genes in free-living cells and in planta. In the course of this work, we isolated a new exo locus, exoT. We have obtained evidence that several of the exo loci may encode membrane proteins. The activity of TnphoA fusions in several exo loci is increased two- to fivefold in the presence of the regulatory mutations exoR95 and exoS96. While examining the regulation of the exo gens by exoR95 and exoS96, we found that certain classes of exo mutations are lethal in an exoR95 or exoS96 background unless a plasmid complementing the exo mutation is present. This result has possible implications for the role of these exo loci in EPS I biosynthesis. We have developed a method for staining nodules specifically for the alkaline phosphatase activity present in the inducing bacteria and used this method to show that an exoF::TnphoA fusion is expressed mainly in the invasion zone of the nodule.

Alkaline Phosphatase↗
PubMed1991

The exoD gene of Rhizobium meliloti encodes a novel function needed for alfalfa nodule invasion.

During the symbiotic interaction between alfalfa and the nitrogen-fixing bacterium Rhizobium meliloti, the bacterium induces the formation of nodules on the plant roots and then invades these nodules. Among the bacterial genes required for nodule invasion are the exo genes, involved in production of an extracellular polysaccharide, and the ndv genes, needed for production of a periplasmic cyclic glucan. Mutations in the exoD gene result in altered exopolysaccharide production and in a nodule invasion defect. In this work we show that the stage of symbiotic arrest of exoD mutants is similar to that of other exo and ndv mutants. However, the effects of exoD mutations on exopolysaccharide production and growth on various media are different from the effects of other exo and ndv mutations. Finally, exoD mutations behave differently from other exo mutations in their ability to be suppressed or complemented extracellularly. The results suggest that exoD represents a new class of Rhizobium genes required for nodule invasion, distinct from the other exo genes and the ndv genes. We discuss models for the function of exoD.

Chromosomes, Bacterial↗
PubMed1991

Fatigue and depression in brain-injured patients correlated with quadriceps strength and endurance.

Many brain-injured (BI) patients complain of persistent fatigue that may alter their lifestyles. In order to assess muscular strength and endurance after brain injury, 22 BI men, greater than 9 months postinjury and aged 20 to 51 years, were separated into two groups based on a complaint of fatigue (fatigue n = 13, nonfatigue n = 9); each performed one maximal isometric knee extension at 60 degrees and 20 maximal isokinetic contractions at 20 rpm using a Cybex II dynamometer. A third group of age-matched, able-bodied men (n = 10) were used as controls. A battery of tests assessing the presence of fatigue (using a symptom checklist and two rating scales), depression, anxiety, and health status were given at the time of isokinetic/isometric testing. The mean fatigue rating, a subjective score, for the fatigue group of BI subjects was significantly worse than the other groups (p less than .01). There was a positive correlation between clinically significant Zung depression scores and fatigue rating (r = .46) and between Zung anxiety scores and fatigue rating. The depression scores for those who complained of fatigue were significantly higher than the other groups (p less than .005). Maximal isometric contractions were no different among the three groups. The mean maximal isokinetic torque during the 20 repetitions was greater in controls than in the BI groups, but did not reach statistical significance (p less than .25). There were no significant declines in isokinetic torque in the 20 repetitions for any of the groups, and the fatigue index was nearly equal for all three groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Structural studies of a novel exopolysaccharide produced by a mutant of Rhizobium meliloti strain Rm1021.

The structure of a novel expolysaccharide obtained from a mutant of Rhizobium meliloti strain Rm1021 was elucidated by a combination of enzymic, chemical, and spectroscopic methods. The polysaccharide is composed of a disaccharide repeating-unit, beta-D-Glcp-(1----3)-alpha-D-Galp-(1----3), having a 6-O-acetyl group attached to most D-glucose residues and a 4,6-O-(1-carboxyethylidene) group attached to every D-galactose residue.

Carbohydrate Conformation↗
PubMed1990

Mutations altering heat shock specific subunit of RNA polymerase suppress major cellular defects of E. coli mutants lacking the DnaK chaperone.

An Escherichia coli mutant lacking HSP70 function, delta dnaK52, is unable to grow at both high and low temperatures and, at intermediate temperature (30 degrees C), displays defects in major cellular processes such as cell division, chromosome segregation and regulation of heat shock gene expression that lead to poor growth and genetic instability of the cells. In an effort to understand the roles of molecular chaperones such as DnaK in cellular metabolism, we analyzed secondary mutations (sid) that suppress the growth defects of delta dnaK52 mutants at 30 degrees C and also permit growth at low temperature. Of the five suppressors we analyzed, four were of the sidB class and mapped within rpoH, which encodes the heat shock specific sigma subunit (sigma 32) of RNA polymerase. The sidB mutations affected four different regions of the sigma 32 protein and, in one case, resulted in a several fold reduction in the cellular concentration of sigma 32. Presence of any of the sidB mutations in delta dnaK52 mutants as well as in dnaK+ cells caused down-regulation of heat shock gene expression at 30 degrees C and decreased induction of the heat shock response after shift to 43.5 degrees C. These findings suggest that the physiologically most significant function of DnaK in the metabolism of unstressed cells is its function in heat shock gene regulation.

Bacterial Proteins↗
PubMed1990

Genetic analyses of cellular functions required for UV mutagenesis in Escherichia coli.

In Escherichia coli, most UV and chemical mutagenesis is not a passive process and requires the participation of the umuD and umuC gene products. However, the molecular mechanism of UV mutagenesis is not yet understood and the roles of the UmuD and UmuC proteins have not been elucidated. The umuDC operon is induced by UV irradiation and regulated as part of the SOS response. Genetic evidence now indicates that RecA-mediated cleavage activates UmuD for its role in mutagenesis. The COOH-terminal fragment of UmuD is both necessary and sufficient for this role. The RecA protein appears to have a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD at the time of SOS induction. In addition, we have obtained evidence which indicates that the GroEL and GroES proteins also play a role in UV mutagenesis. Similarities of the amino acid sequence of UmuD to the sequence of gene 45 protein of bacteriophage T4 and of the sequence of UmuC to those of the gene 44 and gene 62 proteins suggest possible roles for UmuD and UmuC in mutagenesis that are supported by preliminary evidence.

Bacterial Proteins↗
PubMed1990

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.

Carbohydrate Sequence↗
PubMed1990

A constitutive O6-methylguanine-DNA methyltransferase of Rhizobium meliloti.

We have identified a DNA methyltransferase activity of the nitrogen-fixing bacterium, Rhizobium meliloti, that repairs O6-methylguanine lesions. Repair of the O6-methylguanine residue results in transfer of the methyl group to a cysteine residue of a 28,000-dalton protein. The O6-methyltransferase activity is expressed constitutively and R. meliloti does not exhibit an adaptive response to alkylating agents.

DNA Repair↗
PubMed1990

Dominant negative umuD mutations decreasing RecA-mediated cleavage suggest roles for intact UmuD in modulation of SOS mutagenesis.

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. The UmuD protein shares homology with a family of proteins that includes LexA and several bacteriophage repressors. UmuD is posttranslationally activated for its role in mutagenesis by a RecA-mediated proteolytic cleavage that yields UmuD'. A set of missense mutants of umuD was isolated and shown to encode mutant UmuD proteins that are deficient in RecA-mediated cleavage in vivo. Most of these mutations are dominant to umuD+ with respect to UV mutagenesis yet do not interfere with SOS induction. Although both UmuD and UmuD' form homodimers, we provide evidence that they preferentially form heterodimers. The relationship of UmuD to LexA, lambda repressor, and other members of the family of proteins is discussed and possible roles of intact UmuD in modulating SOS mutagenesis are discussed.

Amino Acid Sequence↗
PubMed1990

LexA-independent expression of a mutant mucAB operon.

pKM101 is a naturally occurring plasmid that carries mucAB, an analog of the umuDC operon, the gene products of which are required for the SOS-dependent processing of damaged DNA necessary for most mutagenesis. Genetic studies have indicated that mucAB expression is controlled by the SOS regulatory circuit, with LexA acting as a direct repressor. pGW16 is a pKM101 derivative obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis that was originally identified on the basis of its ability to cause a modest increase in spontaneous mutation rate. In this report, we show that pGW16 differs from pKM101 in being able to enhance methyl methanesulfonate mutagenesis and to confer substantial resistance to UV killing in a lexA3 host. The mutation carried by pGW16 is dominant and was localized to a 2.4-kb region of pGW16 that includes the mucAB coding region and approximately 0.6 kb of the 5'-flanking region. We determined the sequence of a 119-bp fragment containing the region upstream of mucAB and identified a single-base-pair change in that region, a G.C-to-A.T transition that alters a sequence homologous to known LexA-binding sites. DNA gel shift experiments indicate that LexA protein binds poorly to a 125-bp fragment containing this mutation, whereas a fragment containing the wild-type sequence is efficiently bound by LexA. This mutation also alters an overlapping sequence that is homologous to the -10 region of Escherichia coli promoters, moving it closer to the consensus sequence. The observation that the synthesis of pGW16-encoded mucAB proteins in maxicells is increased relative to that of pKM101-encoded mucAB proteins even in the absence of a lexA+ plasmid suggests that this mutation also increases the activity of the mucAB promoter.

Bacterial Proteins↗
PubMed1990

The Escherichia coli polB gene, which encodes DNA polymerase II, is regulated by the SOS system.

The dinA (damage inducible) gene was previously identified as one of the SOS genes with no known function; it was mapped near the leuB gene, where the polB gene encoding DNA polymerase II was also mapped. We cloned the chromosomal fragment carrying the dinA region from the ordered Escherichia coli genomic library and mapped the dinA promoter precisely on the physical map of the chromosome. The cells that harbored multicopy plasmids with the dinA region expressed very high levels of DNA polymerase activity, which was sensitive to N-ethylmaleimide, an inhibitor of DNA polymerase II. Expression of the polymerase activity encoded by the dinA locus was regulated by SOS system, and the dinA promoter was the promoter of the gene encoding the DNA polymerase. From these data we conclude that the polB gene is identical to the dinA gene and is regulated by the SOS system. The product of the polB (dinA) gene was identified as an 80-kDa protein by the maxicell method.

DNA Polymerase II↗
PubMed1989

A novel exopolysaccharide can function in place of the calcofluor-binding exopolysaccharide in nodulation of alfalfa by Rhizobium meliloti.

We have found that R. meliloti strain Rm1021, which is known to synthesize a Calcofluor-binding exopolysaccharide (EPS I), also has a cryptic capacity to synthesize a second exopolysaccharide (EPS II). Structural analysis of EPS II has shown that it differs in many respects from EPS I. Genetic analysis indicates that EPS II synthesis requires the products of at least seven loci on the second symbiotic megaplasmid of R. meliloti, and is induced by a mutation, expR101, which causes increased transcription of these genes. Synthesis of EPS II suppresses the symbiotic defects of EPS I-deficient strains on Medicago sativa (alfalfa), but not on four other plants that are normally hosts for Rm1021. These observations suggest that structural features of bacterial exopolysaccharides are involved in the determination of host range. The implications of these results for models of exopolysaccharide function, such as serving as signals to the plant or shielding the bacteria from plant defense responses, are discussed.

Benzenesulfonates↗
PubMed1989

Nucleotide sequence of the Salmonella typhimurium mutL gene required for mismatch repair: homology of MutL to HexB of Streptococcus pneumoniae and to PMS1 of the yeast Saccharomyces cerevisiae.

The mutL gene of Salmonella typhimurium LT2 is required for dam-dependent methyl-directed DNA mismatch repair. We have cloned and sequenced the mutL gene of S. typhimurium LT2 and compared its sequence with those of the hexB gene product of the gram-positive bacterium Streptococcus pneumoniae and the PMS1 gene product of the yeast Saccharomyces cerevisiae. MutL was found to be quite similar to the HexB mismatch repair protein of S. pneumoniae and to the mismatch repair protein PMS1 of the yeast S. cerevisiae. The significant similarities among these proteins were confined to their amino-terminal regions and suggest common evolution of the mismatch repair machinery in those organisms. The DNA sequence for mutL predicted a gene encoding a protein of 618 amino acid residues with a molecular weight of 67,761. The assignment of reading frame was confirmed by the construction of a chimeric protein consisting of the first 30 amino acids of LacZ fused to residues 53 through 618 of MutL. Interestingly, the presence of excess amounts of this fusion protein in wild-type mutL+ cells resulted in a trans-dominant effect causing the cell to exhibit a high spontaneous mutation frequency.

Amino Acid Sequence↗