Diffusible signal factor-dependent quorum sensing in pathogenic bacteria and its exploitation for disease control.

Cell-to-cell signals of the diffusible signal factor (DSF) family are cis-2-unsaturated fatty acids of differing chain length and branching pattern. DSF signalling has been described in diverse bacteria to include plant and human pathogens where it acts to regulate functions such as biofilm formation, antibiotic tolerance and the production of virulence factors. DSF family signals can also participate in interspecies signalling with other bacteria and interkingdom signalling such as with the yeast Candida albicans. Interference with DSF signalling may afford new opportunities for the control of bacterial disease. Such strategies will depend in part on detailed knowledge of the molecular mechanisms underlying the processes of signal synthesis, perception and turnover. Here, I review both recent progress in understanding DSF signalling at the molecular level and prospects for translating this knowledge into approaches for disease control.

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.

Lipopolysaccharides and plant innate immunity.

Plants posses an innate immune system that has many parallels with those found in mammals and insects. A range of molecules of microbial origin called Microbe Associated Molecular Patterns (MAMPs) act to trigger basal defense responses in plants. These elicitors include lipopolysaccharides (LPS) from diverse Gram-negative bacteria. Both core oligosaccharide and the lipid A moieties of LPS as well as synthetic O-antigen oligosaccharides have activity in inducing defense responses in the model plant Arabidopsis thaliana. Very little is known of the mechanism of LPS perception by plants, although plant receptors for other MAMPs such as flagellin have been described. Recent work has implicated the Arabidopsis syntaxin PEN1 as a potential actor in LPS induction of plant defenses, which may suggest a role for vesicle trafficking in the signalling process.

Mechanical devices for urinary incontinence in women.

BACKGROUND: Incontinence can have a devastating effect on the lives of sufferers with significant economic implications. Non-surgical treatments such as pelvic floor muscle training and the use of mechanical devices are usually the first line of management. The latter more so when a person did not want surgery or when considered unfit for surgery. Mechanical devices are inexpensive and do not compromise future surgical treatment. OBJECTIVES: To determine the effects of mechanical devices in the management of adult female urinary incontinence. SEARCH STRATEGY: We searched the Cochrane Incontinence Group Specialised Trials Register (7 December 2005). The register contains trials identified from MEDLINE, the Cochrane Central Register of Controlled Trials (CENTRAL), CINAHL and handsearching of journals and conference proceedings. SELECTION CRITERIA: All randomised or quasi-randomised controlled trials of mechanical devices in the management of adult female urinary incontinence determined either by symptom classification or by urodynamic diagnosis. DATA COLLECTION AND ANALYSIS: Three reviewers assessed the identified studies for eligibility and methodological quality and independently extracted data from the included studies. Data analysis was performed using RevMan software (version 4.2). MAIN RESULTS: There were six trials involving a total of 286 women. Two small trials compared a mechanical device with no treatment and although they suggested that use of a mechanical device might be better than no treatment, the evidence for this was inconclusive. Five trials compared one mechanical device with another. Quantitative synthesis of data from these trials was not possible because different mechanical devices were compared in each trial using different outcome measures. Data from the individual trials showed no clear difference between devices, but with wide confidence intervals. There were no trials comparing a mechanical device with another type of treatment. AUTHORS' CONCLUSIONS: The place of mechanical devices in the management of urinary incontinence remains in question. Currently there is little evidence from controlled trials on which to judge whether their use is better than no treatment and a large well-conducted trial is required for clarification. There was also insufficient evidence in favour of one device over another and no evidence to compare mechanical devices with other forms of treatment.

Mechanical devices for urinary incontinence in women.

BACKGROUND: Incontinence can have a devastating effect on the lives of sufferers with significant economic implications. Non-surgical treatments such as pelvic floor muscle training and the use of mechanical devices are usually the first line of management. The latter more so when a person did not want surgery or when considered unfit for surgery. Mechanical devices are inexpensive and do not compromise future surgical treatment. OBJECTIVES: To determine the effects of mechanical devices in the management of adult female urinary incontinence. SEARCH STRATEGY: We searched the Cochrane Incontinence Group Specialised Trials Register (7 December 2005). The register contains trials identified from MEDLINE, the Cochrane Central Register of Controlled Trials (CENTRAL), CINAHL and handsearching of journals and conference proceedings. SELECTION CRITERIA: All randomised or quasi-randomised controlled trials of mechanical devices in the management of adult female urinary incontinence determined either by symptom classification or by urodynamic diagnosis. DATA COLLECTION AND ANALYSIS: Three reviewers assessed the identified studies for eligibility and methodological quality and independently extracted data from the included studies. Data analysis was performed using RevMan software (version 4.2). MAIN RESULTS: There were six trials involving a total of 286 women. Two small trials compared a mechanical device with no treatment and although they suggested that use of a mechanical device might be better than no treatment, the evidence for this was inconclusive. Five trials compared one mechanical device with another. Quantitative synthesis of data from these trials was not possible because different mechanical devices were compared in each trial using different outcome measures. Data from the individual trials showed no clear difference between devices, but with wide confidence intervals. There were no trials comparing a mechanical device with another type of treatment. AUTHORS' CONCLUSIONS: The place of mechanical devices in the management of urinary incontinence remains in question. Currently there is little evidence from controlled trials on which to judge whether their use is better than no treatment and a large well-conducted trial is required for clarification. There was also insufficient evidence in favour of one device over another and no evidence to compare mechanical devices with other forms of treatment.

Serotonin and noradrenaline reuptake inhibitors (SNRI) for stress urinary incontinence in adults.

BACKGROUND: To date, standard recommendations for the management of stress urinary incontinence (SUI) would be either pelvic floor muscle training (PFMT) or surgery. A new form of drug treatment with a serotonin-noradrenaline reuptake inhibitor (SNRI), duloxetine, may now have a place in treatment of this condition. OBJECTIVES: To determine whether a SNRI is better than placebo (or no treatment, other pharmacological and non-pharmacological therapies, or surgery) in the treatment of women with SUI, or mixed urinary incontinence that includes stress incontinence (MUI), or both and which doses should be used. SEARCH STRATEGY: We searched the Cochrane Incontinence Group specialised register (searched 1 December 2004), (CENTRAL) (Issue 2, 2004), MEDLINE (January 1966 to September 2004), PREMEDLINE (11 March 2004), Dissertation Abstracts and the reference lists of relevant articles. SELECTION CRITERIA: All randomised or quasi-randomised controlled trials of treatment for SUI or MUI, in which at least one management arm involved a SNRI. DATA COLLECTION AND ANALYSIS: Two authors evaluated the trials for appropriateness for inclusion and methodological quality. Three authors performed the data extraction using predetermined criteria. Analyses were performed using the Cochrane Review Manager software, RevMan. MAIN RESULTS: Nine randomised trials were included, involving 3327 adults with predominantly SUI, randomised to receive duloxetine or placebo. Both arms in individual trials were comparable for various baseline characteristics. Treatment duration was between three weeks and 12 weeks. Duloxetine was significantly better than placebo in terms of improving patients' quality of life (WMD 5.26, 95%CI 3.84 to 6.68. P< 0.00001) and perception of improvement. Individual studies demonstrated a significant reduction in the Incontinence Episode Frequency (IEF) by approximately 50% during treatment with duloxetine. With regard to objective cure, however, meta-analysis of stress pad test and 24 hour pad weight change failed to demonstrate a benefit for duloxetine over placebo though data were relatively few. Subjective cure favoured duloxetine, albeit with a small effect size (3%). One trial suggested that duloxetine was better than pelvic floor muscle training alone in reducing IEF (P < 0.05) based on median percentage decrease in IEF per week. Although significant side effects were commonly associated with duloxetine, they were reported as acceptable. AUTHORS' CONCLUSIONS: The available evidence suggests that duloxetine treatment can significantly improve the quality of life of patients with stress urinary incontinence, but it is unclear whether or not benefits are sustainable. Adverse effects are common but not serious. About one in three participants allocated duloxetine reported adverse effects (most commonly nausea) related to treatment, and about one in eight allocated duloxetine stopped treatment as a consequence.

Induction of extracellular matrix glycoproteins in Brassica petioles by wounding and in response to Xanthomonas campestris.

A panel of monoclonal antibodies that recognize plant extracellular matrix glycoproteins previously implicated in plant-microbe interactions was used to study the effects of pathogen inoculation and wounding on glycoproteins in petioles of Brassica campestris. The panel of monoclonals comprised two sets: JIM11, JIM12, and JIM20 recognize epitopes carried on hydroxyproline-rich glycoproteins (HRGPs) (M. Smallwood, A. Beven, N. Donovan, S. J. Neitl, J. Peart, K. Roberts, and J. P. Knox, Plant J. 5:237-246, 1994); MAC204 and MAC265 recognize glycoproteins of the Rhizobium infection thread (K. A. VandenBosch, D. J. Bradley, S. Perotto, G. W. Butcher, and N. J. Brewin, EMBO J. 8:335-342, 1989). Wounding or inoculation of petioles with avirulent strains of pathovars of Xanthomonas campestris induced the synthesis of two new groups of antigens: gp160 ran as a smear on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with apparent molecular mass from 120 to 200 kDa and was recognized by JIM20 and MAC204; gpS remained in the stacking gel on SDS-PAGE and was recognized by JIM11, JIM20, and MAC204. The response to virulent strains of pathovars of X. campestris was either less pronounced or absent. gpS comprised several components that were resolved by cation-exchange chromatography. Some of these components were characterized as extensin-like HRGPs. The level of induction of the gpS group of antigens by virulent strains was not altered by mutation of a number of genes required for basic pathogenicity or by heat-killing the bacteria.

The activity of lipid A and core components of bacterial lipopolysaccharides in the prevention of the hypersensitive response in pepper.

Pre-treatment of leaves of pepper (Capsicum annuum) with lipopolysaccharide (LPS) preparations from enteric bacteria and Xanthomonas campestris could prevent the hypersensitive response caused by an avirulent X. campestris strain. By use of a range of deep-rough mutants, the minimal structure in Salmonella LPS responsible for the elicitation of this effect was determined to be lipid A attached to a disaccharide of 2-keto-3-deoxyoctulosonate; lipid A alone and the free core oligosaccharide from a Salmonella Ra mutant were not effective. For Xanthomonas, the core oligosaccharide alone had activity although lipid A was not effective. The results suggest that pepper cells can recognize different structures within bacterial LPS to trigger alterations in plant response to avirulent pathogens.

A metalloprotease from Xanthomonas campestris that specifically degrades proline/hydroxyproline-rich glycoproteins of the plant extracellular matrix.

Culture supernatants of Xanthomonas campestris pv. campestris contain an enzymic activity capable of degrading gp120, a proline-rich glycoprotein associated with the extracellular matrix of the vascular bundles in petioles of turnip (Brassica campestris). This activity did not reside in any of the three previously characterized proteases of X. campestris pv. campestris that were identified by their action against the model substrate beta-casein. The novel enzyme was purified by ion-exchange and size-exclusion high-performance liquid chromatography (HPLC). The enzyme, which has no activity against beta-casein, is active against some plant glycoproteins of the hydroxyproline-rich class such as extensin from potato and tomato and gpS-3, a glycoprotein induced in B. campestris petioles by wounding. Other hydroxyproline-rich glycoproteins, such as the solanaceous lectins, were not substrates however. Studies of the products released upon degradation of tomato extensin suggested that the degradative mechanism was proteolysis. Inhibitor studies suggested that the enzyme was a zinc-requiring metalloprotease. Extracellular matrix glycoproteins of the proline-rich and hydroxyproline-rich classes have been implicated in plant resistance to microbial attack, hence their degradation by X. campestris pv. campestris may have considerable significance for black rot pathogenesis.

Lipopolysaccharide from Xanthomonas campestris induces defense-related gene expression in Brassica campestris.

Purified lipopolysaccharide (LPS) from Xanthomonas campestris pv. campestris induced accumulation of transcript for beta-1,3-glucanase in turnip at concentrations of 1 micrograms/ml. The lipid A-inner core structure was required for activity but the O-antigen had no role. We suggest that release of LPS in planta triggers expression of at least some defense-related genes.

Expression of the gum operon directing xanthan biosynthesis in Xanthomonas campestris and its regulation in planta.

The gum gene cluster of Xanthomonas campestris pv. campestris comprises 12 genes whose products are involved in the biosynthesis of the extracellular polysaccharide xanthan. These genes are expressed primarily as an operon from a promoter upstream of the first gene, gumB. Although the regulation of xanthan synthesis in vitro has been well studied, nothing is known of its regulation in planta. A reporter plasmid was constructed in which the promoter region of the gum operon was fused to gusA. In liquid cultures, the expression of the gumgusA reporter was correlated closely with the production of xanthan, although a low basal level of beta-glucuronidase activity was seen in the absence of added carbon sources when xanthan production was very low. The expression of the gumgusA fusion also was subject to positive regulation by rpfF, which is responsible for the synthesis of the diffusible signal factor (DSF). The expression of the gumgusA fusion in bacteria recovered from inoculated turnip leaves was maximal at the later phases of growth and was subject to regulation by rpfF. These results provide indirect support for the operation of the DSF regulatory system in bacteria in planta.

A locus determining pathogenicity of Xanthomonas campestris is involved in lipopolysaccharide biosynthesis.

A pathogenicity locus in Xanthomonas campestris pv. campestris has been shown to comprise two genes which mediate biosynthesis of the bacterial lipopolysaccharide (LPS) but not extracellular polysaccharide. Mutants with Tn5 insertions in either gene showed alterations in the electrophoretic patterns of both water-soluble and phenol-soluble LPS forms, which suggested defects in the biosynthesis of the core oligosaccharide component. On gel chromatography, core oligosaccharides of the mutants were of apparently lower molecular weight than those from the wild type. Furthermore, the content of mannose and glucose, sugars characteristic of the core oligosaccharide, were significantly lower in the water-soluble LPS of the mutants. Because of their role in LPS core biosynthesis, the two genes were called rfaX and rfaY. rfaX mutants show altered behavior in a range of host and non-host plants such that the number of recoverable bacteria drop within the first 24 h after inoculation. In contrast, the behavior of rfaY mutants only differed from the wild type in Datura, a non-host plant in which the growth of the wild type is severely attenuated. The predicted protein RfaY showed significant sequence homology to a sub-family of RNA polymerase sigma factors which are involved in extracytoplasmic functions.

A gene for superoxide dismutase from Xanthomonas campestris pv. campestris and its expression during bacterial-plant interactions.

A recombinant plasmid selected from a library of Xanthomonas campestris pv. campestris genomic DNA by functional complementation of a superoxide dismutase (SOD)-deficient strain of Escherichia coli contained a gene encoding the major SOD activity of X. campestris pv. campestris. Inhibition and renaturation studies suggested that manganese was the metal cofactor for this SOD. Examination of the nucleotide sequence of an active subclone revealed a 612-bp open reading frame that encodes a protein with high amino acid sequence homology to a range of SOD enzymes. The sod gene was mutagenized with Tn5-lacZ. None of the insertions that abolished SOD-conferring activity were in the correct orientation for lacZ expression. Repeated attempts to introduce these insertions into the chromosome of X. campestris pv. campestris were unsuccessful and it was concluded that the sod gene may be essential for viability. In order to monitor the expression of the sod gene, a sod-gus transcriptional fusion was constructed. Expression of the sod gene varied according to the growth stage of the organism in culture. In planta, the sod gene was induced within 3 to 4 h of inoculation, with similar kinetics during compatible and incompatible interactions with turnip and pepper, respectively.

Induction of hydroxycinnamoyl-tyramine conjugates in pepper by Xanthomonas campestris, a plant defense response activated by hrp gene-dependent and hrp gene-independent mechanisms.

Inoculation of pepper leaves, Capsicum annuum cv. Early Calwonder ECW 10R, with strains of Xanthomonas campestris led to an accumulation of the phenolic conjugates feruloyltyramine (FT) and p-coumaroyltyramine (CT) 24 h postinoculation in nonhost- and gene-for-gene-determined incompatible interactions with X. campestris pv. campestris and X. campestris pv. vesicatoria, respectively. In contrast, neither compound was detected in compatible interactions with X. campestris pv. vesicatoria. The accumulation of FT and CT was preceded by an increase in the extractable activity of tyrosine decarboxylase as well as increases in the transcription of genes encoding phenylalanine ammonia-lyase and tyramine hydroxycinnamoyl transferase. No such changes were detected in compatible interactions. Very rapid accumulation of FT and CT occurred (4 h postinoculation) in pepper in response to a X. campestris pv. campestris mutant carrying a deletion of the hrp gene cluster. In contrast, hrp mutants of X. campestris pv. vesicatoria failed to elicit the production of FT and CT. These observations suggest the existence of hrp gene-dependent and -independent activation mechanisms of a defense response involving hydroxycinnamoyltyramines.

Defense-related gene induction in Brassica campestris in response to defined mutants of Xanthomonas campestris with altered pathogenicity.

We have studied the induction of beta-1,3-glucanase (BGL) in turnip following inoculation with pathovars of Xanthomonas campestris and derived mutants. BGL transcript accumulated more rapidly in leaves in the incompatible interactions with X. c. pv. armoraciae and X. c. pv. raphani than in the compatible interaction with X. c. pv. campestris. No accumulation was seen in response to wounding or inoculation with water, salicylic acid, or Escherichia coli. Deletion of the hrp cluster from the X. campestris pathovars caused a reduction in the level of transcript accumulation; these effects were much more pronounced in the incompatible than in the compatible interaction, in which bacterial growth was also affected. In the compatible interaction, bacterial growth and BGL transcript accumulation were not altered by mutation of bacterial genes involved in the regulation of the synthesis of extracellular enzymes or their export from the cell, or by mutation of the structural genes for extracellular endoglucanase and serine protease. Mutation of genes involved in the synthesis of extracellular polysaccharide or lipopolysaccharide reduced bacterial survival in planta, so that the numbers were between two and three orders of magnitude lower than the number of wild-type bacteria. However, total BGL transcript accumulation after inoculation with these mutants was about 80% of that seen after inoculation with the wild-type bacteria, suggesting that one aspect of the role of extracellular polysaccharide and lipopolysaccharide in pathogenesis is to mask the presence of bacteria in the plant. Our results are discussed in the context of work on other plant-microbe interactions.

Cloning and characterization of the rpfC gene of Xanthomonas oryzae pv. oryzae: involvement in exopolysaccharide production and virulence to rice.

rpfC is one of a cluster of genes which coordinately regulate the synthesis of extracellular enzymes and exopolysaccharide and pathogenicity in Xanthomonas campestris pv. campestris, the black rot pathogen of brassicas. An rpfC homolog which could functionally complement an rpfC mutant of X. campestris pv. campestris was identified in Xanthomonas oryzae pv. oryzae and the gene was characterized. Mutation of this gene in X. oryzae pv. oryzae had no effect on extracellular enzymes, but exopolysaccharide synthesis and virulence to rice were substantially reduced.

Nucleotide sequence of the engXCA gene encoding the major endoglucanase of Xanthomonas campestris pv. campestris.

The nucleotide sequence of the gene (engXCA) encoding the major extracellular endoglucanase (ENGXCA) of the phytopathogenic bacterium Xanthomonas campestris pv. campestris (X. c. campestris) was determined and compared with the N-terminal amino acid (aa) sequence of the purified enzyme. An open reading frame of 1479 bp encoding 493 aa was identified, of which the N-terminal 25 aa represent a potential signal peptide. Determination of the exact position of a Tn5 insertion within engXCA, which did not reduce the encoded enzyme activity, indicated that the C-terminal region of the protein is not crucial for ENGXCA activity. Comparison of the complete deduced aa sequence with those deduced from other endoglucanase- and exoglucanase-encoding genes revealed a region with a high degree of homology, located towards the C terminus of the protein. These data indicate that the X. c. campestris ENGXCA may have a domain structure similar to that of many other bacterial and fungal cellulolytic enzymes. Hydrophobic cluster analysis was performed on the deduced aa sequence. Comparison of this analysis with those of 30 other cellulase sequences belonging to six different families indicated that the X. c. campestris enzyme can be classified in family A. The two aa residues which had previously been identified as 'potentially catalytic' within this family of cellulases, are conserved in the X. c. campestris ENGXCA.