Biomedical subjects

M F Hynes

Publications and source records attributed to M F Hynes.

At least 19 recordsLinked to original sources

PubMed2001

RpoN of Rhizobium leguminosarum bv. viciae strain VF39SM plays a central role in FnrN-dependent microaerobic regulation of genes involved in nitrogen fixation.

The rpoN gene, which codes for the alternative transcription factor sigma54, was cloned and sequenced from Rhizobium leguminosarum strain VF39SM. Construction of a rpoN mutant allowed analysis of the role of RpoN as a transcriptional regulator of genes carrying lacZ reporter fusions. Analysis of a rpoN::lacZ transcriptional fusion in the rpoN background revealed that this gene was negatively autoregulated. Site-directed mutagenesis was used to demonstrate that this autoregulation was dependent on a reverse complement RpoN binding site located upstream of the rpoN gene. rpoN was shown to be required for full microaerobic expression of both copies of fixGHIS, as well as of fixNOQP, despite the absence of apparent rpoN binding sites upstream of fixG. Moreover, rpoN was found to be required for full microaerobic expression of fnrN, which in turn is absolutely required for microaerobic induction of fixGHIS. This suggests that the reduced fixG::lacZ expression seen in the rpoN background is due to the dependence of fnrN expression on RpoN.

Bacterial Proteins↗

PubMed2001

Analysis of the genetic region encoding a novel rhizobiocin from Rhizobium leguminosarum bv. viciae strain 306.

Cross-testing of a number of strains of Rhizobium leguminosarum for bacteriocin production revealed that strain 306 produced at least two distinct bacteriocins. Further analysis involving plasmid transfer to Agrobacterium and other hosts demonstrated that there were bacteriocin determinants on plasmids pRle306b and pRle306c, as well as a third bacteriocin. The bacteriocin encoded by pRle306b was indistinguishable from the bacteriocin encoded by strain 248, whereas the bacteriocin encoded by plasmid pRle306c had a distinctive spectrum of activity against susceptible strains, as well as different physical properties from other bacteriocins that we have studied in our lab. Two mutants altered in production of the pRle306c bacteriocin were generated by transposon Tn5 mutagenesis, and the DNA flanking the transposon inserts in these mutants was cloned and characterized. DNA sequence analysis suggested that the pRle306c bacteriocin was a large protein belonging to the RTX family, and that a type I secretion system involving an ABC type transporter was required for export of the bacteriocin. A mutant unable to produce this bacteriocin was unaltered in its competitive properties, both in broth and in nodulation assays, suggesting that the bacteriocin may not play a major role in determining the ecological success of this strain.

Bacterial Proteins↗

PubMed2000

Proteome analysis demonstrates complex replicon and luteolin interactions in pSyma-cured derivatives of Sinorhizobium meliloti strain 2011.

Sinorhizobium meliloti was studied by proteomic analysis to investigate the contribution made by plasmid-encoded functions on the intracellular regulation of this bacterium. Protein profiles of strain 2011 were compared with those from its mutant strains which were either cured of their pRme2011a (also called pSyma) plasmid (strain 818), or contained an extensive deletion of this plasmid (strain SmA146). Plasmid pSyma contains the nodulation and nitrogen fixation genes and is 1.4 Mbp with an estimated coding potential of 1,400 proteins. However, under the growth conditions used we could detect 60 differences between the parent strain and its pSyma-cured derivative, strain 818. While the majority of these differences were due to regulatory changes, such as up- and downregulation, some proteins were totally missing in some strains. These 60 proteins were classified into 21 subgroups, A to U, based on their measured protein levels when the cells were grown in the presence or absence of luteolin. Comparisons were made between the different strains to assess the possible interactions of the different proteins of the subgroups and plasmid pSyma. These results suggest that pSyma has a role in the regulation of the expression of genes from the other replicons (3.5 Mbp chromosome and the 1.7 Mbp pSymB plasmid) present in the S. meliloti cells. Proteome analysis provides a sensitive tool to examine the functional organisation of the S. meliloti genome and the intracellular gene interactions between replicons and will provide a powerful analytical tool to complement the genome sequencing of strain 1021.

Bacterial Proteins↗

PubMed2000

Megaplasmid pRme2011a of Sinorhizobium meliloti is not required for viability.

We report the curing of the 1,360-kb megaplasmid pRme2011a from Sinorhizobium meliloti strain Rm2011. With a positive selection strategy that utilized Tn5B12-S containing the sacB gene, we were able to cure this replicon by successive rounds of selecting for deletion formation in vivo. Subsequent Southern blot, Eckhardt gel, and pulsed-field gel electrophoresis analyses were consistent with the hypothesis that the resultant strain was indeed missing pRme2011a. The cured derivative grew as well as the wild-type strain in both complex and defined media but was unable to use a number of substrates as a sole source of carbon on defined media.

Blotting, Southern↗

PubMed1999

Cloning and characterization of a Rhizobium leguminosarum gene encoding a bacteriocin with similarities to RTX toxins.

A 3-kb region containing the determinant for bacteriocin activity from Rhizobium leguminosarum 248 was isolated and characterized by Tn5 insertional mutagenesis and DNA sequencing. Southern hybridizations showed that this bacteriocin was encoded on the plasmid pRL1JI and that homologous loci were not found in other unrelated R. leguminosarum strains. Tn5 insertional mutagenesis showed that mutations in the C-terminal half of the bacteriocin open reading frame apparently did not abolish bacteriocin activity. Analysis of the deduced amino acid sequence revealed that, similarly to RTX proteins (such as hemolysin and leukotoxin), this protein contains a characteristic nonapeptide repeated up to 18 times within the protein. In addition, a novel 19- to 25-amino-acid motif that occurred every 130 amino acids was detected. Bacteriocin bioactivity was correlated with the presence of a protein of approximately 100 kDa in the culture supernatants, and the bacteriocin bioactivity demonstrated a calcium dependence in both R. leguminosarum and Sinorhizobium meliloti. A mutant of strain 248 unable to produce this bacteriocin was found to have a statistically significant reduction in competitiveness for nodule occupancy compared to two test strains in coinoculation assays. However, this strain was unable to compete any more successfully with a third test strain, 3841, than was wild-type 248.

Amino Acid Sequence↗

PubMed1998

Decreased symbiotic effectiveness of Rhizobium leguminosarum strains carrying plasmid RP4.

The presence of derivatives of the broad host range plasmid RP4 in strains of Rhizobium leguminosarum biovar viciae severely inhibited nitrogen fixation by these strains in nodules on cultivars of pea (Pisum sativum). The strains formed small white nodules. Yield and total nitrogen values were comparable with those obtained for plants inoculated with a non-nodulating mutant. Strains carrying the same derivatives gave rise to nitrogen fixing nodules when inoculated on cultivars of lentils (Lens culinaris). Similar results were observed with plasmid R702 but not with R751, suggesting that the effect is limited to plasmids of the IncP alpha classification. Histological examination of nodules induced by strains carrying RP4 indicated that there are fewer infected cells and starch granules are organised unusually in the infected cells. Tn5 mutagenesis of plasmid RP4-4 was undertaken and Tn5 inserts were screened for abolition of the effect on nitrogen fixation. Eight mutants, having no effect on nitrogen fixation, were isolated. Seven of these had lost the ability to transfer by conjugation and the eighth was greatly reduced in conjugation frequency. Physical analysis of the transposon inserts revealed that they were located in the Tra regions of RP4.

DNA Transposable Elements↗

PubMed1998

Rhizobium tropici teu genes involved in specific uptake of Phaseolus vulgaris bean-exudate compounds.

Rhizobium tropici nodulates and fixes nitrogen in bean. In the R. tropici strain CFN299 we identified and characterized teu genes (tropici exudate uptake) induced by bean root exudates, localized by insertion of a promoter-less Tn5-gusA1 transposon. teu genes are present on a plasmid of around 185 kb that is conserved in all R. tropici strains. Proteins encoded by teu genes show similarity to ABC transporters, specifically to ribose transport proteins. No induction of the teu genes was obtained by treatment with root exudates from any of several other plants tested, with the exception of Macroptilium atropurpureum, which is also a host plant for R. tropici. It appears that the inducing compound is characteristic of bean and closely related legumes. It is present in root exudates, but not in seeds. This compound is removed, presumably by metabolism, from the exudates by the majority of bean-nodulating rhizobia (such as R. etli, R. leguminosarum bv. phaseoli and R. giardinii). The principal inducing compound has not been identified, but some induction was obtained using trigonelline. The CFN299 strain seems to have an additional uptake system, as no phenotype is observed in two different mutants. R. tropici strain CIAT899, on the other hand, must have only one uptake system, since a mutant bearing an insertion in the teu genes could not remove the compound from the exudates as efficiently as the wild type, and it showed diminished nodulation competitiveness.

ATP-Binding Cassette Transporters↗

PubMed1998

Expression of a Bacillus thuringiensis delta-endotoxin gene by Bacillus pumilus.

The delta-endotoxin genes from Bacillus thuringiensis were introduced into a rhizosphere-inhabiting Bacillus pumilus isolate to create a delta-endotoxin expression and delivery system for subterranean feeding insects such as the larvae of pale western cutworm (Agrotis orthogonia Morrison (Lepidoptera: Noctuidae)). Preliminary experiments indicated that Bacillus thuringiensis subsp. kurstaki cultures were toxic to pale western cutworm larvae. Three different cry genes from Bacillus thuringiensis subsp. kurstaki were cloned into high and low copy number vectors and mated into Bacillus pumilus RB8. When carried on high copy number vectors, cry genes appeared to inhibit sporulation and delta-endotoxin production in Bacillus pumilus RB8 cultures, since microscopic examination of these cultures revealed that < 0.1% of the cells of late stationary phase cultures had sporulated and produced parasporal inclusions. On low copy number vectors, the cry genes did not inhibit sporulation; however, production of delta-endotoxins was undetectable. Using a heat shock regime for enrichment of sporogenous crystalliferous variants, a Bacillus pumilus isolate, carrying cryIA(c) on a high copy number plasmid, was obtained in which high level delta-endotoxin production occurred concomitant with sporulation. Synthesis of functional delta-endotoxin by this strain was confirmed by Western blot analysis and bioassay with pale western cutworm larvae. These results show that rhizosphere-inhabiting bacilli are indeed a potential route for introduction of delta-endotoxins to the root environment for biocontrol purposes.

Animals↗

PubMed1997

Transposon-like structure of a new plasmid-encoded restriction-modification system in Rhizobium leguminosarum VF39SM.

Total DNA isolated from Rhizobium leguminosarum VF39SM cells is resistant to cleavage by the restriction endonuclease PstI. Plasmid curing and transfer studies localized this phenotype to pRleVF39b, the second smallest of six plasmids found in this bacterium. In vitro selection for vector modification was employed to isolate a presumptive methylase gene (M.Rle39BI) from a plasmid gene library. Total and plasmid DNAs isolated from E. coli containing M.RleBI were resistant to digestion by PstI. Sequence data suggested that a putative restriction endonuclease (R.Rle39BI) was also encoded on the same fragment. The two genes were flanked by identical copies of a putative insertion sequence, which was also present in several copies elsewhere in the VF39SM genome. The presence of this element in other strains examined suggested that this element is indeed an insertion sequence. The differences in G/C content between the DNA coding for the R/M system and that of the IS element suggest that this DNA region may have been acquired by horizontal transfer.

Amino Acid Sequence↗

PubMed1997

Functional and regulatory analysis of the two copies of the fixNOQP operon of Rhizobium leguminosarum strain VF39.

DNA corresponding to two copies of the Rhizobium leguminosarum bv. viciae strain VF39 fixNOQP operon coding for a putative symbiotic terminal oxidase of the heme-copper oxidase superfamily was cloned, sequenced, and genetically analyzed. The first copy is located upstream of the fixK-fixL region on plasmid pRleVF39c, whereas the second copy resides on the nodulation plasmid pRleVF39d. Insertional mutagenesis with antibiotic resistance cassettes confirmed that both copies were functional, and that the presence of at least one functional copy was required for nitrogen fixation. The deduced amino acid sequences of both fixN genes are highly similar (95% identity) and contain 15 putative transmembrane helices, suggesting that the fixN gene products are integral membrane proteins. Furthermore, six histidine residues predicted to be the ligands for a heme-copper binuclear center and a low-spin heme b are conserved in both R. leguminosarum fixN proteins. The deduced fixO and fixP gene products show characteristics of membrane-bound monoheme and diheme cytochrome c, respectively. Upstream of both fixN copies putative Fnr-consensus binding sites (anaeroboxes) were found that differ in certain base pairs. As R. leguminosarum VF39 possesses two members of the Fnr/FixK regulator family, FnrN and FixK, the possible differential regulation of both fixN copies was analyzed with fixN-gusA reporter gene fusions. Both fixN fusions were induced under free-living microaerobic conditions and in the symbiotic zone of the root nodule. Induction of the expression of fixNc and fixNd was highly reduced in a fnrN mutant background and in a fixL mutant background, whereas fixK was only marginally involved in fixN regulation. Residual expression of fixN was observed in an fnrN/fixK double mutant.

Amino Acid Sequence↗

PubMed1996

Rhizobium leguminosarum as a plant growth-promoting rhizobacterium: direct growth promotion of canola and lettuce.

Early seedling root growth of the nonlegumes canola (Brassica campestris cv. Tobin, Brassica napus cv. Westar) and lettuce (Lactuca sativa cv. Grand Rapids) was significantly promoted by inoculation of seeds with certain strains of Rhizobium leguminosarum, including nitrogen- and nonnitrogen-fixing derivatives under gnotobiotic conditions. The growth-promotive effect appears to be direct, with possible involvement of the plant growth regulators indole-3-acetic acid and cytokinin. Auxotrophic Rhizobium mutants requiring tryptophan or adenosine (precursors for indole-3-acetic acid and demonstrate a new facet of the Rhizobium-plant relationship and that Rhizobium leguminosarum can be considered a plant growth-promoting rhizobacterium (PGPR).

Indoleacetic Acids↗

PubMed1994

A conjugative transfer system for the rumen bacterium, Butyrivibrio fibrisolvens, based on Tn916-mediated transfer of the Staphylococcus aureus plasmid pUB110.

A limitation of genetic studies of the rumen bacterium, Butyrivibrio fibrisolvens, has been the availability of suitable vectors and transfer systems. Using the conjugative tetracycline resistant transposon, Tn916, the Staphylococcus aureus plasmid, pUB110, and the pUB110-based shuttle vector, pUBLRS, a conjugative transfer system was developed for B. fibrisolvens. B fibrisolvens donor strains H17c2 and H17c12, containing Tn916 and pUB110 or pUBLRS, respectively, were used in mating experiments with selected B. fibrisolvens strains. Kanamycin resistant transconjugants, containing pUB110, of strains 193, 194, and 195 were detected at a combined average frequency of 7.78 x 10(-7) per donor and 1.11 x 10(-5) per recipient. Transconjugants of strains 193 and 194, containing pUBLRS, were detected at an average frequency of 1.22 x 10(-6) per donor and 4.70 x 10(-8) per recipient. Southern hybridization analysis confirmed the presence of pUB110 and pUBLRS in transconjugants. Results indicated that Tn916 was necessary for mobilization of pUB110 as transconjugants were not detected when the transposon was absent from the donor strains. The ability to mobilize pUB110 and pUBLRS between B. fibrisolvens strains provides a conjugative transfer system that circumvents problems encountered with electroporation.

Animals↗

PubMed1993

Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria.

A set of vector plasmids which greatly facilitate gene replacement and reverse genetics in many Gram-negative bacteria was constructed. These vectors are based on the P15A origin of replication (ori) and incorporate sacB from Bacillus subtilis, which is inducible by sucrose and is lethal when expressed in Gram-negative bacteria. The vectors also have a convenient antibiotic-resistance marker (gentamicin resistance) and the lacZ alpha system which allows blue/white selection of cloned fragments. Three different multiple cloning sites, allowing several distinct cloning and gene replacement strategies, are available in the 5' end of lacZ on different vectors. One of these cloning sites, which we synthesised, contains only a NotI-SmaI-NotI sequence; this allows access to most of the restriction sites within the cloned fragment for the purpose of insertion of various cassettes and interposons. The vectors carry the mob region from the broad-host-range plasmid RP4 and are thus mobilizable by conjugation into a wide range of Gram-negative bacteria; since they will not replicate in bacteria other than enterobacteria, they function as 'suicide' vectors. Variants of the vectors carrying the phage lambda cos site were also constructed. We have used these vectors to carry out gene replacement experiments in the fixN region of Rhizobium leguminosarum and have demonstrated that they are extremely useful in eliminating long and tedious screening procedures.

Amino Acid Sequence↗

PubMed1992

Cloning and expression of an amylase gene from Streptococcus bovis in Escherichia coli.

An amylase gene was identified in a Streptococcus bovis 033 lambda gtWES lambda B genomic library. Using a starch overlay and a Congo red-iodine staining procedure, amylase positive clones could be identified by zones of clearing. Ten amylase positive clones were identified using this procedure. The clone chosen for further study, lambda SBA105, contained an insert of approximately 7.5 kb. The insert was mapped, and subcloning localized the amylase gene to a region of approximately 3.1 kb. Cloning of the 3.1 kb amylase fragment into pUC18 in both orientations revealed that the amylase gene was transcribed from its own promoter. Amylase activity was expressed by the Escherichia coli subclones and was found to be largely associated with the cytoplasmic fraction. Southern hybridization of genomic DNA from the amylolytic strains, S. bovis 033, S. bovis 077, Butyrivibrio fibrisolvens 194 and 195 revealed a single hybridizing band in S. bovis 033 DNA only. This indicates that the amylase gene from S. bovis may differ from the amylases of these other amylolytic bacteria.

Amylases↗

PubMed1992

Utilization of carbon substrates, electrophoretic enzyme patterns, and symbiotic performance of plasmid-cured clover rhizobia.

Plasmids in Rhizobium spp. are relatively large, numerous, and difficult to cure. Except for the symbiotic plasmid, little is known about their functions. The primary objective of our investigation was to obtain plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii by using a direct selection system and to determine changes in the phenotype of the cured strains. Three strains of rhizobia were utilized that contained three, four, and five plasmids. Phenotypic effects observed after curing of plasmids indicated that the plasmids were involved in the utilization of adonitol, arabinose, catechol, glycerol, inositol, lactose, malate, rhamnose, and sorbitol and also influenced motility, lipopolysaccharide production, and utilization of nitrate. Specific staining of 26 enzymes electrophoretically separated on starch gels indicated that superoxide dismutase, hexokinase, and carbamate kinase activities were affected by curing of plasmids. Curing of cryptic plasmids also influenced nodulation and growth of plants on nitrogen-deficient media. The alteration in the ability to utilize various substrates after curing of plasmids suggests that the plasmids may encode genes that contribute significantly to the saprophytic competence of rhizobia in soil.

Journal Article↗

PubMed1992

Partial characterization of the gonadotropin-releasing hormone (GnRH) gene transcript in the rat ovary.

It has been hypothesized that GnRH or a GnRH-like peptide is produced in the rat ovary, but the presence of GnRH in the ovary has not been unequivocally demonstrated. This study was undertaken to determine whether the GnRH gene is expressed in the rat ovary and to compare the GnRH gene transcripts from the ovary and the hypothalamus. Twelve samples of total RNA from ovaries of individual rats were screened by reverse transcription-polymerase chain reaction (RT-PCR) for the presence of GnRH gene transcripts. Fragments of GnRH cDNA were amplified using pairs of specific primers. GnRH transcripts were detected in all the ovaries examined, and differed from hypothalamic GnRH transcripts in two ways: first, in the ovaries a greater proportion of GnRH transcripts contained intronic sequences; second, the major transcription start utilized in the ovary differed from that used in the hypothalamus. Although fully processed GnRH gene transcripts were detected by RT-PCR in both, ovary and hypothalamus, they were not detected in the ovary by Northern blot. The GnRH probe hybridized specifically to the predicted 0.6 kb transcript in the hypothalamus, and to a 3.3 kb transcript in the ovary. We conclude that in the ovary, most GnRH gene transcripts retain intronic sequences.

Animals↗

PubMed1990

Two plasmids other than the nodulation plasmid are necessary for formation of nitrogen-fixing nodules by Rhizobium leguminosarum.

A system which allows direct selection for curing of plasmids in Gram-negative bacteria was used to generate derivatives of Rhizobium leguminosarum VF39 cured of each of six plasmids present in this strain. Phenotypes could be correlated with the absence of five of the six plasmids. The smallest plasmid, pRleVF39a, carries genes for the production of a melanin-like pigment as has been previously reported. Plasmid pRleVF39d carries nodulation and nitrogen fixation genes. Curing of the plasmids pRleVF39c and pRleVF39e gave rise to strains which formed Fix- nodules on peas, lentils, and faba beans. The nodules formed by the strains cured of pRleVF39c contained few, if any, bacteria. Analysis of washed cells by SDS-PAGE showed that this strain is defective in lipopolysaccharide (LPS) production; the defect could be complemented by introducing plasmids from several other R. leguminosarum strains, and by the R. leguminosarum biovar phaseoli LPS gene clones pCos126 and pDel27. The nodules formed by the strain cured of pRleVF39e had a reduced symbiotic zone, an enlarged senescence zone, and an abundance of starch granules. This strain grew at a much slower rate than the wild type, was unable to grow on minimal medium, and no longer produced melanin. These defects could be complemented by at least one other Rhizobium plasmid, pRle336e, a plasmid of strain 336 which is distinct from the nodulation plasmid (pRle336c) and the plasmid (pRle336d) which could complement the LPS defect associated with the loss of pRleVF39c. This demonstrates that genes necessary for symbiosis can be carried on at least three different plasmids in R. leguminosarum.

DNA Transposable Elements↗

PubMed1990

Mobilization of closely related plasmids pUB110 and pBC16 by Bacillus plasmid pXO503 requires trans-acting open reading frame beta.

Genetic analysis of the closely related nonconjugative plasmids pUB110 and pBC16 has demonstrated that the open reading frame beta (ORF-beta) region in pUB110 and the corresponding homologous region in pBC16 are essential for mobilization of these plasmids by pLS20 or its derivatives. Deletions in this region or insertions that interrupted ORF-beta severely impaired or eliminated the mobilization of pUB110::pUC18 and pBC16::pUC18 hybrids. In contrast, a hybrid in which pUC18 was inserted into pBC16 at a point outside ORF-beta transferred at a frequency comparable to that of intact pUB110 or pBC16 (10(-4) transcipients per donor cell). The defect of most transfer-deficient (Mob-) hybrid plasmids could be complemented by an intact sister plasmid (i.e., pBC16 for pUB110::pUC18 Mob- hybrids). The inability to complement certain constructs suggested that the origin of transfer might be located in an area 5' to ORF-beta. Furthermore, cloning the region 5' to ORF-beta onto a nonmobilizable pC194::pUC18 construct resulted in a hybrid plasmid, pUCCoriTBC16, that could be mobilized with complementation. These results indicate that mobilization of pUB110 and pBC16 by conjugative helper plasmids requires ORF-beta in trans and at least one other region, including the RSA sequence, which presumably functions as an origin of transfer, in cis.

Bacillus↗