Biomedical subjects

Martin Wiedmann

Publications and source records attributed to Martin Wiedmann.

At least 55 records · Page 3Linked to original sources

PubMed2004

The cost and benefit of Listeria monocytogenes food safety measures.

The objectives of this study were to evaluate economic techniques used to determine the cost and benefit of Listeria monocytogenes control and to estimate the economic optimum of L. monocytogenes food safety measures. The level of food safety measures is optimal if marginal benefit and marginal cost equate. Estimates of benefit and cost of L. monocytogenes food safety measures, from available published literature, are derived from different methods of economic analysis (willingness to pay, cost of illness, cost function, and event study methods). The estimated annual benefit and cost of L. monocytogenes food safety measures range from $2.3 billion to $22 billion and from $.01 billion to $2.4 billion, respectively. The estimated marginal benefit exceeds the estimated marginal cost, which implies that more food safety measures are warranted before the optimal level of L. monocytogenes food safety can be reached. However, due to considerable lack of data, the optimal level of L. monocytogenes food safety measures could not be estimated. When better data become available, this study can serve as a template for estimating the optimal level of food safety. The understanding of the economic optimum of food safety level will contribute to designing a control program that is economical and acceptable for US society.

Cost of Illness↗

PubMed2004

sigmaB-dependent gene induction and expression in Listeria monocytogenes during osmotic and acid stress conditions simulating the intestinal environment.

Listeria monocytogenes must overcome a variety of stress conditions in the host digestive tract to cause foodborne infections. The alternative sigma factor sigma(B), encoded by sigB, is responsible for regulating transcription of several L. monocytogenes virulence and stress-response genes, including genes that contribute to establishment of gastrointestinal infections. A quantitative RT-PCR assay was used to measure mRNA transcript accumulation for the virulence genes inlA and bsh, the stress-response genes opuCA and lmo0669 (encoding a carnitine transporter and an oxidoreductase, respectively) and the housekeeping gene rpoB. Assays were conducted on mid-exponential phase L. monocytogenes cells exposed to conditions reflecting osmotic (0.3 M NaCl) or acid (pH 4.5) conditions typical for the human intestinal lumen. In exponential-phase cells, as well as under osmotic and acid stress, inlA, opuCA and bsh showed significantly lower absolute expression levels in a L. monocytogenes DeltasigB null mutant compared to wild-type. A statistical model that normalized target gene expression relative to rpoB showed that accumulation of inlA, opuCA and bsh transcripts was significantly increased in the wild-type strain within 5 min of acid and osmotic stress exposure; lmo0669 transcript accumulation increased significantly only after acid exposure. It was concluded that sigma(B) is essential for rapid induction of the tested stress-response and virulence genes under conditions typically encountered during gastrointestinal passage. As inlA, bsh and opuCA are critical for gastrointestinal infections in animal models, the data also suggest that sigma(B) contributes to the ability of L. monocytogenes to cause foodborne infections.

Acids↗

PubMed2004

Listeria monocytogenes isolates from foods and humans form distinct but overlapping populations.

A total of 502 Listeria monocytogenes isolates from food and 492 from humans were subtyped by EcoRI ribotyping and PCR-restriction fragment length polymorphism analysis of the virulence gene hly. Isolates were further classified into genetic lineages based on subtyping results. Food isolates were obtained through a survey of selected ready-to-eat food products in Maryland and California in 2000 and 2001. Human isolates comprised 42 isolates from invasive listeriosis cases reported in Maryland and California during 2000 and 2001 as well as an additional 450 isolates from cases that had occurred throughout the United States, predominantly from 1997 to 2001. Assignment of isolates to lineages and to the majority of L. monocytogenes subtypes was significantly associated with the isolate source (food or human), although most subtypes and lineages included both human and food isolates. Some subtypes were also significantly associated with isolation from specific food types. Tissue culture plaque assay characterization of the 42 human isolates from Maryland and California and of 91 representative food isolates revealed significantly higher average infectivity and cell-to-cell spread for the human isolates, further supporting the hypothesis that food and human isolates form distinct populations. Combined analysis of subtype and cytopathogenicity data showed that strains classified into specific ribotypes previously linked to multiple human listeriosis outbreaks, as well as those classified into lineage I, are more common among human cases and generate larger plaques than other subtypes, suggesting that these subtypes may represent particularly virulent clonal groups. These data will provide a framework for prediction of the public health risk associated with specific L. monocytogenes subtypes.

Animals↗

PubMed2004

Multilocus sequence typing of Listeria monocytogenes by use of hypervariable genes reveals clonal and recombination histories of three lineages.

In an attempt to develop a method to discriminate among isolates of Listeria monocytogenes, the sequences of all of the annotated genes from the fully sequenced strain L. monocytogenes EGD-e (serotype 1/2a) were compared by BLASTn to a file of the unfinished genomic sequence of L. monocytogenes ATCC 19115 (serotype 4b). Approximately 7% of the matching genes demonstrated 90% or lower identity between the two strains, and the lowest observed identity was 80%. Nine genes (hisJ, cbiE, truB, ribC, comEA, purM, aroE, hisC, and addB) in the 80 to 90% identity group and two genes (gyrB and rnhB) with approximately 97% identity were selected for multilocus sequence analysis in two sets of L. monocytogenes isolates (a 15-strain diversity set and a set of 19 isolates from a single food-processing plant). Based on concatenated sequences, a total of 33 allotypes were differentiated among the 34 isolates tested. Population genetics analyses revealed three lineages of L. monocytogenes that differed in their history of apparent recombination. Lineage I appeared to be completely clonal, whereas representatives of the other lineages demonstrated evidence of horizontal gene transfer and recombination. Although most of the gene sequences for lineage II strains were distinct from those of lineage I, a few strains with the majority of genes characteristic of lineage II had some that were characteristic of lineage I. Genes from lineage III organisms were mostly similar to lineage I genes, with instances of genes appearing to be mosaics with lineage II genes. Even though lineage I and lineage II generally demonstrated very distinct sequences, the sequences for the 11 selected genes demonstrated little discriminatory power within each lineage. In the L. monocytogenes isolate set obtained from one food-processing plant, lineage I and lineage II were found to be almost equally prevalent. While it appears that different lineages of L. monocytogenes can share habitats, they appear to differ in their histories of horizontal gene transfer.

Animals↗

PubMed2004

Evaluation of "Helicobacter heilmannii" subtypes in the gastric mucosas of cats and dogs.

Infection with candidatus "Helicobacter heilmannii" is associated with gastritis and mucosa-associated lymphoid tissue lymphoma in people. Infection with "H. heilmannii" type 1 predominates (80%) and is thought to be acquired from dogs, cats, or pigs. We further examined the zoonotic potential of dogs and cats by amplifying gastric DNA from cats (n = 45) and dogs (n = 10) with primers against "H. heilmannii" ureB and 16S rRNA genes and sequencing the products. Fluorescence in situ hybridization (FISH) with eubacterial and "H. heilmannii"-specific probes was employed to directly visualize "H. heilmannii" types and their intragastric distribution. ureB sequences of "H. heilmannii" amplicons clustered with human and feline isolates of "H. heilmannii" and were distinct from the "H. heilmannii"-like organisms (HHLO) H. felis, H. salomonis, and H. bizzozeronii. 16S ribosomal DNA sequences in 20 "H. heilmannii"-infected cats and dogs were distinct from "H. heilmannii" type 1 and "H. suis" and clustered with "H. heilmannii" types 2 and 4. FISH confirmed the presence of "H. heilmannii" types 2 and 4 in dogs but failed to definitively characterize the "H. heilmannii" types present in cats. In infected dogs, "H. heilmannii" inhabited the gastric mucus and glands, and in dogs coinfected with other HHLO it shared the same gastric niche. The results indicate that dogs and cats are predominantly colonized by "H. heilmannii" bacteria that are distinct from type 1 and from "H. suis." As "H. heilmannii" type 1 predominates in people, the zoonotic risk posed by dogs and cats is likely small.

Animals↗

PubMed2004

Eye infections due to Listeria monocytogenes in three cows and one horse.

A retrospective study was conducted to determine case histories, microbiological characteristics, and molecular subtypes associated with Listeria monocytogenes infections of the eye in large animals. For selected cases, environmental L. monocytogenes contamination patterns on case farms were also evaluated to probe for potential sources and spread of listerial eye infections. Records of 170 L. monocytogenes isolates from animal infections were reviewed to determine the fraction of isolates associated with eye infections (conjunctivitis, keratitis, and uveitis) of animals and to gather information on the clinical history of these cases. Overall, 4 of 170 Listeria monocytogenes isolates were associated with eye infections; 3 of these had occurred in cows and 1 in a horse. Molecular subtyping (by EcoRI ribotying) showed that 4 different L. monocytogenes subtypes were isolated from these 4 cases; the same ribotypes had previously been found among invasive animal listeriosis infections. Although a variety of L. monocytogenes subtypes were isolated from environmental sources, on 1 farm, the same ribotype associated with the eye infection was also isolated from a fecal sample of a healthy animal and from a soil sample. The data reported in this study further suggest that L. monocytogenes can be a cause of eye infections in several animal species. Listerial eye infections do not seem to require specific pathogen-related virulence characteristics but rather seem to be a function of environmental or host factors, such as direct exposure of the eyes of susceptible animals to high numbers of the pathogen. Although listerial eye infections are rarely diagnosed because of its ubiquitous nature, L. monocytogenes may have to be considered more commonly as a causative agent of eye infections in ruminants and horses.

Animals↗

PubMed2004

Longitudinal studies on Listeria in smoked fish plants: impact of intervention strategies on contamination patterns.

Four ready-to-eat smoked fish plants were monitored for 2 years to study Listeria contamination patterns and the impact of plant-specific Listeria control strategies, including employee training and targeted sanitation procedures, on Listeria contamination patterns. Samples from the processing plant environment and from raw and finished product were collected monthly and tested for Listeria spp. and Listeria monocytogenes. Before implementation of intervention strategies, 19.2% of raw product samples (n = 276), 8.7% of finished product samples (n = 275), and 26.1% of environmental samples (n = 617) tested positive for Listeria spp. During and after implementation of Listeria control strategies, 19.0% of raw product samples (n = 242), 7.0% of finished product samples (n = 244), and 19.5% of environmental samples (n = 527) were positive for Listeria spp. In one of the four fish plants (plant 4), no environmental samples were positive for L. monocytogenes, and this plant was thus excluded from statistical analyses. Based on data pooled from plants 1, 2, and 3, environmental Listeria spp. prevalence was significantly lower (P < 0.05) for nonfood contact surfaces and the finished product area and for the overall core environmental samples after implementation of control strategies. Listeria prevalence for floor drains was similar before and after implementation of controls (49.6 and 54.2%, respectively). Regression analysis revealed a significant positive relationship (P < 0.05) between L. monocytogenes prevalence in the environment and in finished products before implementation of control strategies; however, this relationship was absolved by implementation of Listeria control strategies. Molecular subtyping (EcoRI ribotyping) revealed that specific L. monocytogenes ribotypes persisted in three processing plants over time. These persistent ribotypes were responsible for all six finished product contamination events detected in plant 1. Ribotype data also indicated that incoming raw material is only rarely a direct source of finished product contamination. While these data indicate that plant-specific Listeria control strategies can reduce cross-contamination and prevalence of Listeria spp. and L. monocytogenes in the plant environment, elimination of persistent L. monocytogenes strains remains a considerable challenge.

Animals↗

PubMed2004

Mathematical model of Listeria monocytogenes cross-contamination in a fish processing plant.

Listeriosis is a foodborne disease caused by the bacterium Listeria monocytogenes. The food industry and government agencies devote considerable resources to reducing contamination of ready-to-eat foods with L. monocytogenes. Because inactivation treatments can effectively eliminate L. monocytogenes present on raw materials, postprocessing cross-contamination from the processing plant environment appears to be responsible for most L. monocytogenes food contamination events. An improved understanding of cross-contamination pathways is critical to preventing L. monocytogenes contamination. Therefore, a plant-specific mathematical model of L. monocytogenes cross-contamination was developed, which described the transmission of L. monocytogenes contamination among food, food contact surfaces, employees' gloves, and the environment. A smoked fish processing plant was used as a model system. The model estimated that 10.7% (5th and 95th percentile, 0.05% and 22.3%, respectively) of food products in a lot are likely to be contaminated with L. monocytogenes. Sensitivity analysis identified the most significant input parameters as the frequency with which employees' gloves contact food and food contact surfaces, and the frequency of changing gloves. Scenario analysis indicated that the greatest reduction of the within-lot prevalence of contaminated food products can be achieved if the raw material entering the plant is free of contamination. Zero contamination of food products in a lot was possible but rare. This model could be used in a risk assessment to quantify the potential public health benefits of in-plant control strategies to reduce cross-contamination.

Animals↗

PubMed2004

Tracking of Listeria monocytogenes in smoked fish processing plants.

Four smoked fish processing plants were used as a model system to characterize Listeria monocytogenes contamination patterns in ready-to-eat food production environments. Each of the four plants was sampled monthly for approximately 1 year. At each sampling, four to six raw fish and four to six finished product samples were collected from corresponding lots. In addition, 12 to 14 environmental sponge samples were collected several hours after the start of production at sites selected as being likely contamination sources. A total of 234 raw fish, 233 finished products, and 553 environmental samples were tested. Presumptive Listeria spp. were isolated from 16.7% of the raw fish samples, 9.0% of the finished product samples, and 27.3% of the environmental samples. L. monocytogenes was isolated from 3.8% of the raw fish samples (0 to 10%, depending on the plant), 1.3% of the finished product samples (0 to 3.3%), and 12.8% of the environmental samples (0 to 29.8%). Among the environmental samples, L. monocytogenes was found in 23.7% of the samples taken from drains, 4.8% of the samples taken from food contact surfaces, 10.4% of the samples taken from employee contact surfaces (aprons, hands, and door handles), and 12.3% of the samples taken from other nonfood contact surfaces. Listeria spp. were isolated from environmental samples in each of the four plants, whereas L. monocytogenes was not found in any of the environmental samples from one plant. Overall, the L. monocytogenes prevalence in the plant environment showed a statistically significant (P < 0.0001) positive relationship with the prevalence of this organism in finished product samples. Automated EcoRI ribotyping differentiated 15 ribotypes among the 83 L. monocytogenes isolates. For each of the three plants with L. monocytogenes-positive environmental samples, one or two ribotypes seemed to persist in the plant environment during the study period. In one plant, a specific L. monocytogenes ribotype represented 44% of the L. monocytogenes-positive environmental samples and was also responsible for one of the two finished product positives found in this plant. In another plant, a specific L. monocytogenes ribotype persisted in the raw fish handling area. However, this ribotype was never isolated from the finished product area in this plant, indicating that this operation has achieved effective separation of raw and finished product areas. Molecular subtyping methods can help identify plant-specific L. monocytogenes contamination routes and thus provide the knowledge needed to implement improved L. monocytogenes control strategies.

Animals↗

PubMed2004

Prevalence and growth of Listeria on naturally contaminated smoked salmon over 28 days of storage at 4 degrees C.

Only limited data are available on the growth characteristics of Listeria in naturally contaminated ready-to-eat foods. To evaluate Listeria contamination patterns and growth in smoked salmon, 72 smoked salmon product samples from two processing plants were tested for Listeria spp. and L. monocytogenes. Samples were divided into four approximately equal portions: one portion was tested on receipt, and the other three were vacuum sealed and stored at 4 degrees C for 7, 14, and 28 days. Listeria testing was performed using both an enrichment procedure and direct plating to enumerate Listeria in samples that contained >2 to 10 CFU/g. Five samples were positive for Listeria spp., including one sample that was positive for L. monocytogenes. Most samples yielded only sporadic positive results among the portions tested on days 0, 7, 14, and 28. Only one sample contained Listeria spp. in numbers above the detection limit for enumeration. For this sample, the portions tested on days 7 and 28 contained 46 and 52 CFU/g, respectively, whereas the portion tested on day 14 was negative. Overall, our data indicate that there is considerable heterogeneity in Listeria spp. distribution within a single positive smoked fish sample. Even with refrigerated storage for 28 days, none of the naturally contaminated samples reached Listeria spp. numbers >100 CFU/g, which indicates that Listeria growth was limited within a 4-week storage period. However, because of the apparent heterogeneity of Listeria distribution within samples, the interpretation of growth data collected on naturally contaminated samples is difficult.

Animals↗

PubMed2004

Impact of intervention strategies on Listeria contamination patterns in crawfish processing plants: a longitudinal study.

Two ready-to-eat crawfish processing plants were monitored for 2 years to study the impact of Listeria control strategies, including employee training and targeted sanitation procedures, on Listeria contamination. Environmental, raw material, and finished product samples were collected weekly during the main processing months (April to June) and tested for Listeria spp. and Listeria monocytogenes. Before implementation of control strategies (year 1), the two processing plants showed Listeria spp. prevalences of 29.5% (n = 78) in raw, whole crawfish, 5.2% (n = 155) in the processing plant environment, and 0% (n = 78) in finished products. In year 2, after plant-specific Listeria control strategies were implemented, Listeria spp. prevalence increased in raw crawfish (57.5%, n = 101), in the processing plant environment (10.8%, n = 204), and in the finished product (1.0%, n = 102). Statistical analysis showed a significant increase in Listeria spp. prevalence (P < 0.0001) and a borderline nonsignificant increase in L. monocytogenes prevalence (P = 0.097) on raw material in year 2. Borderline nonsignificant increases were also observed for Listeria spp. prevalence in environmental samples (P = 0.082). Our data showed that Listeria spp. prevalence in raw crawfish can vary significantly among seasons. However, the increased contamination prevalence for raw materials only resulted in a limited Listeria prevalence increase for the processing plant environment with extremely low levels of finished product contamination. Heat treatment of raw materials combined with Listeria control strategies to prevent cross-contamination thus appears to be effective in achieving low levels of finished product contamination, even with Listeria spp. prevalences for raw crawfish of more than 50%.

Animals↗

PubMed2004

Distribution of Listeria monocytogenes molecular subtypes among human and food isolates from New York State shows persistence of human disease--associated Listeria monocytogenes strains in retail environments.

While there is considerable information available regarding Listeria monocytogenes contamination patterns in food processing plants, our understanding of L. monocytogenes contamination and transmission in retail operations is limited. We characterized 125 food, 40 environmental, and 342 human clinical L. monocytogenes isolates collected in New York State from 1997 to 2002 using automated ribotyping and hly allelic variation. All environmental isolates were obtained from retail establishments and the majority of food isolates (98 isolates) were obtained from foods that were prepared or handled at retail. Overall, food and/or environmental isolates from 50 different retail establishments were characterized. The 125 food and 40 environmental isolates were differentiated into 29 and 10 ribotypes, respectively. For 16 retail establishments, we found evidence for persistence of one or more specific L. monocytogenes strains as indicated by isolation of the same EcoRI ribotype from food or environmental samples collected in a given establishment on different days. The human isolates were differentiated into 48 ribotypes. Statistical analyses showed that two ribotypes were significantly (P < 0.0001) more common among food isolates as compared with human isolates. However, a total of 17 ribotypes found among the human clinical isolates were also found among the food and environmental isolates. We conclude that L. monocytogenes, including subtypes that have been linked to human disease, can persist in retail environments. Implementation of Listeria control procedures in retail operations, which process and handle products that permit the growth of L. monocytogenes, are thus a critical component of a farm-to-table L. monocytogenes control program.

Animals↗

PubMed2003

Oncogenic Ras and Akt signaling contribute to glioblastoma formation by differential recruitment of existing mRNAs to polysomes.

In order to determine the global effects of oncogenic Ras and Akt signaling pathways on translational efficiencies, we compared the gene expression profiles of total cellular mRNA and mRNA associated with polysomes. We found that the immediate effect of Ras and Akt signaling blockade on transcription was relatively modest; however, the profile of mRNA associated with polysomes was substantially altered. These observations indicate that the immediate effect of Ras and Akt signaling regulates the recruitment of specific mRNAs to ribosomes to a far greater extent than they regulate the production of mRNAs by transcriptional effects. The mRNAs most affected are those encoding proteins that regulate growth, transcription regulation, cell to cell interactions, and morphology. These data support a model whereby Ras and Akt signaling primarily lead to cellular transformation by altering the transcriptome and producing a radical shift in the composition of mRNAs associated with actively translating polysomes.

Animals↗

PubMed2003

Polypeptide-binding proteins mediate completion of co-translational protein translocation into the mammalian endoplasmic reticulum.

The first step in the secretion of most mammalian proteins is their transport into the lumen of the endoplasmic reticulum (ER). Transport of pre-secretory proteins into the mammalian ER requires signal peptides in the precursor proteins and a protein translocase in the ER membrane. In addition, hitherto unidentified lumenal ER proteins have been shown to be required for vectorial protein translocation. This requirement was confirmed in this study by using proteoliposomes that were made from microsomal detergent extracts and contained either low or high concentrations of lumenal ER proteins. Furthermore, immunoglobulin-heavy-chain-binding protein (BiP) was shown to be able to substitute for the full set of lumenal proteins and, in the case of biotinylated precursor proteins, avidin was found to be able to substitute for lumenal proteins. Thus, the polypeptide-chain-binding protein BiP was identified as one lumenal protein that is involved in efficient vectorial protein translocation into the mammalian ER.

Animals↗

PubMed2003

Sigma(B)-dependent expression patterns of compatible solute transporter genes opuCA and lmo1421 and the conjugated bile salt hydrolase gene bsh in Listeria monocytogenes.

Listeria monocytogenes is a food-borne pathogen that can persist and grow under a wide variety of environmental conditions including low pH and high osmolarity. The alternative sigma factor sigma(B) contributes to L. monocytogenes survival under extreme conditions. The purpose of this study was to identify and confirm specific sigma(B)-dependent genes in L. monocytogenes and to characterize their expression patterns under various stress conditions. opuCA, lmo1421 and bsh were identified as putative sigma(B)-dependent genes based on the presence of a predicted sigma(B)-dependent promoter sequence upstream of each gene. opuCA and lmo1421 encode known and putative compatible solute transporter proteins, respectively, and bsh encodes a conjugated bile salt hydrolase (BSH). Reporter fusions and semi-quantitative RT-PCR techniques were used to confirm sigma(B)-dependent regulation of these stress-response genes and to determine their expression patterns in response to environmental stresses. RT-PCR demonstrated that opuCA, lmo1421 and bsh transcript levels are reduced in stationary-phase L. monocytogenes deltasigB cells relative to levels present in wild-type cells. Furthermore, BSH activity is abolished in a L. monocytogenes deltasigB strain. RT-PCR confirmed growth-phase-dependent expression of opuCA, with highest levels of expression in stationary-phase cells. The L. monocytogenes wild-type strain exhibited two- and threefold induction of opuCA expression and seven- and fivefold induction of lmo1421 expression following 10 and 15 min exposure to 0.5 M KCl, respectively, as determined by RT-PCR, suggesting rapid induction of sigma(B) activity in exponential-phase L. monocytogenes upon exposure to salt stress. Single-copy chromosomal opuCA-gus reporter fusions also showed significant induction of opuCA expression following exposure of exponential-phase cells to increased salt concentrations (0.5 M NaCl or 0.5 M KCl). In conjunction with recent findings that indicate a role for opuCA and bsh in L. monocytogenes virulence, the data presented here provide further evidence of specific sigma(B)-mediated contributions to both environmental stress resistance and intra-host survival in L. monocytogenes.

ATP-Binding Cassette Transporters↗

PubMed2003

Role of sigmaB in regulating the compatible solute uptake systems of Listeria monocytogenes: osmotic induction of opuC is sigmaB dependent.

The regulation of the compatible solute transport systems in Listeria monocytogenes by the stress-inducible sigma factor sigma(B) was investigated. Using wild-type strain 10403S and an otherwise isogenic strain carrying an in-frame deletion in sigB, we have examined the role of sigma(B) in regulating the ability of cells to utilize betaine and carnitine during growth under conditions of hyperosmotic stress. Cells lacking sigma(B) were defective for the utilization of carnitine but retained the ability to utilize betaine as an osmoprotectant. When compatible solute transport studies were performed, the initial rates of uptake of both betaine and carnitine were found to be reduced in the sigB mutant; carnitine transport was almost abolished, whereas betaine transport was reduced to approximately 50% of that of the parent strain. Analysis of the cytoplasmic pools of compatible solutes during balanced growth revealed that both carnitine and betaine steady-state pools were reduced in the sigB mutant. Transcriptional reporter fusions to the opuC (which encodes an ABC carnitine transporter) and betL (which encodes an a secondary betaine transporter) operons were generated by using a promoterless copy of the gus gene from Escherichia coli. Measurement of beta-glucuronidase activities directed by opuC-gus and betL-gus revealed that transcription of opuC is largely sigma(B) dependent, consistent with the existence of a potential sigma(B) consensus promoter motif upstream from opuCA. The transcription of betL was found to be sigB independent. Reverse transcriptase PCR experiments confirmed these data and indicated that the transcription of all three known compatible solute uptake systems (opuC, betL, and gbu), as well as a gene that is predicted to encode a compatible solute transporter subunit (lmo1421) is induced in response to elevated osmolarity. The osmotic induction of opuCA and lmo1421 was found to be strongly sigma(B) dependent. Together these observations suggest that sigma(B) plays a major role in the regulation of carnitine utilization by L. monocytogenes but is not essential for betaine utilization by this pathogen.

ATP-Binding Cassette Transporters↗

PubMed2003

Genome diversification in phylogenetic lineages I and II of Listeria monocytogenes: identification of segments unique to lineage II populations.

Thirteen different serotypes of Listeria monocytogenes can be distinguished on the basis of variation in somatic and flagellar antigens. Although the known virulence genes are present in all serotypes, greater than 90% of human cases of listeriosis are caused by serotypes 1/2a, 1/2b, and 4b and nearly all outbreaks of food-borne listeriosis have been caused by serotype 4b strains. Phylogenetic analysis of these three common clinical serotypes places them into two different lineages, with serotypes 1/2b and 4b belonging to lineage I and 1/2a belonging to lineage II. To begin examining evolution of the genome in these serotypes, DNA microarray analysis was used to identify lineage-specific and serotype-specific differences in genome content. A set of 44 strains representing serotypes 1/2a, 1/2b, and 4b was probed with a shotgun DNA microarray constructed from the serotype 1/2a strain 10403s. Clones spanning 47 different genes in 16 different contiguous segments relative to the lineage II 1/2a genome were found to be absent in all lineage I strains tested (serotype 4b and 1/2b) and an additional nine were altered exclusively in 4b strains. Southern hybridization confirmed that conserved alterations were, in all but two loci, due to absence of the segments from the genome. Genes within these contiguous segments comprise five functional categories, including genes involved in synthesis of cell surface molecules and regulation of virulence gene expression. Phylogenetic reconstruction and examination of compositional bias in the regions of difference are consistent with a model in which the ancestor of the two lineages had the 1/2 somatic serotype and the regions absent in the lineage I genome arose by loss of ancestral sequences.

Biological Transport↗

PubMed2003

Listeria monocytogenes sigma B regulates stress response and virulence functions.

While the stress-responsive alternative sigma factor sigma(B) has been identified in different species of Bacillus, Listeria, and Staphylococcus, the sigma(B) regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify sigma(B)-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate sigma(B)-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted sigma(B)-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and Delta sigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant sigma(B)-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting sigma(B)-dependent expression, 54 were preceded by a sequence resembling the sigma(B) promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the sigma(B)-dependent nature of a subset of eight selected promoter regions. Notably, the sigma(B)-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, sigma(B) also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that sigma(B) contributes to L. monocytogenes gene expression during infection.

Bacterial Proteins↗