Swarm motility of Salmonella enterica serovar Typhimurium is inhibited by compounds from fruit peel extracts.

UNLABELLED: Controlling spread of human pathogens on fresh produce is a top priority for public health reasons. Isolation of compounds from agricultural waste that would control spread of human pathogens was explored using Salmonella enterica serovar Typhimurium as a model organism. In the environment, micro-organisms migrate as a 'community' especially when they move on moist surfaces. This type of motility is characterized as swarming motility. We examined extracts from agricultural waste such as soya bean husk, peels of orange, pineapple, avocado and pomegranate for antiswarming activity. Avocado and pineapple peels showed moderate (~40%) inhibition of swarming motility while pomegranate peel extract had high antiswarming activity (~85% inhibition) and was examined in further detail. Although the pomegranate peel extract was acidic, swarm-inhibitory activity was not due to low pH and the peel extract did not inhibit growth of Salmonella. Among the key swarm motility regulatory genes, class II (fliF, fliA, fliT and fliZ) and class III (fliC and fliM) regulators were downregulated upon exposure to pomegranate peel extract. Pomegranate peels offer great potential as a bioactive repellent for pathogenic micro-organisms on moist surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: Controlling the spread of food-borne pathogens in moist environments is an important microbial food safety issue. Isolation of compounds from agricultural waste (such as fruit peels) that would control spread of human pathogens was explored using Salmonella enterica serovar Typhimurium as a model organism. Pomegranate peels offer great potential as a bioactive repellent for pathogenic micro-organisms.

Length polymorphisms of simple sequence repeat DNA in soybean.

The objective of this work was to ascertain the presence and degree of simple sequence repeat (SSR) DNA length polymorphism in the soybean [Glycine max (L.) Merr.]. A search of GenBank revealed no (CA)n or (GT)n SSRs with n greater than 8 in soybean. In contrast, 5 (AT)n and 1 (ATT)n SSRs with n ranging from 14 to 27 were detected. Polymerase chain reaction (PCR) primers to regions flanking the six SSR loci were used in PCR amplification of DNA from 43 homozygous soybean genotypes. At three loci, amplification produced one PCR product per genotype and revealed 6, 7 and 8 product length variants (alleles) at the three loci, respectively. F1 hybrids between parents carrying different alleles produced two PCR products identical to the two parents. Codominant segregation of alleles among F2 progeny was demonstrated at each locus. A soybean DNA library was screened for the presence of (CA/GT)n SSRs. Sequencing of positive clones revealed that the longest such SSR was (CA)9. Thus, (CA)n SSRs with n of 15 or more are apparently much less common in soybean than in the human genome. In contrast to humans, (CA)n SSRs will probably not provide an abundant source of genetic markers in soybean. However, the apparent abundance of long (AT)n sequences should allow this SSR to serve as a source of highly polymorphic genetic markers in soybean.

Nodule development induced by mutants of Bradyrhizobium japonicum defective in cyclic B-glucan synthesis.

The soybean response to inoculation with B. japonicum mutants defective in cyclic beta-(1-->3), beta-(1-->6) glucan synthesis was examined by electron microscopy and by monitoring the expression of early and late nodulin genes. Two mutants were examined. Strain AB-14 is an ndvB mutant and is unable to synthesize beta-glucans. Strain AB-1 is an ndvC mutant and produces cyclic glucans containing 95 to 100% beta-(1-->3) glycosidic linkages. Nodules formed by either mutant were defective in nitrogen fixation activity. Soybean plants inoculated with strain AB-14 formed nodules roughly at the same rate as the wild-type strain USDA110, but nodulation by strain AB-1 was significantly delayed. Microscopy of nodules formed by strain AB-14 showed an overall ultrastructure similar to nodules formed by the wild type. However, in some nodules bacteroids were limited to only one part of the infected cells and in others the few bacteroids present showed signs of degradation. Nodulelike structures were formed by strain AB-1 that showed some signs of cellular differentiation. For example, clear parenchyma and sclerenchyma tissue could be seen. However, no infection threads or bacteria were evident in these structures. The expression of early (e.g., ENOD2 and ENOD55) and late (e.g., NOD26 and leghemoglobin) nodulins was examined. Nodules formed by strain AB-14 expressed all of the nodulins tested but at a reduced level. Expression of late nodulins was delayed in strain AB-14-induced nodules. Nodules formed by strain AB-1 were more strongly affected in nodulin expression. Although leghemoglobin synthesis was not detected, infection-specific nodulin transcripts of GmN93 and ENOD55 were detected. However, expression of the early nodulins ENOD2 and ENOD55 was considerably delayed and only apparent when assayed 21 days postinoculation. A low level of expression of intermediate nodulin GmN70 and late nodulin NOD26 could also be detected by 21 days postinoculation. The microscopy data show that tissue differentiation occurs in these nodules even in the absence of active bacterial invasion. These results demonstrate the importance of cyclic beta-(1-->3), beta-(1-->6) glucan synthesis to symbiotic development in soybean.

Induction of H(2)O(2) synthesis by beta-glucan elicitors in soybean is independent of cytosolic calcium transients.

Soybean cell suspension cultures have been used to investigate the role of the elevation of the cytosolic Ca(2+) concentration in beta-glucan elicitors-induced defence responses, such as H(2)O(2) and phytoalexin production. The intracellular Ca(2+) concentration was monitored in transgenic cells expressing the Ca(2+)-sensing aequorin. Two lines of evidence showed that a transient increase of the cytosolic Ca(2+) concentration is not necessarily involved in the induction of H(2)O(2) generation: (i) a Bradyrhizobium japonicum cyclic beta-glucan induced the H(2)O(2) burst without increasing the cytosolic Ca(2+) concentration; (ii) two ion channel blockers (anthracene-9-carboxylate, A9C; 5-nitro-2-(3-phenylpropylamino)-benzoate, NPPB) could not prevent a Phytophthora soja beta-glucan elicitor-induced H(2)O(2) synthesis but did prevent a cytosolic Ca(2+) concentration increase. Moreover, A9C and NPPB inhibited P. sojae beta-glucan-elicited defence-related gene inductions as well as the inducible accumulation of phytoalexins, suggesting that the P. sojae beta-glucan-induced transient cytosolic Ca(2+) increase is not necessary for the elicitation of H(2)O(2) production but is very likely required for phytoalexin synthesis.

Identification and cloning of a cyclic beta-(1-->3), beta-(1-->6)-D-glucan synthesis locus from Bradyrhizobium japonicum.

A cosmid clone complementing a cyclic beta-(1-->2)-glucan biosynthesis (ndvB) mutant of Rhizobium meliloti was isolated from a Bradyrhizobium japonicum gene library. This clone specified synthesis of beta-(1-->3), beta-(1-->6)-linked glucans in R. meliloti. The complemented strain was osmotically tolerant and symbiotically competent on alfalfa. Thus, beta-(1-->3), beta-(1-->6)-glucans can substitute functionally for beta-(1-->2)-glucans in R. meliloti.

Cyclolaminarinose. A new biologically active beta-(1-->3) cyclic glucan.

A unique glucan has been isolated from a recombinant strain of a Rhizobium meliloti TY7, a cyclic beta-(1-->2) glucan mutant carrying a locus specifying beta-(1-->3; 1-->6) glucan synthesis from Bradyrhizobium japonicum USDA110. This compound, which appears to have considerable hydrophobic affinity, was separated from a perchloric acid cell extract by adsorption to a C-18 silica column. Unlike those cyclic glucans previously isolated from Rhizobium meliloti or Bradyrhizobium japonicum, this molecule contains neither phosphoglycerol nor phosphocholine substituents, respectively. 2D NMR, FAB mass spectrometric analysis and high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) confirmed that this glucan is a single, cyclic decasaccharide (cyclolaminarinose) in which one of the residues is substituted in its 6-position with beta-laminarabiose. This structural assignment was confirmed by mass spectral and NMR analyses of the product obtained from two consecutive Smith degradations. Unlike the complex 13C spectrum of the unoxidized material, the spectrum of this product consisted of only six resonances due to rapid time averaging of its symmetrical structure on the relatively slow NMR timescale. Synthesis of this newly described cyclic beta-glucan in the R. meliloti ndvB mutant restored the symbiotic and hypoosmotic adaptation characteristics of the R. meliloti wild type strain.

Bradyrhizobium japonicum mutants defective in cyclic beta-glucan synthesis show enhanced sensitivity to plant defense responses.

Susceptibility of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum to inducible plant defense metabolites such as phytoalexin and H2O2, was investigated. On the wild-type strain USDA 110 the soybean phytoalexin, glyceollin, showed bacteriostatic activity. Viable bacteria isolated from intact nodules were adapted to glyceollin. H2O2 in physiological concentrations did not affect wild-type bacteria. B. japonicum mutants defective in the biosynthesis of cyclic beta-(1-->3)-(1-->6)-glucans showed higher susceptibility to both phytoalexin and H2O2.

Fresh-cut lettuce in modified atmosphere packages stored at improper temperatures supports enterohemorrhagic E. coli isolates to survive gastric acid challenge.

Incidences of foodborne outbreaks involving enterohemorrhagic Escherichia coli strains with mutations in a key regulatory gene, rpoS, have been reported. Incentives, if any, for losing this regulatory function are not clear since the RpoS regulator is required for the expression of several environmental stress tolerance genes. RpoS also positively regulates 2 of the 3 acid-resistance systems of E. coli under aerobic growth conditions and enables the pathogen to survive gastric acid challenge. We selected 7 enterohemorrhagic E. coli isolates, 6 of which are known to carry defective rpoS gene, and then analyzed resistance to synthetic gastric juice after the strains were inoculated on fresh-cut lettuce and stored under modified atmosphere packaging (MAP) conditions. Subatmospheric oxygen partial pressures in MAP enabled all 6 rpoS-defective isolates to induce acid resistance over the 8-d storage period if the temperature was >or= 15 degrees C. No acid resistance was induced for MAP-stored lettuce left at temperatures

Rapid detection of Salmonella from hydrodynamic pressure-treated poultry using molecular beacon real-time PCR.

A real-time polymerase chain reaction (PCR) assay was evaluated to detect Salmonella in hydrodynamic pressure (HDP)-treated chicken using molecular beacon probes available as a commercial kit (iQ-Check, Bio-Rad Laboratories). The sensitivity and accuracy of the assay were compared with the conventional USDA microbiological procedure using artificially contaminated minced chicken. Chicken fillets were irradiated at 10 kGy to completely destroy any naturally occurring Salmonella. These fillets were minced and inoculated with as low as 2+/-1 cfu of S. typhimurium per 25 g chicken. The minced chicken samples were vacuum packed in multi-layer barrier bags, heat shrunk, and treated with HDP. Results showed that all inoculated samples (n=36) were detected by the PCR assay and conventional USDA procedure. Similarly, all uninoculated controls (n=11) were negative by both PCR assay and USDA procedure. As few as 2+/-1 cfu could be detected from 25 g HDP-treated chicken following 16-18 h enrichment in buffered peptone water. Real-time PCR proved to be an effective method for Salmonella detection in HDP-treated chicken with high sensitivity and more importantly, a rapid and high-throughput detection in 18 h, compared to 3-8 days for the conventional microbiological methods. HDP treatment, which has been reported to reduce spoilage bacteria in various meats, was unable to kill pathogenic Salmonella in minced chicken.

Identification and cloning of Bradyrhizobium japonicum genes expressed strain selectively in soil and rhizosphere.

The growth of Bradyrhizobium japonicum USDA 110 and USDA 438 in soil extract-supplemented medium led to transcription of a large amount of DNA not expressed in basal medium. Strain USDA 438 was more competitive for the nodulation of soybean than strain USDA 110. To identify and isolate DNA regions which were expressed specifically in strain USDA 438 but not in strain USDA 110 in response to soil extract or soybean root exudate, we developed a subtractive RNA hybridization procedure. Several cosmid clones which showed strain-specific gene expression were isolated from a USDA 438 gene library. Two clones enhanced competitive nodulation when mobilized to USDA 110. The method described may be useful for identifying genes expressed in response to environmental stimuli or genes expressed differently in related microbial strains.

Protective effects of cold temperature and surface-contact on acid tolerance of Salmonella spp.

AIMS: To determine the effect of cold-storage temperature and surface contact on the survival of Salmonella spp. during acid challenge. To determine the contribution of sigma transcriptional factor (encoded by rpoS) in surface contact-mediated acid tolerance. METHODS AND RESULTS: Salmonella serovar Typhimurium cells in exponential and stationary growth phase were subjected to acid challenge in planktonic and surface-associated states. Low temperatures offered protection against acid challenge to cells which were in stationary growth phase (but not to those in exponential growth phase). The cells from stationary, as well as logarithmic growth phase, acquired increased acid tolerance upon surface contact with various surfaces, such as fresh-cut apples, agar and polyethersulphone membranes. The alternative sigma transcription factor was not required to acquire surface contact-mediated acid tolerance. CONCLUSIONS: Salmonella spp. take advantage of low temperature and surface association to overcome acid challenge. Some of the acid tolerance mechanisms are independent of the sigma transcription factor regulon. SIGNIFICANCE AND IMPACT OF THE STUDY: Effective control measures during produce processing must take into account the different susceptibilities of planktonic vs surface-associated food-borne pathogens.

Comparative analysis of proteins induced by heat shock, salinity, and osmotic stress in the nitrogen-fixing cyanobacterium Anabaena sp. strain L-31.

Heat, salinity, or osmotic stress influenced protein synthesis in nitrogen-fixing Anabaena sp. strain L-31. Salinity and osmotic stresses were identical and specifically induced 15 polypeptides. Four polypeptides were unique to heat shock, and four other polypeptides were induced under every stress. The results demonstrate a commonality and a stress specificity of protein synthesis regulation.

Salinity-stress-induced proteins in two nitrogen-fixing Anabaena strains differentially tolerant to salt.

Salinity altered the protein synthesis patterns in two cyanobacterial strains: Anabaena torulosa, a salt-tolerant brackish water strain, and Anabaena sp. strain L-31, a salt-sensitive freshwater strain. The cyanobacterial response to salinity was very rapid, varied with time, and was found to be correlated with the external salt (NaCl) concentration during stress. Salinity induced three prominent types of modification. First, the synthesis of several proteins was inhibited, especially in the salt-sensitive strain; second, the synthesis of certain proteins was significantly enhanced; and third, synthesis of a specific set of proteins was induced de novo by salinity stress. Proteins which were selectively synthesized or induced de novo during salt stress, tentatively called the salt-stress proteins, were confined to an isoelectric pI range of 5.8 to 7.5 and were distributed in a molecular mass range of 12 to 155 kilodaltons. These salt-stress proteins were unique to each Anabaena strain, and their expression was apparently regulated coordinately during exposure to salt stress. In Anabaena sp. strain L-31, most of the salt-stress-induced proteins were transient in nature and were located mainly in the cytoplasm. In A. torulosa, salt-stress-induced proteins were evenly distributed in the membrane and cytoplasmic fractions and were persistent, being synthesized at high rates throughout the period of salinity stress. These initial studies reveal that salinity-induced modification of protein synthesis, as has been demonstrated in higher plant species, also occurs in cyanobacteria and that at least some of the proteins preferentially synthesized during salt stress may be important to cyanobacterial osmotic adaptation.

Legume-Rhizobium interactions: cowpea root exudate elicits faster nodulation response by Rhizobium species.

Preinfection events in legume-Rhizobium symbiosis were analyzed by studying the different nodulation behaviors of two rhizobial strains in cowpeas (Vigna sinensis). Log-phase cultures of Rhizobium sp. strain 1001, an isolate from the plant nodule, initiated host responses leading to infection within 2 h after inoculation, whereas log-phase cultures of Rhizobium sp. strain 32H1 took at least 7 h to trigger a discernible response. The delay observed with strain 32H1 could be eliminated by incubating the rhizobial suspension, before inoculation, for 4.5 h either in the cowpea rhizosphere/rhizoplane condition or in the root exudate of cowpea plants, grown without NH(4) in the rooting medium. The delay could not be eliminated by incubating the rhizobial suspension in the rooting medium of plants grown in the presence of 5 mM NH(4), indicating that there is a regulatory role of combined nitrogen in triggering preinfection events by the legume. The substance(s) in the root exudate which elicited the faster nodulation response by Rhizobium sp. strain 32H1 could be separated into a high-molecular-weight fraction by Sephadex G-100 gel filtration. The data support the notion that legume roots release substances that favor the development of rhizobial features essential for infection and nodulation.

Isolation and characterization of an ndvB locus from Rhizobium fredii.

A gene (ndvB) in Rhizobium meliloti that is essential for nodule development in Medicago sativa (alfalfa), specifies synthesis of a large membrane protein. This protein appears to be an intermediate in beta-1,2-glucan synthesis by the microsymbiont. Southern hybridization analysis showed strong homology between an ndvB (chvB) probe and genomic DNA of R. fredii but not from Bradyrhizobium japonicum. A cosmid clone containing the putative ndvB locus was isolated from a Rhizobium fredii gene library. The cosmid clone which complemented R. meliloti ndvB mutants for synthesis of beta-1,2-glucans and effective nodulation of alfalfa was mapped and subcloned. Fragment-specific Tn5 mutagenesis followed by homologous recombination into the R. fredii genome indicated that the region was essential for beta-1,2-glucan synthesis and for formation of an effective symbiosis with Glycine max (soybean).

Isolation and Characterization of a Competition-Defective Bradyrhizobium japonicum Mutant.

Tn5 mutagenesis was coupled with a competition assay to isolate mutants of Bradyrhizobium japonicum defective in competitive nodulation. A double selection procedure was used, screening first for altered extracellular polysaccharide production (nonmucoid colony morphology) and then for decreased competitive ability. One mutant, which was examined in detail, was deficient in acidic polysaccharide and lipopolysaccharide production. The wild-type DNA region corresponding to the Tn5 insertion was isolated, mapped, and cloned. A 3.6-kb region, not identified previously as functioning in symbiosis, contained the gene(s) necessary for complementation of the mutation. The mutant was motile, grew normally on minimal medium, and formed nodules on soybean plants which fixed almost as much nitrogen as the wild type during symbiosis.

Availability of glutamate and arginine during acid challenge determines cell density-dependent survival phenotype of Escherichia coli strains.

The cell density-dependent acid sensitivity phenotypes of Escherichia coli strains K-12 and O157:H7 were examined with reference to three possible mechanisms of acid resistance. There was no evidence of any diffusible substance released from dead cells which could influence the cell density-dependent acid survival phenotype. Instead, cell density-dependent acid survival phenotype was associated with induction of glutamate- and arginine-decarboxylase acid survival pathways and concomitant availability of glutamate and arginine during acid challenge.

Transient susceptibility of root cells in four common legumes to nodulation by rhizobia.

Root cells of four common legumes were found to remain susceptible to nodulation by rhizobia for only a short period of time. Delayed inoculation experiments conducted with these legume hosts indicated that the initially susceptible region of the root became progressively less susceptible if inoculations were delayed by a few hours. Profiles of the frequency of nodule formation relative to marks indicating the regions of root and root hair development at the time of inoculation indicated that nodulation of Vigna sinensis (L.) Endl. cv California Black Eye and Medicago sativa L. cvs Moapa and Vernal roots was inhibited just below the region that was most susceptible at the time of inoculation. This result suggests the existence of a fast-acting regulatory mechanism in these hosts that prevents overnodulation. Nodulation in white clover may occur in two distinct phases. In addition to the transient susceptibility of preemergent and developing root hair cells, there appeared to be an induced susceptibility of mature clover root hair cells. A cell-free bacterial exudate preparation from Rhizobium trifolii cells was found to render mature root hair cells of white clover more rapidly susceptible to nodulation.

Simple sequence repeat DNA markers in alfalfa and perennial and annual Medicago species.

Simple sequence repeat (SSR) or microsatellite DNA markers have been shown to function well in plant and mammalian species for genetic map construction and genotype identification. The objectives of the work reported here were to search GenBank for the presence of SSR-containing sequences from the genus Medicago, to assess the presence and frequency of SSR DNA in the alfalfa (Medicago sativa (L.) L. &L.) genome, and to examine the function of selected markers in a spectrum of perennial and annual Medicago species. The screening of an alfalfa genomic DNA library and sequencing of clones putatively containing SSRs indicated approximately 19 000 (AT)n + (CT)n + (CA)n + (ATT)n SSRs in the tetraploid genome. Inheritance was consistent with Mendelian expectations at four selected SSR loci with different core motifs. Additionally, genotypes of a range of Medicago species, including 10 perennial subspecies of the M. sativa complex and other perennial and annual Medicago species, were analyzed at each of the loci to ascertain the presence, number, and size of SSR alleles at each locus in each genotype. These studies indicate that SSR markers can function in alfalfa for the construction of genetic maps and will also be useful in a range of Medicago species for purposes of assessing genetic relatedness and taxonomic relationships, and for genotype identification.

Beta-glucan synthesis in Bradyrhizobium japonicum: characterization of a new locus (ndvC) influencing beta-(1-->6) linkages.

Bradyrhizobium japonicum synthesizes periplasmic cyclic beta-(1-->3),beta-(1-->6)-D-glucans during growth in hypoosmotic environments, and evidence is growing that these molecules may have a specific function during plant-microbe interactions in addition to osmoregulation. Site-directed Tn5 mutagenesis of the DNA region upstream of ndvB resulted in identification of a new gene (ndvC) involved in beta-(1--> 3), beta-(1-->6)-glucan synthesis and in nodule development. The predicted translation product was a polypeptide (ca. 62 kDa) with several transmembrane domains. It contained a sequence characteristic of a conserved nucleoside-sugar-binding motif found in many bacterial enzymes and had 51% similarity with a beta-glucanosyltransferase from Candida albicans. B. japonicum carrying a Tn5 insertion in ndvC resulted in synthesis of altered cyclic beta-glucans composed almost entirely of beta-(1--> 3)-glycosyl linkages. The mutant strain was only slightly sensitive to hypoosmotic growth conditions compared with the ndvB mutant, but it was severely impaired in symbiotic interactions with soybean (Glycine max). Nodulation was delayed by 8 to 10 days, and many small nodule-like structures apparently devoid of viable bacteria were formed. This finding suggests that the structure of the beta-glucan molecule is important for a successful symbiotic interaction, and beta-glucans may have a specific function in addition to their role in hypoosmotic adaptation.