Calcium modulation of bacterial wilt disease on potato.

Ralstonia solanacearum species complex (RSSC) is a phytopathogenic bacterial group that causes bacterial wilt in several crops, being potato (Solanum tuberosum) one of the most important hosts. The relationship between the potato plant ionome (mineral and trace elements composition) and the resistance levels to this pathogen has not been addressed until now. Mineral content of xylem sap, roots, stems and leaves of potato genotypes with different levels of resistance to bacterial wilt was assessed in this work, revealing a positive correlation between divalent calcium (Ca) cation concentrations and genotype resistance. The aim of this study was to investigate the effect of Ca on bacterial wilt resistance, and on the growth and virulence of RSSC. Ca supplementation significantly decreased the growth rate of Ralstonia pseudosolanacearum GMI1000 in minimal medium and affected several virulence traits such as biofilm formation and twitching motility. We also incorporate for the first time the use of microfluidic chambers to follow the pathogen growth and biofilm formation in conditions mimicking the plant vascular system. By using this approach, a reduction in biofilm formation was observed when both, rich and minimal media, were supplemented with Ca. Assessment of the effect of Ca amendments on bacterial wilt progress in potato genotypes revealed a significant delay in disease progress, or a complete absence of wilting symptoms in the case of partially resistant genotypes. This work contributes to the understanding of Ca effect on virulence of this important pathogen and provides new strategies for an integrated control of bacterial wilt on potato. IMPORTANCE: Ralstonia solanacearum species complex (RSSC) includes a diverse group of bacterial strains that cause bacterial wilt. This disease is difficult to control due to pathogen aggressiveness, persistence, wide range of hosts, and wide geographic distribution in tropical, subtropical, and temperate regions. RSSC causes considerable losses depending on the pathogen strain, host, soil type, environmental conditions, and cultural practices. In potato, losses of $19 billion per year have been estimated for this pathogen worldwide. In this study, we report for the first time the mineral composition found in xylem sap and plant tissues of potato germplasm with different levels of resistance to bacterial wilt. This study underscores the crucial role of calcium (Ca) concentration in the xylem sap and stem in relation to the resistance of different genotypes. Our in vitro experiments provide evidence of Ca's inhibitory effect on the growth, biofilm formation, and twitching movement of the model RSSC strain R. pseudosolanacearum GMI1000. This study introduces a novel element, the Ca concentration, which should be included into the integrated disease control management strategies for bacterial wilt in potatoes.

The Ptr1 Locus of Solanum lycopersicoides Confers Resistance to Race 1 Strains of Pseudomonas syringae pv. tomato and to Ralstonia pseudosolanacearum by Recognizing the Type III Effectors AvrRpt2 and RipBN.

Race 1 strains of Pseudomonas syringae pv. tomato, which cause bacterial speck disease of tomato, are becoming increasingly common and no simply inherited genetic resistance to such strains is known. We discovered that a locus in Solanum lycopersicoides, termed Pseudomonas tomato race 1 (Ptr1), confers resistance to race 1 P. syringae pv. tomato strains by detecting the activity of type III effector AvrRpt2. In Arabidopsis, AvrRpt2 degrades the RIN4 protein, thereby activating RPS2-mediated immunity. Using site-directed mutagenesis of AvrRpt2, we found that, like RPS2, activation of Ptr1 requires AvrRpt2 proteolytic activity. Ptr1 also detected the activity of AvrRpt2 homologs from diverse bacteria, including one in Ralstonia pseudosolanacearum. The genome sequence of S. lycopersicoides revealed no RPS2 homolog in the Ptr1 region. Ptr1 could play an important role in controlling bacterial speck disease and its future cloning may shed light on an example of convergent evolution for recognition of a widespread type III effector.

Anaerobic digestion in mesophilic and room temperature conditions: Digestion performance and soil-borne pathogen survival.

Tomato plant waste (TPW) was used as the feedstock of a batch anaerobic reactor to evaluate the effect of anaerobic digestion on Ralstonia solanacearum and Phytophthora capsici survival. Batch experiments were carried out for TS (total solid) concentrations of 2%, 4% and 6% respectively, at mesophilic (37±1°C) and room (20-25°C) temperatures. Results showed that higher digestion performance was achieved under mesophilic digestion temperature and lower TS concentration conditions. The biogas production ranged from 71 to 416L/kg VS (volatile solids). The inactivation of anaerobic digestion tended to increase as digestion performance improved. The maximum log copies reduction of R. solanacearum and P. capsici detected by quantitative PCR (polymerase chain reaction) were 3.80 and 4.08 respectively in reactors with 4% TS concentration at mesophilic temperatures. However, both in mesophilic and room temperature conditions, the lowest reduction of R. solanacearum was found in the reactors with 6% TS concentration, which possessed the highest VFA (volatile fatty acid) concentration. These findings indicated that simple accumulation of VFAs failed to restrain R. solanacearum effectively, although the VFAs were considered poisonous. P. capsici was nearly completely dead under all conditions. Based on the digestion performance and the pathogen survival rate, a model was established to evaluate the digestate biosafety.

High-density genome-wide association mapping implicates an F-box encoding gene in Medicago truncatula resistance to Aphanomyces euteiches.

• The use of quantitative disease resistance (QDR) is a promising strategy for promoting durable resistance to plant pathogens, but genes involved in QDR are largely unknown. To identify genetic components and accelerate improvement of QDR in legumes to the root pathogen Aphanomyces euteiches, we took advantage of both the recently generated massive genomic data for Medicago truncatula and natural variation of this model legume. • A high-density (≈5.1 million single nucleotide polymorphisms (SNPs)) genome-wide association study (GWAS) was performed with both in vitro and glasshouse phenotyping data collected for 179 lines. • GWAS identified several candidate genes and pinpointed two independent major loci on the top of chromosome 3 that were detected in both phenotyping methods. Candidate SNPs in the most significant locus (σ(A)²= 23%) were in the promoter and coding regions of an F-box protein coding gene. Subsequent qRT-PCR and bioinformatic analyses performed on 20 lines demonstrated that resistance is associated with mutations directly affecting the interaction domain of the F-box protein rather than gene expression. • These results refine the position of previously identified QTL to specific candidate genes, suggest potential molecular mechanisms, and identify new loci explaining QDR against A. euteiches.

The noncommensal bacterium Methylococcus capsulatus (Bath) ameliorates dextran sulfate (Sodium Salt)-Induced Ulcerative Colitis by influencing mechanisms essential for maintenance of the colonic barrier function.

Dietary inclusion of a bacterial meal has recently been shown to efficiently abolish soybean meal-induced enteritis in Atlantic salmon. The objective of this study was to investigate whether inclusion of this bacterial meal in the diet could abrogate disease development in a murine model of epithelial injury and colitis and thus possibly have therapeutic potential in human inflammatory bowel disease. C57BL/6N mice were fed ad libitum a control diet or an experimental diet containing 254 g/kg of body weight BioProtein, a bacterial meal consisting of Methylococcus capsulatus (Bath), together with the heterogenic bacteria Ralstonia sp., Brevibacillus agri, and Aneurinibacillus sp. At day 8, colitis was induced by 3.5% dextran sulfate sodium (DSS) ad libitum in the drinking water for 6 days. Symptoms of DSS treatment were less profound after prophylactic treatment with the diet containing the BioProtein. Colitis-associated parameters such as reduced body weight, colon shortening, and epithelial damage also showed significant improvement. Levels of acute-phase reactants, proteins whose plasma concentrations increase in response to inflammation, and neutrophil infiltration were reduced. On the other, increased epithelial cell proliferation and enhanced mucin 2 (Muc2) transcription indicated improved integrity of the colonic epithelial layer. BioProtein mainly consists of Methylococcus capsulatus (Bath) (88%). The results that we obtained when using a bacterial meal consisting of M. capsulatus (Bath) were similar to those obtained when using BioProtein in the DSS model. Our results show that a bacterial meal of the noncommensal bacterium M. capsulatus (Bath) has the potential to attenuate DSS-induced colitis in mice by enhancing colonic barrier function, as judged by increased epithelial proliferation and increased Muc2 transcription.

Bacterial swimming, swarming and chemotactic response to heavy metal presence: which could be the influence on wastewater biotreatment efficiency?

Fixed-bed reactors are usually designed for wastewater biotreatments, where the biofilm establishment and maintenance play the most important roles. Biofilm development strictly relies on different types of bacterial motility: swimming, swarming, and chemotaxis, which can be altered by the microenvironment conditions. The aim of this work is to do an integrated study on the effects of Cu(II), Cd(II), Zn(II) and Cr(VI) on swimming, swarming and chemotaxis of Pseudomonas veronii 2E, Delftia acidovorans AR and Ralstonia taiwanensis M2 to improve biofilm development and maintenance for metal loaded wastewater biotreatment in fixed-bed bioreactors. Swimming, swarming and chemotactic response evaluation experiments were carried out at different metal concentrations. P. veronii 2E motility was not affected by metal presence, being this strain optimal for fixed-bed reactors. D. acidovorans AR swarming was inhibited by Cd and Zn. Although R. taiwanensis M2 showed high resistance to Cu, Cd, Cr and Zn, motility was definitively altered, so further studies on R. taiwanensis M2 resistance mechanisms would be particularly interesting.

Light modulates important physiological features of Ralstonia pseudosolanacearum during the colonization of tomato plants.

Ralstonia pseudosolanacearum GMI1000 (Rpso GMI1000) is a soil-borne vascular phytopathogen that infects host plants through the root system causing wilting disease in a wide range of agro-economic interest crops, producing economical losses. Several features contribute to the full bacterial virulence. In this work we study the participation of light, an important environmental factor, in the regulation of the physiological attributes and infectivity of Rpso GMI1000. In silico analysis of the Rpso genome revealed the presence of a Rsp0254 gene, which encodes a putative blue light LOV-type photoreceptor. We constructed a mutant strain of Rpso lacking the LOV protein and found that the loss of this protein and light, influenced characteristics involved in the pathogenicity process such as motility, adhesion and the biofilms development, which allows the successful host plant colonization, rendering bacterial wilt. This protein could be involved in the adaptive responses to environmental changes. We demonstrated that light sensing and the LOV protein, would be used as a location signal in the host plant, to regulate the expression of several virulence factors, in a time and tissue dependent way. Consequently, bacteria could use an external signal and Rpsolov gene to know their location within plant tissue during the colonization process.

Strains of internal biofilm in aerobic granular membrane bioreactors.

This study isolated strains in suspended liquor, the surface fouling layer, and biofilm inside hollow-fiber membranes of a membrane bioreactor (MBR); analyzed their distributions, sizes, surface charges, and growth behaviors; and determined the quantities of extracellular polymeric substances (EPS) secreted by these strains under different organic loadings. Three strains, which may penetrate the microfiltration membranes, were close relatives of the Ralstonia mannitolilytica strain SDV (GenBank Accession No. GU451066), Arthrobacter sp. BJQ-2 (GenBank Accession No. GU451067), and Actinobacterium DS3 (GenBank Accession No. GU451068). Among these three strains, only Arthrobacter sp. developed an internal biofilm. The relatively short length of Arthrobacter sp. minimizes resistance to cells moving through the membrane matrix, thereby enhancing its ability to build a biofilm in the interior surface of membranes.

Biocontrol of Tomato Bacterial Wilt by Foliar Spray Application of a Novel Strain of Endophytic Bacillus sp.

The aim of the present study was to identify a strain of endophytic Bacillus species that control tomato bacterial wilt by foliar spray application. Fifty heat-tolerant endophytic bacteria were isolated from the surface-sterilized foliar tissues of symptomless tomato plants that had been pre-inoculated with the pathogen Ralstonia pseudosolanacearum. In the primary screening, we assessed the suppressive effects of a shoot-dipping treatment with bacterial strains against bacterial wilt on tomato seedlings grown on peat pellets. Bacillus sp. strains G1S3 and G4L1 significantly suppressed the incidence of tomato bacterial wilt. In subsequent pot experiments, the biocontrol efficacy of foliar spray application was examined under glasshouse conditions. G4L1 displayed consistent and significant disease suppression, and, thus, was selected as a biocontrol candidate. Moreover, the pathogen population in the stem of G4L1-treated plants was markedly smaller than that in control plants. A quantitative real-time PCR analysis revealed that the foliar spraying of tomato plants with G4L1 up-regulated the expression of PR-1a and LoxD in stem and GluB in roots upon the pathogen inoculation, implying that the induction of salicylic acid-, jasmonic acid-, and ethylene-dependent defenses was involved in the protective effects of this strain. In the re-isolation experiment, G4L1 efficiently colonized foliar tissues for at least 4| |weeks after spray application. Collectively, the present results indicate that G4L1 is a promising biocontrol agent for tomato bacterial wilt. Furthermore, to the best of our knowledge, this is the first study to report the biocontrol of bacterial wilt by the foliar spraying with an endophytic Bacillus species.

Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco.

A new member of the AP2/ERF transcription factor family, GmERF3, was isolated from soybean. Sequence analysis showed that GmERF3 contained an AP2/ERF domain of 58 amino acids and two putative nuclear localization signal (NLS) domains. It belonged to a group IV protein in the ERF (ethylene response factor) subfamily as typified by a conserved N-terminal motif [MCGGAI(I/L)]. Expression of GmERF3 was induced by treatments with high salinity, drought, abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and soybean mosaic virus (SMV), whereas there was no significant GmERF3 mRNA accumulation under cold stress treatment. GmERF3 could bind to the GCC box and DRE/CRT element, and was targeted to the nucleus when transiently expressed in onion epidermal cells. The GmERF3 protein fused to the GAL4 DNA-binding domain to activate transcription of reporter genes in yeast. Ectopic expression of the GmERF3 gene in transgenic tobacco plants induced the expression of some PR genes and enhanced resistance against infection by Ralstonia solanacearum, Alternaria alternata, and tobacco mosaic virus (TMV), and gave tolerance to high salinity and dehydration stresses. Furthermore, overexpression of GmERF3 in transgenic tobacco led to higher levels of free proline and soluble carbohydrates compared to wild-type plants under drought conditions. The overall results suggested that GmERF3 as an AP2/ERF transcription factor may play dual roles in response to biotic and abiotic stresses in plants.

Association between gentamicin resistance and stress tolerance in water isolates of Ralstonia pickettii and R. mannitolilytica.

Members of the species Ralstonia pickettii and R. mannitolilytica, although ubiquitous and lacking major virulence factors, have been associated with nosocomial outbreaks. Tolerance to metals, antibiotics, and disinfectants may represent an advantage for their ubiquity and opportunistic pathogenic potential. In this study, we compared five strains that differed on the origin (hospital effluent, tap water, mineral water) and in the susceptibility to aminoglycosides, regarding their tolerance to metals and disinfection. The growth kinetics and biofilm formation capacity were tested in four R. pickettii strains and one R. mannitolilytica at sub-inhibitory concentrations of aminoglycosides or arsenite. The survival to UV radiation, chlorine, or hydrogen peroxide was also compared in aminoglycoside resistant and susceptible strains. Aminoglycoside-resistant strains presented a higher tolerance to arsenite than the susceptible ones and either aminoglycosides or arsenite was observed to stimulate the biofilm formation. Sub-inhibitory concentrations of the aminoglycoside gentamicin or arsenite significantly decreased the growth rate and yield, but only arsenite caused a significant increase of the lag phase. Hydrogen peroxide presented higher disinfection effectiveness against aminoglycoside susceptible than against resistant strains, an effect that was not observed for UV or chlorine. Although this conclusion needs validation based on a larger number of isolates, including clinical, the results suggest that aminoglycoside resistance may be associated with traits that influence Ralstonia spp. fitness in the environment.

Ralstonia eutropha H16 flagellation changes according to nutrient supply and state of poly(3-hydroxybutyrate) accumulation.

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE), in combination with matrix-assisted laser desorption ionization-time of flight analysis, and the recently revealed genome sequence of Ralstonia eutropha H16 were employed to detect and identify proteins that are differentially expressed during different phases of poly(3-hydroxybutyric acid) (PHB) metabolism. For this, a modified protein extraction protocol applicable to PHB-harboring cells was developed to enable 2D PAGE-based proteome analysis of such cells. Subsequently, samples from (i) the exponential growth phase, (ii) the stationary growth phase permissive for PHB biosynthesis, and (iii) a phase permissive for PHB mobilization were analyzed. Among several proteins exhibiting quantitative changes during the time course of a cultivation experiment, flagellin, which is the main protein of bacterial flagella, was identified. Initial investigations that report on changes of flagellation for R. eutropha were done, but 2D PAGE and electron microscopic examinations of cells revealed clear evidence that R. eutropha exhibited further significant changes in flagellation depending on the life cycle, nutritional supply, and, in particular, PHB metabolism. The results of our study suggest that R. eutropha is strongly flagellated in the exponential growth phase and loses a certain number of flagella in transition to the stationary phase. In the stationary phase under conditions permissive for PHB biosynthesis, flagellation of cells admittedly stagnated. However, under conditions permissive for intracellular PHB mobilization after a nitrogen source was added to cells that are carbon deprived but with full PHB accumulation, flagella are lost. This might be due to a degradation of flagella; at least, the cells stopped flagellin synthesis while normal degradation continued. In contrast, under nutrient limitation or the loss of phasins, cells retained their flagella.

Validation of reference genes for quantitative gene expression analysis in Ralstonia pseudosolanacearum CQPS-1 under environment stress.

Quantitative real-time reverse transcriptase PCR (qRT-PCR) has become the method choice for quantification of gene expression changes, however, the accuracy of the method depends on the stability of reference genes. Ralstonia pseudosolanacearum (R. pseudosolanacearum) is an important plant pathogen, infecting >450 plant species and causing bacterial wilt. In order to identify stable reference genes in R. pseudosolanacearum CQPS-1 under different environment stresses. We used five tools (△Ct method, GeNorm, NormFinder, BestKeeper, and RefFinder) to evaluate the stability of seven candidate reference genes including phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 16S ribosomal RNA (16S), cell division protein ftsZ (ftsZ), DNA gyrase subunit A (gyrA), Ribosomal protein L13 (rplM), and phosphoserine aminotransferase (serC) under biotic (growth phases) and abiotic stress (temperature, hydroxycoumarins, nutrition). Overall, gyrA and serC were the most stable genes under different growth phases, while serC, gyrA and ftsZ during temperature stress, gyrA, ftsZ and 16S under hydroxycoumarins stress, and serC and 16S under nutrition stress conditions. This study provides useful resources for normalizing expression changes of target genes in R. pseudosolanacearum subjected to environment stress.

Identification of boric acid as a novel chemoattractant and elucidation of its chemoreceptor in Ralstonia pseudosolanacearum Ps29.

Chemotaxis enables bacteria to move toward more favorable environmental conditions. We observed chemotaxis toward boric acid by Ralstonia pseudosolanacearum Ps29. At higher concentrations, the chemotactic response of R. pseudosolanacearum toward boric acid was comparable to or higher than that toward L-malate, indicating that boric acid is a strong attractant for R. pseudosolanacearum. Chemotaxis assays under different pH conditions suggested that R. pseudosolanacearum recognizes B(OH)3 (or B(OH3) + B(OH)4-) but not B(OH)4- alone. Our previous study revealed that R. pseudosolanacearum Ps29 harbors homologs of all 22R. pseudosolanacearum GMI1000 mcp genes. Screening of 22 mcp single-deletion mutants identified the RS_RS17100 homolog as the boric acid chemoreceptor, which was designated McpB. The McpB ligand-binding domain (LBD) was purified in order to characterize its binding to boric acid. Using isothermal titration calorimetry, we demonstrated that boric acid binds directly to the McpB LBD with a K D (dissociation constant) of 5.4 µM. Analytical ultracentrifugation studies revealed that the McpB LBD is present as a dimer that recognizes one boric acid molecule.

Surface active properties of bacterial strains isolated from petroleum hydrocarbon-bioremediated soil.

Two bacterial strains identified as Ralstonia picketti (BP 20) and Alcaligenes piechaudii (CZOR L-1B) were isolated from petroleum hydrocarbon-contaminated soil following bioremediation treatment. The surface active properties, e.g. surface tension, emulsification and foamability of their culture filtrates were evaluated. Bacterial cell-surface hydrophobicity (BAH) as measured by analyzing cell affinity towards aliphatic and aromatic compounds was also determinated. The bacteria grew in liquid cultures containing 1% (v/v) of crude oil as carbon and energy source at 30 degrees C under aerobic conditions. The surface tensions were reduced to 61 mN/m and 55 mN/m by Ralstonia picketti and Alcaligenes piechaudii, respectively. The emulsification index (EI24) was almost 100% for all tested compounds except diesel oil. The stability of the emulsions was deteminated at 4 degrees C, 45 degrees C and 65 degrees C. The emulsions were stable at 4 degrees C. Ralstonia picketti was better foam inducer (FV = 50 ml) compared to Alcaligenes piechaudii (FV = 10 ml). The BAH measurements revealed higher adhesion of Alcaligenes piechaudii cells towards different hydrocarbons compared to Ralstonia picketti cells. The strains were found to have a surface hydrophobicity in the following order: aliphatic hydrocarbons, BTEX, and PAHs. The ability to adhere to bulk hydrocarbon is mostly a characteristic of hydrocarbon-degrading bacteria. The strains were found to be better emulsifiers than surface tension reducers. They produce water-soluble extracellular bioemulsifiers. Both bacterial isolates have good properties to use them, mainly in the petroleum industry, e.g. in enhanced oil recovery and in bioremediation processes-primarily due to their emulsification property, i.e. emulsion forming and stabilizing capacity.

Effects of environmental parameters on the dual-species biofilms formed by Escherichia coli O157:H7 and Ralstonia insidiosa, a strong biofilm producer isolated from a fresh-cut produce processing plant.

Biofilm-forming bacteria resident to food processing facilities are a food safety concern due to the potential of biofilms to harbor foodborne bacterial pathogens. When cultured together, Ralstonia insidiosa, a strong biofilm former frequently isolated from produce processing environments, has been shown to promote the incorporation of Escherichia coli O157:H7 into dual-species biofilms. In this study, interactions between E. coli O157:H7 and R. insidiosa were examined under different incubating conditions. Under static culture conditions, the incorporation of E. coli O157:H7 into biofilms with R. insidiosa was not significantly affected by either low incubating temperature (10°C) or by limited nutrient availability. Greater enhancement of E. coli O157:H7 incorporation in dual-species biofilms was observed by using a continuous culture system with limited nutrient availability. Under the continuous culture conditions used in this study, E coli O157:H7 cells showed a strong tendency of colocalizing with R. insidiosa on a glass surface at the early stage of biofilm formation. As the biofilms matured, E coli O157:H7 cells were mostly found at the bottom layer of the dual-species biofilms, suggesting an effective protection by R. insidiosa in the mature biofilms.

Nodulation of Mimosa spp. by the beta-proteobacterium Ralstonia taiwanensis.

Several beta-proteobacteria have been isolated from legume root nodules and some of these are thought to be capable of nodulating and fixing N2. However, in no case has there been detailed studies confirming that they are the active symbionts. Here, Ralstonia taiwanensis LMG19424, which was originally isolated from Mimosa pudica nodules, was transformed to carry the green fluorescent protein (gfp) reporter gene before being used to inoculate axenically-grown seedlings of M. pudica and M. diplotricha. Plants were harvested at various intervals for 56 days after inoculation, then examined for evidence of infection and nodule formation. Nodulation of both Mimosa spp. was abundant, and acetylene reduction assays confirmed that nodules had nitrogenase activity. Confocal laser scanning microscopy (CLSM) showed that fresh M. pudica nodules with nitrogenase activity had infected cells containing bacteroids expressing gfp. In parallel, fixed and embedded nodules from both Mimosa spp. were sectioned for light and electron microscopy, followed by immunogold labeling with antibodies raised against gfp and nitrogenase Fe (nifH) protein. Significant immunolabeling with these antibodies confirmed that R. taiwanensis LMG19424 is an effective N2-fixing symbiont of Mimosa spp. Both species were infected via root hairs and, in all respects, the nodule ontogeny and development was similar to that described for other mimosoid legumes. The nodules were indeterminate with a persistent meristem, an invasion zone containing host cells being invaded via prominent infection threads, and an N2-fixing zone with infected cells containing membrane-bound symbiosomes.

Antibacterial enzymes from the functional screening of metagenomic libraries hosted in Ralstonia metallidurans.

Phenotype-based screening of bacterial metagenomic libraries provides an avenue for the discovery of novel genes, enzymes, and metabolites that have a variety of potential clinical and industrial uses. Here, we report the identification of a functionally diverse collection of antibacterially active enzymes from the phenotypic screening of 700 000 cosmid clones prepared from Arizona soil DNA and hosted in Ralstonia metallidurans. Environmental DNA clones surrounded by zones of growth inhibition in a bacterial overlay assay were found, through bioinformatics and functional analyses, to encode enzymes with predicted peptidase, lipase, and glycolytic activities conferring antibiosis. The antibacterial activities observed in our R. metallidurans-based assay could not be replicated with the same clones in screens using Escherichia coli as a heterologous host, suggesting that the large-scale screening of metagenomic libraries for antibiosis using phylogenetically diverse hosts should be a productive strategy for identifying enzymes with functionally diverse antibacterial activities.

T3_MM: a Markov model effectively classifies bacterial type III secretion signals.

MOTIVATION: Type III Secretion Systems (T3SSs) play important roles in the interaction between gram-negative bacteria and their hosts. T3SSs function by translocating a group of bacterial effector proteins into the host cytoplasm. The details of specific type III secretion process are yet to be clarified. This research focused on comparing the amino acid composition within the N-terminal 100 amino acids from type III secretion (T3S) signal sequences or non-T3S proteins, specifically whether each residue exerts a constraint on residues found in adjacent positions. We used these comparisons to set up a statistic model to quantitatively model and effectively distinguish T3S effectors. RESULTS: In this study, the amino acid composition (Aac) probability profiles conditional on its sequentially preceding position and corresponding amino acids were compared between N-terminal sequences of T3S and non-T3S proteins. The profiles are generally different. A Markov model, namely T3_MM, was consequently designed to calculate the total Aac conditional probability difference, i.e., the likelihood ratio of a sequence being a T3S or a non-T3S protein. With T3_MM, known T3S and non-T3S proteins were found to well approximate two distinct normal distributions. The model could distinguish validated T3S and non-T3S proteins with a 5-fold cross-validation sensitivity of 83.9% at a specificity of 90.3%. T3_MM was also shown to be more robust, accurate, simple, and statistically quantitative, when compared with other T3S protein prediction models. The high effectiveness of T3_MM also indicated the overall Aac difference between N-termini of T3S and non-T3S proteins, and the constraint of Aac exerted by its preceding position and corresponding Aac. AVAILABILITY: An R package for T3_MM is freely downloadable from: http://biocomputer.bio.cuhk.edu.hk/softwares/T3_MM. T3_MM web server: http://biocomputer.bio.cuhk.edu.hk/T3DB/T3_MM.php.