Sima Yaron

Transmission of Escherichia coli O157:H7 from Contaminated Manure and Irrigation Water to Lettuce Plant Tissue and Its Subsequent Internalization

ABSTRACT The transmission of Escherichia coli O157:H7 from manure-contaminated soil and irrigation water to lettuce plants was demonstrated using laser scanning confocal microscopy, epifluorescence microscopy, and recovery of viable cells from the inner tissues of plants. E. coli O157:H7 migrated to internal locations in plant tissue and was thus protected from the action of sanitizing agents by virtue of its inaccessibility. Experiments demonstrate that E. coli O157:H7 can enter the lettuce plant through the root system and migrate throughout the edible portion of the plant.

Vesicle-mediated transfer of virulence genes from Escherichia coli O157:H7 to other enteric bacteria.

ABSTRACT Membrane vesicles are released from the surfaces of many gram-negative bacteria during growth. Vesicles consist of proteins, lipopolysaccharide, phospholipids, RNA, and DNA. Results of the present study demonstrate that membrane vesicles isolated from the food-borne pathogen Escherichia coli O157:H7 facilitate the transfer of genes, which are then expressed by recipient Salmonella enterica serovar Enteritidis or E. coli JM109. Electron micrographs of purified DNA from E. coli O157:H7 vesicles showed large rosette-like structures, linear DNA fragments, and small open-circle plasmids. PCR analysis of vesicle DNA demonstrated the presence of specific genes from host and recombinant plasmids (hly, L7095, mobA, andgfp), chromosomal DNA (uidA andeaeA), and phage DNA (stx1 andstx2). The results of PCR and the Vero cell assay demonstrate that genetic material, including virulence genes, is transferred to recipient bacteria and subsequently expressed. The cytotoxicity of the transformed enteric bacteria was sixfold higher than that of the parent isolate (E. coli JM109). Utilization of the nonhost plasmid (pGFP) permitted the evaluation of transformation efficiency (ca. 103 transformants μg of DNA−1) and demonstrated that vesicles can deliver antibiotic resistance. Transformed E. coli JM109 cells were resistant to ampicillin and fluoresced a brilliant green. The role vesicles play in genetic exchange between different species in the environment or host has yet to be defined.

Effect of Heat, Acidification, and Chlorination on Salmonella enterica Serovar Typhimurium Cells in a Biofilm Formed at the Air-Liquid Interface

ABSTRACT Bacterial biofilms have great significance for public health, since biofilm-associated microorganisms exhibit dramatically decreased susceptibility to antimicrobial agents and treatments. To date most attention has focused on biofilms that arise from the colonization of solid-liquid or solid-air interfaces. It is of interest that colonization of the interface between air and liquid, which can be selectively advantageous for aerobic or facultative aerobic bacteria, has been rarely studied, although it may present a major problem in industrial aquatic systems. In this work we investigated the role of a biofilm at the interface between air and liquid (pellicle) in the susceptibility of Salmonella enterica serovar Typhimurium to stress conditions. For a control we used a mutant that had lost its ability to synthesize cellulose and thin aggregative fimbriae and thus did not produce the pellicle. Resistance of bacteria from the pellicle to heat, acidification, and chlorination was compared to resistance of planktonic cells from the logarithmic and stationary phases of growth. Pellicle cells were significantly more resistant to chlorination, and thus the surrounding matrix conferred protection against the reactive sodium hypochlorite. However, the stress management of pellicle cells in response to heat and low pH was not enhanced compared to that of stationary-phase cells. A long-period of incubation resulted in endogenous hydrolysis of the pellicle matrix. This phenomenon provides a potential new approach to combat microbial cells in biofilms.

Expression, purification and subunit-binding properties of cohesins 2 and 3 of the Clostridium thermocellum cellulosome.

The enzymatic subunits of the cellulosome of Clostridium thermocellum are integrated into the complex by a major non‐catalytic polypeptide, called scaffoldin. Its numerous functional domains include a single cellulose‐binding domain (CBD) and nine subunit‐binding domains, or cohesin domains. Two of the cohesin domains, together with the adjacent CBD, have been cloned and expressed in Escherichia coli, and the recombinant constructs were purified by affinity chromatography on a cellulosic matrix. Both cohesin domains, which differ by about 30% in their primary structure, showed a similar binding profile to the cellulosomal subunits. Calcium ions enhanced dramatically this binding. Under the conditions of the assay, only one major catalytic subunit of the cellulosome failed to bind to either cohesin domain. The results indicate a lack of selectivity in the binding of cohesin domains to the catalytic subunits and also suggest that additional mechanisms may be involved in cellulosome assembly.

Heat Tolerance of Salmonella enterica Serovars Agona, Enteritidis, and Typhimurium in Peanut Butter

Recent large foodborne outbreaks caused by Salmonella enterica serovars have been associated with consumption of foods with high fat content and reduced water activity, even though their ingredients usually undergo pasteurization. The present study was focused on the heat tolerance of Salmonella enterica serovars Agona, Enteritidis, and Typhimurium in peanut butter. The Salmonella serovars in the peanut butter were resistant to heat, and even at a temperature as high as 90 degrees C only 3.2-log reduction in CFU was observed. The obtained thermal inactivation curves were upwardly concave, indicating rapid death at the beginning (10 min) followed by lower death rates and an asymptotic tail. The curves fitted the nonlinear Weibull model with beta parameters < 1, indicating that the remaining cells have a lower probability of dying. beta at 70 degrees C (0.40 +/- 0.04) was significantly lower than beta at 80 degrees C (0.73 +/- 0.19) and 90 degrees C (0.69 +/- 0.17). Very little decrease in the viable population (less than 2-log decrease) was noted in cultures that were exposed to a second thermal treatment. Peanut butter is a highly concentrated colloidal suspension of lipid and water in a peanut meal phase. We hypothesized that differences in the local environments of the bacteria, with respect to fat content or water activity, explained the observed distribution and high portion of surviving cells (0.1%, independent of the initial cell number). These results demonstrate that thermal treatments are inadequate to consistently destroy Salmonella in highly contaminated peanut butter and that the pasteurization process cannot be improved significantly by longer treatment or higher temperatures.

CelI, a Noncellulosomal Family 9 Enzyme from Clostridium thermocellum, Is a Processive Endoglucanase That Degrades Crystalline Cellulose

The family 9 cellulase gene celI of Clostridium thermocellum, was previously cloned, expressed, and characterized (G. P. Hazlewood, K. Davidson, J. I. Laurie, N. S. Huskisson, and H. J. Gilbert, J. Gen. Microbiol. 139:307-316, 1993). We have recloned and sequenced the entire celI gene and found that the published sequence contained a 53-bp deletion that generated a frameshift mutation, resulting in a truncated and modified C-terminal segment of the protein. The enzymatic properties of the wild-type protein were characterized and found to conform to those of other family 9 glycoside hydrolases with a so-called theme B architecture, where the catalytic module is fused to a family 3c carbohydrate-binding module (CBM3c); CelI also contains a C-terminal CBM3b. The intact recombinant CelI exhibited high levels of activity on all cellulosic substrates tested, with pH and temperature optima of 5.5 and 70 degrees C, respectively, using carboxymethylcellulose as a substrate. Native CelI was capable of solubilizing filter paper, and the distribution of reducing sugar between the soluble and insoluble fractions suggests that the enzyme acts as a processive cellulase. A truncated form of the enzyme, lacking the C terminal CBM3b, failed to bind to crystalline cellulose and displayed reduced activity toward insoluble substrates. A truncated form of the enzyme, in which both the cellulose-binding CBM3b and the fused CBM3c were removed, failed to exhibit significant levels of activity on any of the substrates examined. This study underscores the general nature of this type of enzymatic theme, whereby the fused CBM3c plays a critical accessory role for the family 9 catalytic domain and changes its character to facilitate processive cleavage of recalcitrant cellulose substrates.

A cohesin domain from Clostridium thermocellum: the crystal structure provides new insights into cellulosome assembly.

BACKGROUND The scaffoldin component of the cellulolytic bacterium Clostridium thermocellum is a non-hydrolytic protein which organizes the hydrolytic enzymes in a large complex, called the cellulosome. Scaffoldin comprises a series of functional domains, amongst which is a single cellulose-binding domain and nine cohesin domains which are responsible for integrating the individual enzymatic subunits into the complex. The cohesin domains are highly conserved in their primary amino acid sequences. These domains interact with a complementary domain, termed the dockerin domain, one of which is located on each enzymatic subunit. The cohesin-dockerin interaction is the crucial interaction for complex formation in the cellulosome. The determination of structural information about the cohesin domain will provide insights into cellulosome assembly and activity. RESULTS We have determined the three-dimensional crystal structure of one of the cohesin domains from C. thermocellum (cohesin 2) at 2.15 A resolution. The domain forms a nine-stranded beta sandwich with a jelly-roll topology, somewhat similar to the fold displayed by its neighboring cellulose-binding domain. CONCLUSIONS The compact nature of the cohesin structure and its lack of a defined binding pocket suggests that binding between the cohesin and dockerin domains is characterized by interactions between exposed surface residues. As the cohesin-dockerin interaction appears to be rather nonselective, the binding face would presumably be characterized by surface residues which exhibit both intraspecies conservation and interspecies dissimilarity. Within the same species, unconserved surface residues may reflect the position of a given cohesin domain within the scaffoldin subunit, its orientation and interactions with neighboring domains.

Transfer of Salmonella enterica serovar Typhimurium from contaminated irrigation water to parsley is dependent on curli and cellulose, the biofilm matrix components.

Enteric pathogens can contaminate fresh produce, and this contaminated produce can be a significant potential source of human illness. The objective of this study was to determine a possible mode of transfer of Salmonella Typhimurium from contaminated irrigation water to mature parsley plants and to investigate the role of bacterial cellulose and curli. Parsley plants were drip irrigated with water containing green fluorescent protein-labeled Salmonella Typhimurium. Stems and leaves were harvested 1 day after the third irrigation and examined for the presence of Salmonella Typhimurium. Three weeks after harvesting, the presence of Salmonella was again confirmed in the regrown plants. During this period, bacterial numbers on leaves declined from 4.1 (+/- 0.3) to 2.3 (+/- 0.1) log CFU g(-1) (P < 0.05). Numbers in the soil were constant (5 log CFU g(-1)). Results demonstrated the ability of Salmonella Typhimurium to transfer from irrigation water to the edible parts of the plants. Confocal laser scanning microscopic images revealed that Salmonella Typhimurium formed aggregates at a depth of 8 to 32 microm beneath the leaf surface. Penetration might be achieved through the roots or the phyllosphere. The importance of the bacterial cellulose and curli was determined by comparing the wild-type strain with its mutants, which lack the ability to synthesize cellulose and curli. Counts of the double mutant were 2-log higher in the soil but 1-log lower in the leaves (P < 0.05). Deletion of the agfBA gene (for curli) was more effective than deletion of bcsA (for cellulose). Thus, curli and cellulose play a role in the transfer or survival of Salmonella Typhimurium in the plant, as they do for plant pathogens.

Use of green fluorescent protein expressing Salmonella Stanley to investigate survival, spatial location, and control on alfalfa sprouts.

Laser scanning confocal microscopy (LSCM) was used to observe the interaction of Salmonella Stanley with alfalfa sprouts. The green fluorescent protein (gfp) gene was integrated into the chromosome of Salmonella Stanley for constitutive expression, thereby eliminating problems of plasmid stability and loss of signal. Alfalfa seeds were inoculated by immersion in a suspension of Salmonella Stanley (ca. 10(7) CFU/ml) for 5 min at 22 degrees C. Epifluorescence microscopy demonstrated the presence of target bacteria on the surface of sprouts. LSCM demonstrated bacteria present at a depth of 12 microm within intact sprout tissue. An initial population of ca. 10(4) CFU/g seed increased to 7.0 log CFU/g during a 24-h germination period and then decreased to 4.9 log CFU/g during a 144-h sprouting period. Populations of Salmonella Stanley on alfalfa seeds decreased from 5.2 to 4.1 log CFU/g and from 5.2 to 2.8 log CFU/g for seeds stored 60 days at 5 and 22 degrees C, respectively. The efficacy of 100, 200, 500, or 2,000 ppm chlorine in killing Salmonella Stanley associated with sprouts was determined. Treatment of sprouts in 2,000 ppm chlorine for 2 or 5 min caused a significant reduction in populations of Salmonella Stanley. Influence of storage on Salmonella Stanley populations was investigated by storing sprouts 4 days at 4 degrees C. The initial population (7.76 log CFU/g) of Salmonella Stanley on mature sprouts decreased (7.67 log CFU/g) only slightly. Cross-contamination during harvest was investigated by harvesting contaminated sprouts, then directly harvesting noncontaminated sprouts. This process resulted in the transfer of ca. 10(5) CFU/g Salmonella Stanley to the noncontaminated sprouts.

Cohesin-Dockerin Recognition in Cellulosome Assembly: Experiment Versus Hypothesis

The cohesin‐dockerin interaction provides the basis for incorporation of the individual enzymatic subunits into the cellulosome complex. In a previous article ( Pagés et al. , Proteins 1997;29:517–527) we predicted that four amino acid residues of the ∼70‐residue dockerin domain would serve as recognition codes for binding to the cohesin domain. The validity of the prediction was examined by site‐directed mutagenesis of the suspected residues, whereby the species‐specificity of the cohesin‐dockerin interaction was altered. The results support the premise that the four residues indeed play a role in biorecognition, while additional residues may also contribute to the specificity of the interaction. Proteins 2000;39:170–177.