Biomedical subjects

F O'Gara

Publications and source records attributed to F O'Gara.

At least 19 recordsLinked to original sources

PubMed2006

Exploiting new systems-based strategies to elucidate plant-bacterial interactions in the rhizosphere.

The rhizosphere is the site of intense interactions between plant, bacterial, and fungal partners. In plant-bacterial interactions, signal molecules exuded by the plant affect both primary initiation and subsequent behavior of the bacteria in complex beneficial associations such as biocontrol. However, despite this general acceptance that plant-root exudates have an effect on the resident bacterial populations, very little is still known about the influence of these signals on bacterial gene expression and the roles of genes found to have altered expression in plant-microbial interactions. Analysis of the rhizospheric communities incorporating both established techniques, and recently developed "omic technologies" can now facilitate investigations into the molecular basis underpinning the establishment of beneficial plant-microbial interactomes in the rhizosphere. The understanding of these signaling processes, and the functions they regulate, is fundamental to understanding the basis of beneficial microbial-plant interactions, to overcoming existing limitations, and to designing improved strategies for the development of novel Pseudomonas biocontrol strains.

Bacterial Physiological Phenomena↗

PubMed2005

LST1 and NCR3 expression in autoimmune inflammation and in response to IFN-gamma, LPS and microbial infection.

Many genes in the central region of the major histocompatibility complex (MHC) encode proteins involved in immune and inflammatory responses. In this study, we have further characterized two genes in the MHC class IV region, leucocyte-specific transcript (LST) 1 and natural cytotoxicity-triggering receptor 3 (NCR3) (also known as 1C7 and natural killer (NK)p30). The specific function of LST1 is not known, although expression analysis and functional data suggest an immunomodulatory role. The LST1 gene undergoes extensive alternative splicing, giving rise to both membrane-bound (encoded by exon 3) and soluble isoforms. The NCR3 protein is involved in NK-mediated cytotoxicity and plays a role in NK/dendritic cell crosstalk. Expression of these genes was examined, by real-time reverse transcriptase-polymerase chain reaction, in autoimmune-induced inflammation, specifically rheumatoid-arthritis-affected blood and synovium, and in response to stimulation with inflammatory mediators and bacterial agents. The expression of LST1, specifically splice variants encoding soluble isoforms and NCR3, was increased in rheumatoid-arthritis-affected blood and synovium and was associated with more severe inflammation in the synovium. Furthermore, both genes were significantly up-regulated in response to lipopolysaccharide, interferon (IFN)-gamma and bacterial infection. These findings suggest that NCR3 and soluble isoforms of LST1 may play a role in inflammatory and infectious diseases.

Arthritis, Rheumatoid↗

PubMed2003

Residual impact of the biocontrol inoculant Pseudomonas fluorescens F113 on the resident population of rhizobia nodulating a red clover rotation crop.

A field trial was previously conducted in which sugarbeet seeds were either untreated, inoculated with the biocontrol strain Pseudomonas fluorescens F113Rif, or treated with chemical fungicides. Following harvest of sugarbeet, the field site was sown with uninoculated red clover. The aim of this study was to assess the residual impact of the microbial inoculant (and the fungicide treatment) on the diversity of resident rhizobia nodulating the red clover rotation crop. The percentage of nodules yielding rhizobial isolates after surface disinfection was 67% in the control and 70% in the P. fluorescens F113Rif treatment, but only 23% in the chemical treatment. Isolates were characterized by RAPD analysis. The main RAPD cluster (arbitrarily defined at 70% similarity) was prevalent in all three treatments. In addition, the distribution of RAPD clusters followed a log series model, regardless of the treatment applied, indicating that neither the microbial inoculant nor the fungicide treatment had caused a strong perturbation of the rhizobial population. When the P. fluorescens F113Rif and control treatments were compared using diversity indices, however, it appeared that the genetic diversity of rhizobia was significantly less in the inoculated treatment. The percentage of rhizobia sensitive to 2,4-diacetylphloroglucinol (Phl; the antimicrobial metabolite produced by P. fluorescens F113Rif) fluctuated according to field site heterogeneity, and treatments had no effect on this percentage. Yet, the proportion of Phl-sensitive isolates in the main RAPD cluster was lower in the P. fluorescens F113Rif treatment compared with the control, raising the possibility that the residual impact of the inoculant could have been partly mediated by production of Phl. This impact on the rhizobial population took place without affecting the functioning of the Rhizobium-clover symbiosis.

DNA, Bacterial↗

PubMed2001

Pseudomonas for biocontrol of phytopathogens: from functional genomics to commercial exploitation.

Pseudomonas spp. that can colonise the roots of crop plants and produce antifungal metabolites represent a real alternative to the application of chemical fungicides. Presently, much research is aimed at understanding, at the molecular level, the mechanisms that enable Pseudomonas strains to act as efficient biological control agents. This approach is facilitating the development of novel strains with modified traits for enhanced biocontrol efficacy. However, without solving some inherent problems associated with the effective delivery of microbial inoculants to seeds and without knowledge on the biosafety aspects of novel biocontrol agents, the commercial potential of Pseudomonas spp. for plant disease control will not be realised.

Fungi↗

PubMed2001

Tumour necrosis factor 5' promoter single nucleotide polymorphisms influence susceptibility to rheumatoid arthritis (RA) in immunogenetically defined multiplex RA families.

Tumour necrosis factor (TNF) plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA) and it has been shown that the TNF-lymphotoxin (TNF-LT) region influences susceptibility to RA. To investigate the role of the TNF-LT locus further, inheritance of TNF 5' promoter alleles was determined in multiplex RA families. Six previously defined TNF promoter single nucleotide polymorphisms (SNPs) (-238, -308, -376, -857, -863, -1031) were observed in these families and in addition, a heretofore undocumented adenine (A) to cytosine (C) substitution at position -572 relative to the transcription start site was defined. TNF 5' promoter SNPs were found to co-segregate with specific TNF microsatellite haplotypes. In particular, the SNP -308A allele was found to be inherited with the TNF a2, b3, c1, d1, e3 (H2) microsatellite haplotype (P < 0.001) which had previously been found to be associated with RA in individuals heterozygous for the HLA-DR 'shared epitope' (SE). When the data were stratified by the presence of the SE with further stratification according to SE DR subtypes and analysed by transmission disequilibrium test (TDT) for which offspring were assumed independent, the -308A and -857T alleles were found to be associated with RA in patients carrying the SE (P = 0.0076 and 0.0063 respectively). The data were further stratified to analyse for association in individuals homozygous or heterozygous for SE alleles. Results showed that the -308A allele was significantly associated with RA susceptibility in individuals heterozygous for the SE (P < 0.001) with the significance only occurring in patients carrying HLA-DR4 (P < 0.001), while the -857T allele was significant in individuals homozygous for the SE (P = 0.0039). Further analysis using the pedigree disequilibrium test (PDT) which conservatively adjusts for all sources of familial correlation except that conferred by linkage disequilibrium still indicated a significant role for the -308A and -857T alleles. These data provide evidence that TNF promoter SNPs may play an independent role in RA susceptibility in specific immunogenetically-defined groups of RA patients.

Arthritis, Rheumatoid↗

PubMed2001

Impact of 2,4-diacetylphloroglucinol-producing biocontrol strain Pseudomonas fluorescens F113 on intraspecific diversity of resident culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings.

The impact of the 2,4-diacetylphloroglucinol-producing biocontrol agent Pseudomonas fluorescens F113Rif on the diversity of the resident community of culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings was evaluated. At 19 days after sowing, the seed inoculant F113Rif had replaced some of the resident culturable fluorescent pseudomonads at the rhizoplane but had no effect on the number of these bacteria in the rhizosphere. A total of 498 isolates of resident fluorescent pseudomonads were obtained and characterized by molecular means at the level of broad phylogenetic groups (by amplified ribosomal DNA restriction analysis) and at the strain level (with random amplified polymorphic DNA markers) as well as phenotypically (55 physiological tests). The introduced pseudomonad induced a major shift in the composition of the resident culturable fluorescent Pseudomonas community, as the percentage of rhizoplane isolates capable of growing on three carbon substrates (erythritol, adonitol, and L-tryptophan) not assimilated by the inoculant was increased from less than 10% to more than 40%. However, the pseudomonads selected did not display enhanced resistance to 2,4-diacetylphloroglucinol. The shift in the resident populations, which was spatially limited to the surface of the root (i.e., the rhizoplane), took place without affecting the relative proportions of phylogenetic groups or the high level of strain diversity of the resident culturable fluorescent Pseudomonas community. These results suggest that the root-associated Pseudomonas community of sugar beet seedlings is resilient to the perturbation that may be caused by a taxonomically related inoculant.

Chenopodiaceae↗

PubMed2000

Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria.

The minimal replicon of the Pseudomonas plasmid pVS1 was genetically defined and combined with the Escherichia coli p15A replicon, to provide a series of new, oligocopy cloning vectors (5.3 to 8.3 kb). Recombinant plasmids derived from these vectors were stable in growing and nongrowing cells of root-colonizing P. fluorescens strains incubated under different environmental conditions for more than 1 month.

Amino Acid Sequence↗

PubMed2000

A regulatory RNA (PrrB RNA) modulates expression of secondary metabolite genes in Pseudomonas fluorescens F113.

The GacS-GacA two-component signal transduction system, which is highly conserved in gram-negative bacteria, is required for the production of exoenzymes and secondary metabolites in Pseudomonas spp. Screening of a Pseudomonas fluorescens F113 gene bank led to the isolation of a previously undefined locus which could restore secondary metabolite production to both gacS and gacA mutants of F113. Sequence analysis of this locus demonstrated that it did not contain any obvious Pseudomonas protein-coding open reading frames or homologues within available databases. Northern analysis indicated that the locus encodes an RNA (PrrB RNA) which is able to phenotypically complement gacS and gacA mutants and is itself regulated by the GacS-GacA two-component signal transduction system. Primer extension analysis of the 132-base transcript identified the transcription start site located downstream of a sigma(70) promoter sequence from positions -10 to -35. Inactivation of the prrB gene in F113 resulted in a significant reduction of 2, 4-diacetylphloroglucinol (Phl) and hydrogen cyanide (HCN) production, while increased metabolite production was observed when prrB was overexpressed. The prrB gene sequence contains a number of imperfect repeats of the consensus sequence 5'-AGGA-3', and sequence analysis predicted a complex secondary structure featuring multiple putative stem-loops with the consensus sequences predominantly positioned at the single-stranded regions at the ends of the stem-loops. This structure is similar to the CsrB and RsmB regulatory RNAs in Escherichia coli and Erwinia carotovora, respectively. Results suggest that a regulatory RNA molecule is involved in GacA-GacS-mediated regulation of Phl and HCN production in P. fluorescens F113.

Bacterial Proteins↗

PubMed1999

Signalling by the fungus Pythium ultimum represses expression of two ribosomal RNA operons with key roles in the rhizosphere ecology of Pseudomonas fluorescens F113.

Pseudomonas fluorescens F113 produces antifungal metabolites that protect the roots of sugarbeet from the fungus Pythium ultimum. The phytopathogen, in turn, has the ability to downregulate the expression of genes fundamental to the rhizosphere competence of the bacterial strain. This paper describes the characterization of two of these genes, which were isolated by screening a mini-Tn5::lacZ mutant bank for differential expression of beta-galactosidase in the presence of P. ultimum. In order to identify the genes affected in reporter mutants SF3 and SF5, the transposons and flanking regions were cloned. Sequence analysis of the regions flanking the transposons in SF3 revealed that mini-Tn5::lacZ had inserted into a tRNA(Ile) gene, which maps within a ribosomal RNA (rrn) operon. In SF5, the transposon inserted between the promoter of a second rrn operon and a gene encoding a 16S rRNA. Southern blot analysis demonstrated that there are five rrn operons in P. fluorescens F113 and that the transposons in SF3 and SF5 had inserted into two different operons. Further characterization of these mutants suggests that their reduced rhizosphere competence is not the result of reduced viability in the short term but may be accounted for partly by reduced growth rates under conditions that support rapid growth. Analysis of lacZ expression in the reporter mutants indicate that the marked rrn operons are regulated differently, suggesting different physiological roles.

Base Sequence↗

PubMed1999

Genetic analysis of the 3' untranslated region of the tumour necrosis factor shows a highly conserved region in rheumatoid arthritis affected and unaffected subjects.

Tumour necrosis factor (TNF) is a key proinflammatory mediator in rheumatoid arthritis (RA). The TNF locus, situated in the class III region of the MHC, is flanked by five microsatellite markers. It has previously been shown that this region influences susceptibility to RA; two TNF microsatellite haplotypes were found to be associated with RA. Evidence from murine studies has indicated that variation in the TNF 3' untranslated region (UTR) could be associated with altered regulation of TNF biosynthesis. In order to identify possible RA associated polymorphisms, more than 800 bp of the TNF 3' UTR was genetically analysed in RA affected and unaffected subjects possessing specific RA and non-RA associated TNF microsatellite haplotypes. The TNF 3' UTR region was analysed using two mutation detection methods, PCR-SSCP and NIRCA analysis and DNA sequencing. No genetic differences were observed in the human TNF 3' UTR between subjects, that is, irrespective of RA status or TNF haplotype, and also compared with previously published TNF sequences from human sources. Therefore it can be concluded that the TNF 3' UTR in this population was highly conserved and did not influence susceptibility to RA.

3' Untranslated Regions↗

PubMed1998

CRP interacts with promoter-bound sigma54 RNA polymerase and blocks transcriptional activation of the dctA promoter.

The cAMP receptor protein (CRP) is an activator of sigma70-dependent transcription. Analysis of the sigma54-dependent dctA promoter reveals a novel negative regulatory function for CRP. CRP can bind to two distant sites of the dctA promoter, sites which overlap the upstream activator sequences for the DctD activator. CRP interacts with Esigma54 bound at the dctA promoter via DNA loop formation. When the CRP-binding sites are deleted, CRP still interacts in a cAMP-dependent manner with the stable Esigma54 closed complex via protein-protein contacts. CRP is able to repress activation of the dctA promoter, even in the absence of specific CRP-binding sites. CRP affects both the final level and the kinetics of activation. The establishment of the repression and its release by the NtrC activator proceed via slow processes. The kinetics suggest that CRP favours a new form of closed complex which interconverts slowly with the classical closed intermediate. Only the latter is capable of interacting with an activator to form an open promoter complex. Thus, Esigma54 promoters are responsive to CRP, a protein unrelated to sigma54 activators, and the repression exerted is the direct result of an interaction between Esigma54 and the CRP-cAMP complex.

Bacterial Proteins↗

PubMed1998

Modelling the major histocompatibility complex susceptibility to RA using the MASC method.

To explain the association between HLA-DRB1 gene and rheumatoid arthritis (RA), two main hypotheses have been proposed. The first, the shared epitope hypothesis, assumes a direct role of DRB1 in RA susceptibility. The second hypothesis assumes a recessive disease susceptibility gene in linkage disequilibrium with DRB1. To investigate these two hypotheses, we analysed data on the HLA-DRB1 and TNF-LT loci in 49 affected sib-pairs. We used the Marker Association Segregation Chi-square (MASC) method in which the genotype distribution of markers among index cases and the haplotype sharing in affected sib-pairs are jointly taken into account. With DRB1 data alone, both hypotheses were shown to fit but with analysis of TNF data, both hypotheses were strongly rejected. Thus the TNF data provided additional information for a better understanding of genetic susceptibility to RA than was previously possible using only HLA-DR data. A theoretical standpoint is addressed here on the advisability of using different linked markers in a candidate region for modelling the contribution of this region in disease susceptibility.

Adolescent↗

PubMed1998

Epidemiology and clinical impact of Pseudomonas aeruginosa infection in cystic fibrosis using AP-PCR fingerprinting.

Arbitrarily primed PCR (AP-PCR) was utilized to genetically fingerprint 252 Pseudomonas aeruginosa strains isolated from the sputa of 50 cystic fibrosis (CF) patients attending the Cork CF clinic over a period of 3 years. Ten distinct P. aeruginosa strains were identified and the distribution, temporal trends and clinical impact of colonization with these individual P. aeruginosa clones was studied. A number of random isolates from each AP-PCR group were analysed using pulsed field gel electrophoresis (PFGE) in order to confirm the discriminatory power of the AP-PCR technique. The majority of patients were colonized with a single strain over the time period of the study, but it was also possible to harbour two or more strains transiently or simultaneously. Four main strains were relatively evenly distributed throughout the CF population, and it was noted that patients from the same family or attending the same school tended to harbour the same P. aeruginosa clone. Disease severity was significantly associated with the age of the patient (P < 0.001), clearly indicating an increase in severity with increase in age. The general clinical status of the CF patients was not significantly associated with the P. aeruginosa variant isolated from their sputa. Lung status was defined by FEV1 measurement and chest X-ray score (CXR). The non parametric Kruskal-Wallis significance test of FEV1, CXR and age by colonizing P. aeruginosa clone indicated that FEV1 (P = 0.017), but not CXR (P = 0.19) or age (P = 0.842), differed significantly across the clones of P. aeruginosa isolated. Patients harbouring P. aeruginosa strains B, F or G clearly had lower FEV1 scores while those harbouring clones A, C, D or H generally had higher FEV1 scores. Thus, the sub-species variant of P. aeruginosa colonizing CF patients may be associated with the severity of progressive lung disease.

Adult↗

PubMed1998

Impact on arbuscular mycorrhiza formation of pseudomonas strains used as inoculants for biocontrol of soil-borne fungal plant pathogens

The arbuscular mycorrhizal symbiosis, a key component of agroecosystems, was assayed as a rhizosphere biosensor for evaluation of the impact of certain antifungal Pseudomonas inoculants used to control soil-borne plant pathogens. The following three Pseudomonas strains were tested: wild-type strain F113, which produces the antifungal compound 2,4-diacetylphloroglucinol (DAPG); strain F113G22, a DAPG-negative mutant of F113; and strain F113(pCU203), a DAPG overproducer. Wild-type strain F113 and mutant strain F113G22 stimulated both mycelial development from Glomus mosseae spores germinating in soil and tomato root colonization. Strain F113(pCU203) did not adversely affect G. mosseae performance. Mycelial development, but not spore germination, is sensitive to 10 &mgr;M DAPG, a concentration that might be present in the rhizosphere. The results of scanning electron and confocal microscopy demonstrated that strain F113 and its derivatives adhered to G. mosseae spores independent of the ability to produce DAPG.

Journal Article↗

PubMed1997

Evidence for signaling between the phytopathogenic fungus Pythium ultimum and Pseudomonas fluorescens F113: P. ultimum represses the expression of genes in P. fluorescens F113, resulting in altered ecological fitness.

There is increasing evidence that communication between members of the same species, as well as members of different species, is important for the survival of microorganisms in diverse ecological niches, such as the rhizosphere. To investigate whether the phytopathogen Pythium ultimum could alter gene expression in the biocontrol strain Pseudomonas fluorescens F113, which protects the roots of sugar beet from the fungus, a screening system was developed to detect differential expression of bacterial genes in the presence of P. ultimum. The transposon Tn5, containing a promoterless lacZ reporter gene, was used to generate a library of transcriptional gene fusions in P. fluorescens F113. By this screening procedure, five P. fluorescens F113 gene clusters were identified and shown to be repressed in the presence of P. ultimum. The ecological fitness of three of the five reporter mutants in the rhizosphere of seed-inoculated sugar beet was lower than that of the wild type. Furthermore, all five mutants were impaired in their ability to subsequently colonize the rhizosphere of uninoculated sugar beet sown repeatedly in the same soil. With the exception of reporter mutant SF10, which was impaired in nitrogen metabolism, the reporter mutants had growth requirements and biocontrol abilities similar to those of the wild type. This is the first reported case of a fungus repressing the expressing of bacterial genes.

Ecology↗

PubMed1996

Regulation of the iron uptake genes in Pseudomonas fluorescens M114 by pseudobactin M114: the pbrA sigma factor gene does not mediate the siderophore regulatory response.

The iron-regulated PbrA sigma factor dictates the production of the siderophore, pseudobactin M114, and its cognate outer membrane receptor, PbuA, in Pseudomonas fluorescens M114. However, the siderophore molecule also has a role in regulating the expression of the siderophore biosynthetic and siderophore receptor genes in P. fluorescens M114. This is based on the fact that beta-galactosidase levels from lacZ fusions of M114 siderophore promoters (biosynthetic and receptor) were reduced in M114 siderophore biosynthetic mutants compared to wild-type M114. Expression of both promoters was increased by the addition of pseudobactin M114 to the growth medium. This effect was widespread and applicable to all but one of the siderophore negative strains of M114 tested. Furthermore, it was demonstrated that transcription of the pbr A sigma factor gene was not reduced in the siderophore biosynthetic mutants. This excludes the possibility that reduced expression of the siderophore biosynthetic and receptor promoters in the siderophore biosynthetic mutants is mediated at the level of expression of the pbr A gene itself. In addition, it was noted that the siderophore regulated response was applicable to promoters with and without the DNA sequence motif, (G/C)CTAAATCCC, which is required for iron-regulated expression of some pseudomonad promoters.

Bacterial Proteins↗

PubMed1996

Modular structure of the Rhizobium meliloti DctB protein.

To investigate the modular structure of the Rhizobium meliloti dicarboxylic acid sensor protein, DctB, three truncated DctB proteins (DctB4, DctB5 and DctB4G) were constructed, overproduced in Escherichia coli and purified. The DctB4G protein was composed of 446 amino acids of the DctB C-terminus and displayed strong autophosphorylation activity in vitro. This activity was sustained when a further 120 amino acids at the N-terminus of the polypeptide were deleted (DctB5). This protein which has an intact transmitter domain exhibits specific but inefficient phospho-transfer capabilities. Removal of 58 amino acids from the DctB4G C-terminus which included blocks F and G2 of the transmitter domain, rendered the resultant protein (DctB4) incompetent in autophosphorylation. Phosphorylation activity was restored to DctB4 through intramolecular complementation with DctB. Therefore, it would appear that the R. meliloti DctB protein is active as a dimer (or higher order oligomer). Furthermore, the intramolecular complementation experiments indicate that the amino acids 171-291, a predicted periplasmic stretch, play an important role in the dimerization process.

Bacterial Proteins↗

PubMed1996

Transcriptional regulation of the iron-responsive sigma factor gene pbrA.

In response to the intracellular iron concentration Pseudomonas fluorescens M114 coordinately regulates the production of pseudobactin M114, its cognate receptor PbuA, and a casein protease. Transcriptional initiation of this coordinate iron-stress response requires the sigma factor PbrA. PbrA is a member of the ECF (Extracytoplasmic function) subgroup of the sigma 70 family of eubacterial RNA polymerase sigma factors. Regulatory studies of the pbrA gene utilising promoter-lacZ transcriptional fusions demonstrate that expression of pbrA dictates the cellular response to iron. pbrA is transcribed in all phases of iron-limited growth but maximally at late-logarithmic to stationary phase. pbrA expression is independent of autoregulatory control but is strictly repressed in iron-rich conditions in a Fur-dependent fashion. Constitutive expression of pbrA from an inducible tac promoter permits the induction of PbrA-dependent transcription and pseudobactin M114 biosynthesis in high-iron conditions. A PbrA consensus sequences was derived from significant DNA sequence homologies observed within the "-25 bp" and "-16 bp" regions conserved among all PbrA-dependent promoters. The predicted PbrA target promoter consensus is homologous for the promoter recognition sites for other environmentally responsive ECF sigma factors.

Amino Acid Sequence↗