Genome mining and characterisation of a novel transaminase with remote stereoselectivity.

Microbial enzymes from pristine niches can potentially deliver disruptive opportunities in synthetic routes to Active Pharmaceutical Ingredients and intermediates in the Pharmaceutical Industry. Advances in green chemistry technologies and the importance of stereochemical control, further underscores the application of enzyme-based solutions in chemical synthesis. The rich tapestry of microbial diversity in the oceanic ecosystem encodes a capacity for novel biotransformations arising from the chemical complexity of this largely unexplored bioactive reservoir. Here we report a novel ω-transaminase discovered in a marine sponge Pseudovibrio sp. isolate. Remote stereoselection using a transaminase has been demonstrated for the first time using this novel protein. Application to the resolution of an intermediate in the synthesis of sertraline highlights the synthetic potential of this novel biocatalyst discovered through genomic mining. Integrated chemico-genomics revealed a unique substrate profile, while molecular modelling provided structural insights into this 'first in class' selectivity at a remote chiral centre.

Bile acids destabilise HIF-1α and promote anti-tumour phenotypes in cancer cell models.

BACKGROUND: The role of the microbiome has become synonymous with human health and disease. Bile acids, as essential components of the microbiome, have gained sustained credibility as potential modulators of cancer progression in several disease models. At physiological concentrations, bile acids appear to influence cancer phenotypes, although conflicting data surrounds their precise physiological mechanism of action. Previously, we demonstrated bile acids destabilised the HIF-1α subunit of the Hypoxic-Inducible Factor-1 (HIF-1) transcription factor. HIF-1 overexpression is an early biomarker of tumour metastasis and is associated with tumour resistance to conventional therapies, and poor prognosis in a range of different cancers. METHODS: Here we investigated the effects of bile acids on the cancer growth and migratory potential of cell lines where HIF-1α is known to be active under hypoxic conditions. HIF-1α status was investigated in A-549 lung, DU-145 prostate and MCF-7 breast cancer cell lines exposed to bile acids (CDCA and DCA). Cell adhesion, invasion, migration was assessed in DU-145 cells while clonogenic growth was assessed in all cell lines. RESULTS: Intracellular HIF-1α was destabilised in the presence of bile acids in all cell lines tested. Bile acids were not cytotoxic but exhibited greatly reduced clonogenic potential in two out of three cell lines. In the migratory prostate cancer cell line DU-145, bile acids impaired cell adhesion, migration and invasion. CDCA and DCA destabilised HIF-1α in all cells and significantly suppressed key cancer progression associated phenotypes; clonogenic growth, invasion and migration in DU-145 cells. CONCLUSIONS: These findings suggest previously unobserved roles for bile acids as physiologically relevant molecules targeting hypoxic tumour progression.

Aspirated bile: a major host trigger modulating respiratory pathogen colonisation in cystic fibrosis patients.

Chronic respiratory infections are a leading global cause of morbidity and mortality. However, the molecular triggers that cause respiratory pathogens to adopt persistent and often untreatable lifestyles during infection remain largely uncharacterised. Recently, bile aspiration caused by gastro-oesophageal reflux (GOR) has emerged as a significant complication associated with respiratory disease, and cystic fibrosis (CF) in particular. Based on our previous finding that the physiological concentrations of bile influence respiratory pathogens towards a chronic lifestyle in vitro, we investigated the impact of bile aspiration on the lung microbiome of respiratory patients. Sputum samples (n = 25) obtained from a cohort of paediatric CF patients were profiled for the presence of bile acids using high-resolution liquid chromatography-mass spectrometry (LC-MS). Pyrosequencing was performed on a set of ten DNA samples that were isolated from bile aspirating (n = 5) and non-bile aspirating (n = 5) patients. Both denaturing gradient gel electrophoresis (DGGE) and pyrosequencing revealed significantly reduced biodiversity and richness in the sputum samples from bile aspirating patients when compared with non-aspirating patients. Families and genera associated with the pervasive CF microbiome dominated aspirating patients, while bacteria associated with the healthy lung were most abundant in non-aspirating patients. Bile aspiration linked to GOR is emerging as a major host trigger of chronic bacterial infections. The markedly reduced biodiversity and increased colonisation by dominant proteobacterial CF-associated pathogens observed in the sputum of bile aspirating patients suggest that bile may play a major role in disease progression in CF and other respiratory diseases.

The non-classical ArsR-family repressor PyeR (PA4354) modulates biofilm formation in Pseudomonas aeruginosa.

PyeR (PA4354) is a novel member of the ArsR family of transcriptional regulators and modulates biofilm formation in Pseudomonas aeruginosa. Characterization of this regulator showed that it has negative autoregulatory properties and binds to a palindromic motif conserved among PyeR orthologues. These characteristics are in line with classical ArsR-family regulators, as is the fact that PyeR is part of an operon structure (pyeR-pyeM-xenB). However, PyeR also exhibits some atypical features in comparison with classical members of the ArsR family, as it does not harbour metal-binding motifs and does not appear to be involved in metal perception or resistance. Hence, PyeR belongs to a subgroup of non-classical ArsR-family regulators and is the second ArsR regulator shown to be involved in biofilm formation.

Characterization of imipenem resistance mechanisms in Pseudomonas aeruginosa isolates from Turkey.

The emergence of carbapenem resistance in Pseudomonas aeruginosa threatens the efficacy of this important anti-pseudomonal antibiotic class. Between 2003 and 2006, an increase in the number of carbapenem-resistant P. aeruginosa isolates at the Zonguldak Karaelmas University Hospital was observed (Zonguldak, Turkey). To assess the imipenem resistance mechanisms emerging in these P. aeruginosa isolates, they were characterized by amplified fragment length polymorphism typing, which revealed diversity among imipenem-resistant isolates as well as two clonally related outbreak groups. The molecular mechanism of carbapenem resistance was characterized in a representative isolate from each clonal group. Mutational disruption of oprD was the most frequently encountered resistance mechanism (23/27 isolates).

Diversity and antibacterial activity of bacteria isolated from the coastal marine sponges Amphilectus fucorum and Eurypon major.

AIMS: To assess the diversity and antimicrobial activity of culturable bacteria associated with two temperate-water marine sponges, Amphilectus fucorum and Eurypon major. METHODS AND RESULTS: Sponge samples were collected in August 2008 and bacteria were cultured on several different media. The 16S rRNA gene of representative strains was sequenced to allow classification. It was found that Proteobacteria were the dominant group of bacteria cultured from both sponges, but overall, the bacterial composition was diverse and distinct between the sponges. The most notable features were the significantly higher proportion of firmicutes in E. major and the low frequency of actinobacteria in both sponges. Four bacterial isolates were identified as potentially novel species and will be characterised in future studies. Approximately 400 cultured bacteria were screened for antimicrobial activity against a collection of indicator strains, with only eight strains, all Pseudovibrio spp., displaying any such activity. These strains were active against Escherichia coli and Bacillus subtilis but not Staphylococcus aureus or a selection of fungal strains. CONCLUSIONS: Diverse and distinct populations of culturable bacteria are present in the coastal sponges A. fucorum and E. major. Only a minority of isolates produce antibacterial metabolites in culture, but this activity is common in Pseudovibrio spp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study illustrates the diversity of sponge-associated bacteria and the need to increase our knowledge about the function of these symbiotic bacteria. The data suggest that production of antibacterial metabolites is restricted to a subset of species, with the majority involved in other functions. The importance of Pseudovibrio as a reservoir of antibacterial metabolites is also highlighted.

Diversity and bioactive potential of endospore-forming bacteria cultured from the marine sponge Haliclona simulans.

AIMS: Despite the frequent isolation of endospore-formers from marine sponges, little is known about the diversity and characterization of individual isolates. The main aims of this study were to isolate and characterize the spore-forming bacteria from the marine sponge Haliclona simulans and to examine their potential as a source for bioactive compounds. METHODS AND RESULTS: A bank of presumptive aerobic spore-forming bacteria was isolated from the marine sponge H. simulans. These represented c. 1% of the total culturable bacterial population. A subgroup of thirty isolates was characterized using morphological, phenotypical and phylogenetic analysis. A large diversity of endospore-forming bacteria was present, with the thirty isolates being distributed through a variety of Bacillus and Paenibacillus species. These included ubiquitous species, such as B. subtilis, B. pumilus, B. licheniformis and B. cereus group, as well as species that are typically associated with marine habitats, such as B. aquimaris, B. algicola and B. hwajinpoensis. Two strains carried the aiiA gene that encodes a lactonase known to be able to disrupt quorum-sensing mechanisms, and various isolates demonstrated protease activity and antimicrobial activity against different pathogenic indicator strains, including Clostridium perfringens, Bacillus cereus and Listeria monocytogenes. CONCLUSIONS: The marine sponge H. simulans harbours a diverse collection of endospore-forming bacteria, which produce proteases and antibiotics. This diversity appears to be overlooked by culture-dependent and culture-independent methods that do not specifically target sporeformers. SIGNIFICANCE AND IMPACT OF STUDY: Marine sponges are an as yet largely untapped and poorly understood source of endospore-forming bacterial diversity with potential biotechnological, biopharmaceutical and probiotic applications. These results also indicate the importance of combining different methodologies for the comprehensive characterization of complex microbial populations such as those found in marine sponges.

Diversity and antimicrobial activities of microbes from two Irish marine sponges, Suberites carnosus and Leucosolenia sp.

AIMS: To evaluate the diversity and antimicrobial activity of bacteria from the marine sponges Suberites carnosus and Leucosolenia sp. METHODS AND RESULTS: Two hundred and thirty-seven bacteria were isolated from the sponges S. carnosus (Demospongiae) and Leucosolenia sp. (Calcarea). Isolates from the phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria were obtained. Isolates of the genus Pseudovibrio were dominant among the bacteria from S. carnosus, whereas Pseudoalteromonas and Vibrio were the dominant genera isolated from Leucosolenia sp. Approximately 50% of the isolates from S. carnosus displayed antibacterial activity, and c. 15% of the isolates from Leucosolenia sp. demonstrated activity against the test fungal strains. The antibacterial activity observed was mostly from Pseudovibrio and Spongiobacter isolates, while the majority of the antifungal activity was observed from the Pseudoalteromonas, Bacillus and Vibrio isolates. CONCLUSIONS: Both sponges possess a diverse range of bioactive and potentially novel bacteria. Differences observed from the sponge-derived groups of isolates in terms of bioactivity suggest that S. carnosus isolates may be a better source of antibacterial compounds, while Leucosolenia sp. isolates appear to be a better source of antifungal compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study in which cultured bacterial isolates from the marine sponges S. carnosus and a Leucosolenia sp. have been evaluated for their antibacterial activity. The high percentage of antibacterial isolates from S. carnosus and of antifungal isolates from Leucosolenia sp. suggests that these two sponges may be good sources for potentially novel marine natural products.

Functional metagenomic strategies for the discovery of novel enzymes and biosurfactants with biotechnological applications from marine ecosystems.

Marine ecosystems are home to bacteria which are exposed to a wide variety of environmental conditions, such as extremes in temperature, salinity, nutrient availability and pressure. Survival under these conditions must have necessitated the adaptation and the development of unique cellular biochemistry and metabolism by these microbes. Thus, enzymes isolated from these microbes have the potential to possess quite unique physiological and biochemical properties. This review outlines a number of function-based metagenomic approaches which are available to screen metagenomic libraries constructed from marine ecosystems to facilitate the exploitation of some of these potentially novel biocatalysts. Functional screens to isolate novel cellulases, lipases and esterases, proteases, laccases, oxidoreductases and biosurfactants are described, together with approaches which can be employed to help overcome some of the typical problems encountered with functional metagenomic-based screens.

In vitro analyses are not reliable predictors of the plant growth promotion capability of bacteria; a Pseudomonas fluorescens strain that promotes the growth and yield of wheat.

AIMS: In this study, we set out to identify bacteria that can be used to promote the growth of cereals, while concurrently investigating the merits of using a range of such tests to preselect bacteria for glasshouse studies. METHODS AND RESULTS: A panel of 15 strains isolated from the rhizosphere and phyllosphere of cereals was tested for the ability to improve the germination of wheat seeds and for production of a range of factors associated with plant growth promotion. In parallel, all bacteria were tested for their ability to improve biomass and grain yield when applied as a soil amendment in glasshouse trials. CONCLUSIONS: There was no significant correlation between growth promotion potential in the glasshouse and the results of either the phenotypic or the germination tests. Glasshouse tests identified that only one strain, Pseudomonas fluorescens strain MKB37, gave a significant increase in head weight and grain yield. SIGNIFICANCE AND IMPACT OF THE STUDY: While this study has identified a candidate for further field tests, it has also highlighted the fact that the modes of action for plant growth-promoting bacteria (PGPB) are still not fully understood, and that there is no efficient and effective screening method for identifying PGPB by laboratory tests.

Diversity and antimicrobial activity of Pseudovibrio spp. from Irish marine sponges.

AIMS: To evaluate the diversity and antimicrobial activity present among Pseudovibrio spp. isolated from marine sponges. METHODS AND RESULTS: Seventy-three bacterial isolates from the marine sponges Polymastia boletiformis, Axinella dissimilis and Haliclona simulans were identified as Pseudovibrio spp. using phylogenetic analysis of 16S rRNA gene sequences. Genetic diversity among these isolates was estimated using random amplification of polymorphic DNA (RAPD), and 33 RAPD types were identified among the 73 Pseudovibrio isolates. These Pseudovibrio spp. were assayed for the production of compounds with antimicrobial activity against various clinically relevant pathogens. Sixty-two (85%) of the isolates showed activity against at least one of the pathogens tested, including Escherichia coli, Salmonella enterica serotype Typhimurium, methicillin-resistant Staphylococcus aureus (MRSA), and Clostridium difficile. PCR screens of the Pseudovibrio isolates also revealed the presence of potential antibiotic-producing polyketide synthase genes. CONCLUSIONS: Marine sponges harbour a diverse population of Pseudovibrio spp., the majority of which demonstrate antimicrobial activity. The identification of several different antimicrobial activity spectra suggests that the Pseudovibrio isolates may produce a suite of antimicrobial compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study in which an extended population of Pseudovibrio isolates from marine sponges has been analysed and establishes the little-studied Pseudovibrio as a potentially important genus in the search for antimicrobial compounds of clinical relevance.

Endoglucanase activities and growth of marine-derived fungi isolated from the sponge Haliclona simulans.

AIMS: The conversion of cheap cellulosic biomass to more easily fermentable sugars requires the use of costly cellulases. We have isolated a series of marine sponge-derived fungi and screened these for cellulolytic activity to determine the potential of this unique environmental niche as a source of novel cellulase activities. METHODS AND RESULTS: Fungi were isolated from the marine sponge Haliclona simulans. Phylogenetic analysis of these and other fungi previously isolated from H. simulans showed fungi from three phyla with very few duplicate species. Cellulase activities were determined using plate-based assays using different media and sea water concentrations while extracellular cellulase activities were determined using 3,5-dinitrosalicylic acid (DNSA)-based assays. Total and specific cellulase activities were determined using a range of incubation temperatures and compared to those for the cellulase overproducing mutant Hypocrea jecorina QM9414. Several of the strains assayed produced total or relative endoglucanase activities that were higher than H. jecorina, particularly at lower reaction temperatures. CONCLUSIONS: Marine sponges harbour diverse fungal species and these fungi are a good source of endoglucanase activities. Analysis of the extracellular endoglucanase activities revealed that some of the marine-derived fungi produced high endoglucanase activities that were especially active at lower temperatures. SIGNIFICANCE AND IMPACT OF THE STUDY: Marine-derived fungi associated with coastal marine sponges are a novel source of highly active endoglucanases with significant activity at low temperatures and could be a source of novel cellulase activities.

High levels of Lymphotoxin-Beta (LT-Beta) gene expression in rheumatoid arthritis synovium: clinical and cytokine correlations.

Lymphotoxin-Beta (LT-Beta) is implicated in lymphoid follicle development, production of pro-inflammatory cytokines, and can enhance the proliferation of fibroblasts and synoviocytes. The objective of this study was to investigate LT-Beta and LT-BetaReceptor (LT-BetaR) gene expression in RA patient synovium and blood samples compared with control individuals, and correlate with LT-Alpha and TNF-Alpha gene expression and disease parameters. RT-PCR was used to investigate the gene expression of LT-Beta, LT-BetaR, TNF-Alpha and LT-Alpha in the blood and synovium of RA patients and a control group of individuals. LT-Beta gene expression was significantly higher in RA patient synovium compared to control synovium (P = 0.005). There was a significant positive correlation between LT-Beta and LT-Alpha gene expression in both the synovium (P = 0.001) and blood (P = 0.002) of RA patients. LT-Beta gene expression was significantly higher in RA patient synovial samples that were inflamed to a moderately severe degree compared to those inflamed to a minimal degree (P = 0.02). Analysis of clinical variables revealed a significant positive correlation between LT-BetaR gene expression in RA patient synovium and Pain VAS Score (P = 0.01) and also HAQ Score (P = 0.01). Increased LT-Beta gene expression occurs in RA synovium and correlates with the degree of inflammation. LT-Beta may play a role in RA disease pathogenesis by contributing to a more intense inflammatory reaction in the synovium.

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.

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.

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.

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.

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.

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.