Molecular characterization of two gene loci required for production of the key pathogenicity factor pectate lyase in Pseudomonas viridiflava.

Four pleiotropic mutants of Pseudomonas viridiflava strain PJ-08-6A that were deficient in production of both pectate lyase (Pel) and protease (Prt) were isolated following transposon mutagenesis. Unlike secretion-defective (Out-) mutants, these four showed no accumulation of enzymes within the cells. Southern hybridization analysis revealed that each mutant had Tn5 inserted in one of two EcoRI genomic fragments. These EcoRI fragments (5.2- and 6.3-kb) appeared to contain two distinct gene loci, designated repA and repB, which were required for production of extracellular enzymes in this bacterium. Cosmid clones carrying the functional repA and repB DNA fragments were identified in a genomic library of strain PJ-08-6A. After analysis of repA+ plasmids by restriction mapping and marker-exchange mutagenesis, the repA gene was located in a joint region between the 1.8-kb EcoRI-HindIII and 2.8-kb EcoRI fragments cloned. Nucleotide sequence analysis of the repA region revealed the presence of an open reading frame consisting of 2,790 bases. The RepA protein predicted from the DNA sequence showed 93% similarity in amino acid sequence to the LemA protein of P. syringae pv. syringae, which was previously identified as a member of a two-component global regulatory system. A plasmid carrying the lemA gene of P. syringae pv. syringae was capable of complementing the RepA- mutation in P. viridiflava. The functions of the repA and lemA genes thus appear to be similar and interchangeable. Mutants of P. viridiflava strain SF312A deficient in production of Pel, Prt, and the exopolysaccharide alginate also were identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Occurrence of alginate gene sequences among members of the pseudomonad rRNA homology groups I-IV.

Total genomic DNA of 13 pseudomonads representing rRNA homology groups I-IV were screened for sequences homologous to four Pseudomonas aeruginosa alginate (alg) genes by Southern hybridization. Biotinylated probes for three structural genes (algA, algC and algD) and one regulatory gene (algR1) were prepared. Genomic DNA of strains representing group I (P. syringae pv. glycinea, P. viridiflava and P. corrugata) hybridized with all four gene probes. Hybridizing fragments were of differing sizes, indicating that evolutionary divergence among group I members has occurred. P. corrugata has not been reported to synthesize alginate. Genomic DNA from representatives of groups II-IV gave no or very weak hybridization with the probes except for algC. This study indicates that the ability to produce alginic acid as an exopolysaccharide among the pseudomonads is restricted to members of rRNA homology group I in agreement with earlier physiological studies.

The structure of the acidic exopolysaccharide of Pseudomonas marginalis strains PF-05-2 and PM-LB-1.

The structure of an acidic exopolysaccharide of two strains of Pseudomonas marginalis, a bacterium which causes soft rots of various vegetables, has been determined to consist of a repeating unit of: ----4) beta-D-Manp-(1----3)alpha-D-Glcp-(1----4)alpha-L-Rhap-(1-. The glucose is pyruvated at O-4 and O-6 and the mannose is acetylated at either O-2 or O-3.

Studies on the primary structure of short polysaccharides using SEC MALDI mass spectroscopy.

The introduction of size-exclusion chromatography (SEC) analysis of polysaccharides prior to MALDI mass spectroscopy accounts for the determination of the molecular mass of the repeating unit when neutral homopolymers are investigated. In the case of natural polysaccharides characterised by more complicated structural features (presence of non-carbohydrate substituents, charged groups, etc.), this mass value usually is in agreement with more than one sugar composition. Therefore, it is not sufficient to give the correct monosaccharidic composition of the polysaccharide investigated. To solve this problem, MALDI spectra were recorded on the permethylated sample and post-source decay experiments were performed on precursor ions. In this way, the composition (in terms of Hex, HexNAc, etc.), size and sequence of the repeating unit were determined.

The structure of the acidic exopolysaccharide produced by Pseudomonas "gingeri" strain Pf9.

The structure of the acidic exopolysaccharide produced by the mushroom pathogen Pseudomonas "gingeri" strain Pf9, a bacterium which causes ginger blotch, was investigated by chemical analysis, mass spectrometry and 1D and 2D NMR spectroscopy. The polysaccharide consists of the linear trisaccharide repeating unit [formula: see text] where the cyclic pyruvic acetal groups at O-4 and O-6 of the mannopyranosyl residues have the S-configuration. Methylation analysis under neutral conditions and NMR data showed that the mannose residues are acetylated at O-2. This exopolysaccharide has the same structure as the E. coli K55 capsular polysaccharide and differs from the Klebsiella K5 capsular polysaccharide only in the position of acetylation (C-2 of the glucopyranose residue).

The structure of the exopolysaccharide of Pseudomonas fluorescens strain H13.

An acidic exopolysaccharide was isolated from P. fluorescens strain H13. The structure of the polysaccharide repeating unit was determined using chemical methods and 1D and 2D NMR techniques. The repeating unit was characterized as a trisaccharide composed of D-glucose, 2-acetamido-2-deoxy-D-glucose and 4-O-acetyl-2-acetamido-2-deoxy-D-mannuronic acid.

Preparation, isolation, and characterization of cutin monomers and oligomers from tomato peels.

Cutin in tomato peels was depolymerized in methanolic base to yield cutin monomers or a mixture of cutin oligomers. These products were isolated by typical solvent extraction methods or by precipitation, and the isolates were characterized by chromatographic and spectroscopic analyses. It was determined that the compositions of the isolates from both isolation procedures were similar, although solvent extraction gave higher yields. However, the precipitation method, which is easy to carry out and avoids the use of undesirable organic solvents, may be preferable in commercial processes for recovering these compounds.

Naturally occurring biofilms on alfalfa and other types of sprouts.

Scanning electron microscopy was used to examine the cotyledons, hypocotyls, and roots of alfalfa, broccoli, clover, and sunflower sprouts purchased from retail outlets as well as alfalfa sprouts grown in the laboratory using a tray system equipped with automatic irrigation. Biofilms were observed on all plant parts of the four types of commercially grown sprouts. Biofilms were also commonly observed on alfalfa sprouts grown in the laboratory by 2 days of growth. Rod-shaped bacteria of various sizes were predominant on all sprouts examined both as free-living cells and as components of biofilms. Occasionally, cocci-shaped bacteria as well as yeast cells were also present in biofilms. The microbes contained in the biofilms appeared to be attached to each other and to the plant surface by a matrix, most likely composed of bacterial exopolysaccharides. Biofilms were most abundant and of the largest dimensions on cotyledons, sometimes covering close to the entire cotyledon surface (approximately 2 mm in length). Naturally occurring biofilms on sprouts may afford protected colonization sites for human pathogens such as Salmonella and Escherichia coli O157:H7, making their eradication with antimicrobial compounds difficult.

Analysis of native microflora and selection of strains antagonistic to human pathogens on fresh produce.

The native microflora of three types of produce (green bell peppers, Romaine lettuce, and prepeeled baby carrots) and two types of sprouting seeds (alfalfa and clover) were investigated. Aerobic plate count (APC) for each produce or seed type as determined on Pseudomonas agar F (PAF) with incubation at 28 degrees C was in the range of 4 to 7 log CFU per g of tissue or seed. There was no significant difference (P > or = 0.05) in APC when the determinations were made with three agar media including PAF, brain heart infusion agar, and plate count agar. However, the APC as determined from plates that were incubated at 28 degrees C was significantly (P < or = 0.05) higher than with incubation at 37 degrees C. Fluorescent pseudomonads accounted for 23 to 73% of APC and 6 to 18% of APC recovered from carrots, pepper, and lettuce were pectolytic. Forty-eight strains of pectolytic bacteria were randomly isolated and identified, respectively, as members of the genera of Pseudomonas, Erwinia, Bacillus, Xanthomonas, or Flavobacterium. Lactic acid bacteria and/or yeast were consistently isolated from baby carrots, lettuce, and sprouting seeds (alfalfa or clover) but not from green bell peppers. Approximately 120 strains of indigenous microflora were tested for their ability to inhibit the growth of Salmonella Chester, Listeria monocytogenes, Escherichia coli, or Erwinia carotovora subsp. carotovora on PAF. Six isolates capable of inhibiting the growth of at least one pathogen were isolated and identified, respectively, as Bacillus spp. (three strains), Pseudomonas aeruginosa (one strain), Pseudomonas fluorescens (strain A3), and yeast (strain D1). When green pepper disks were inoculated with strains A3 and D1, the growth of Salmonella Chester and L. monocytogenes on the disks was reduced by 1 and 2 logs, respectively, over a period of 3 days. Application of strains A3 and D1 as potential biopreservatives for enhancing the quality and safety of fresh produce is discussed.

Efficacy of ozone in killing Listeria monocytogenes on alfalfa seeds and sprouts and effects on sensory quality of sprouts.

A study was done to determine the efficacy of aqueous ozone treatment in killing Listeria monocytogenes on inoculated alfalfa seeds and sprouts. Reductions in populations of naturally occurring aerobic microorganisms on sprouts and changes in the sensory quality of sprouts were also determined. The treatment (10 or 20 min) of seeds in water (4 degrees C) containing an initial concentration of 21.8 +/- 0.1 microg/ml of ozone failed to cause a significant (P < or = 0.05) reduction in populations of L. monocytogenes. The continuous sparging of seeds with ozonated water (initial ozone concentration of 21.3 +/- 0.2 microg/ml) for 20 min significantly reduced the population by 1.48 log10 CFU/g. The treatment (2 min) of inoculated alfalfa sprouts with water containing 5.0 +/- 0.5, 9.0 +/- 0.5, or 23.2 +/- 1.6 microg/ml of ozone resulted in significant (P < or = 0.05) reductions of 0.78, 0.81, and 0.91 log10 CFU/g, respectively, compared to populations detected on sprouts treated with water. Treatments (2 min) with up to 23.3 +/- 1.6 microg/ml of ozone did not significantly (P > 0.05) reduce populations of aerobic naturally occurring microorganisms. The continuous sparging of sprouts with ozonated water for 5 to 20 min caused significant reductions in L. monocytogenes and natural microbiota compared to soaking in water (control) but did not enhance the lethality compared to the sprouts not treated with continuous sparging. The treatment of sprouts with ozonated water (20.0 microg/ml) for 5 or 10 min caused a significant deterioration in the sensory quality during subsequent storage at 4 degrees C for 7 to 11 days. Scanning electron microscopy of uninoculated alfalfa seeds and sprouts showed physical damage, fungal and bacterial growth, and biofilm formation that provide evidence of factors contributing to the difficulty of killing microorganisms by treatment with ozone and other sanitizers.

Influence of substituents on the solution conformation of the exopolysaccharide produced by Pseudomonas 'gingeri' strain Pf9.

The influence of pyruvate ketals and acetyl groups on the conformational behaviour of the exopolysaccharide produced by Pseudomonas 'gingeri' strain Pf9 has been investigated experimentally through studies of intrinsic viscosity and circular dichroism experiments. A conformational variation was detected as a function of the ionic strength. Measurements carried out on the native polymer, as well as on both de-pyruvated and de-acetylated samples, suggested a critical role for the acetyl group on the solution conformation of the polysaccharide. Molecular mechanics calculations indicated the possibility of intramolecular hydrogen bonding between acetyl substituents on the mannose and the C(2)OH group of the preceding saccharidic unit. NMR linewidth measurements, carried out as a function of temperature, on the low molecular weight de-pyruvated sample indicated different polymeric backbone dynamics in aqueous solutions with respect to that observed in 0.3 M NaCl solutions.

Resuscitation of acid-injured Salmonella in enrichment broth, in apple juice and on the surfaces of fresh-cut cucumber and apple.

AIMS: To investigate the resuscitation of acid-injured Salmonella enterica in selected enrichment broths, in apple juice and on cut surfaces of apple and cucumber slices. METHODS AND RESULTS: Following exposure to 2.4% acetic acid for 7 min, S. enterica (serovars Mbandaka, Chester and Newport) cells were used to inoculate enrichment broths, phosphate-buffered saline (PBS), apple juice and fruit slices. Injured Salmonella cells resuscitated and regained the ability to form colonies on selective agar (Xylose-Lysine-Tergitol 4) if they were incubated in lactose broth (LB), universal pre-enrichment broth (UPB) or buffered peptone water (BPW), but not in tetrathionate broth, PBS or apple juice. The resuscitation occurred at a significantly (P > 0.05) faster rate in UPB than in LB or BPW. The resuscitation also occurred on the surfaces of fresh-cut cucumber at 20 degrees C, but not at 4 degrees C. CONCLUSIONS: Acid-injured Salmonella cells resuscitated in nonselective enrichment broths at different rates, but not in selective enrichment broth, apple juice, PBS or on fresh-cut apple. SIGNIFICANCE AND IMPACT OF THE STUDY: Pre-enrichment of food samples in UPB prior to selective enrichment is recommended. Injured Salmonella cells have the ability to resuscitate on fresh-cut surfaces of cucumber when stored at abusive temperatures.

Exopolysaccharides Produced by Phytopathogenic Pseudomonas syringae Pathovars in Infected Leaves of Susceptible Hosts.

Bacterial exopolysaccharide (EPS) was extracted from infected leaves of several host plants inoculated with phytopathogenic strains of Pseudomonas syringae pathovars. Extraction was by a facilitated diffusion procedure or by collection of intercellular fluid using a centrifugation method. The extracted EPS was purified and characterized. All bacterial pathogens which induced watersoaked lesions on their host leaves, a characteristic of most members of this bacterial group, were found to produce alginic acid (a polymer consisting of varying ratios of mannuronic and guluronic acids). Only trace amounts of bacterial EPS could be isolated from leaves inoculated with a pathovar (pv. syringae) which does not induce the formation of lesions with a watersoaked appearance. Guluronic acid was either present in very low amounts or absent in the alginic acid preparations. All bacterial alginates were acetylated (7-11%). Levan (a fructan) was apparently not produced as an EPS in vivo by any of the pathogens tested.

A New Bacterial Agglutinin from Soybean: II. EVIDENCE AGAINST A ROLE IN DETERMINING PATHOGEN SPECIFICITY.

The activity of a bacterial agglutinin from soybean seed [Glycine max (L.) Merrill cv. Clark] against two bacterial pathogens, Pseudomonas glycinea (causal agent of bacterial blight) and Xanthomonas phaseoli var. sojensis (causal agent of bacterial pustule) was determined. The agglutinin was active against several strains of X. phaseoli var. sojensis grown on nutrient agar, but there was no correlation between pathogenicity and agglutination. Agglutination was affected by the age of the bacterial cells and the growth medium used. None of seven strains of P. glycinea was agglutinated.Bacterial agglutination was inhibited by both purified lipopolysaccharide and extracellular polysaccharide from five strains of X. phaseoli var. sojensis. The lipopolysaccharides and extracellular polysaccharides from other species of bacteria were ineffective.Ultrastructural studies showed that an avirulent strain of X. phaseoli var. sojensis was attached to leaf mesophyll cell walls of the susceptible cultivar Clark by 34 hours after vacuum infiltration. Cells of this avirulent strain were enveloped by fibrillar and granular material at the mesophyll cell wall. In contrast, cells of a virulent strain were not attached or enveloped, and they remained free to multiply in the intercellular spaces.

A New Bacterial Agglutinin from Soybean: I. ISOLATION, PARTIAL PURIFICATION, AND CHARACTERIZATION.

A new bacterial agglutinin was isolated from seeds of the soybean cultivar Clark. Purification was carried out by ammonium sulfate precipitation and ion-exchange chromatography. The agglutinin is a heat-labile glycoprotein most active at pH 4.0. Addition of Ca(2+), Mn(2+) and Mg(2+) did not enhance the agglutinating activity of this glycoprotein. Gel electrophoresis in the presence of sodium dodecyl sulfate showed that the agglutinin is composed of two subunits of approximately 50,000 daltons each. In the undissociated state, it agglutinates Xanthomonas phaseoli var. sojensis, the causal agent of bacterial pustule disease of soybean, at concentrations as low as 10 micrograms protein per milliliter but has no hemagglutinating activity. The agglutinin could be distinguished from previously reported soybean lectins on the basis of solubility in ammonium sulfate, lack of hemagglutinating activity, molecular weight, hapten specificity, and immunological determinants.

Structure of an acidic exopolysaccharide of Pseudomonas marginalis HT041B.

The exopolysaccharide of Pseudomonas marginalis HT041B has been characterized as a 1,3-linked galactoglucan in which galactose and glucose are in the alpha- and beta-anomeric configurations, respectively. The polysaccharide is substituted with pyruvate at the 4 and 6 positions of galactose and with succinic acid at either the 2 or 4 position of glucose. This polysaccharide has been given the trivial name marginalan.

Auxin production by plant-pathogenic pseudomonads and xanthomonads.

Pathogenic strains of Xanthomonas campestris pv. glycines which cause hypertrophy of leaf cells of susceptible soybean cultivars and nonpathogenic strains which do not cause hypertrophy were compared for their ability to produce indole compounds, including the plant hormone indole-3-acetic acid (IAA) in liquid media with or without supplementation with l-tryptophan. Several additional strains of plant-pathogenic xanthomonads and pseudomonads were also tested for IAA production to determine whether in vitro production of IAA is related to the ability to induce hypertrophic growth of host tissues. Indoles present in culture filtrates were identified by thin-layer chromatography, high-performance liquid chromatography, UV spectroscopy, mass spectroscopy, and gas chromatography-mass spectrometry and were quantitated by high-performance liquid chromatography. All strains examined produced IAA when liquid media were supplemented with l-tryptophan. The highest levels of IAA were found in culture filtrates from the common bean pathogen Pseudomonas syringae pv. syringae, and this was the only bacterium tested which produced IAA without addition of tryptophan to the medium. Additional indoles identified in culture filtrates of the various strains included indole-3-lactic acid, indole-3-aldehyde, indole-3-acetamide, and N-acetyltryptophan. Pseudomonads and xanthomonads could be distinguished by the presence of N-acetyltryptophan, which was found only in xanthomonad culture filtrates.