Sporulation Strategies and Potential Role of the Exosporium in Survival and Persistence of Clostridium botulinum.

Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains are terrestrial, mesophilic, and produce highly heat-resistant spores, while Group II strains can be terrestrial (type B) or aquatic (type E) and are generally psychrotrophic and produce spores of moderate heat resistance. Group III strains are either terrestrial or aquatic, mesophilic or slightly thermophilic, and the heat resistance properties of their spores are poorly characterized. Here, we analyzed the sporulation dynamics in population, spore morphology, and other spore properties of 10 C. botulinum strains belonging to Groups I-III. We propose two distinct sporulation strategies used by C. botulinum Groups I-III strains, report their spore properties, and suggest a putative role for the exosporium in conferring high heat resistance. Strains within each physiological group produced spores with similar characteristics, likely reflecting adaptation to respective environmental habitats. Our work provides new information on the spores and on the population and single-cell level strategies in the sporulation of C. botulinum.

Specific Isolation of Clostridium botulinum Group I Cells by Phage Lysin Cell Wall Binding Domain with the Aid of S-Layer Disruption.

Clostridium botulinum is a notorious pathogen that raises health and food safety concerns by producing the potent botulinum neurotoxin and causing botulism, a potentially fatal neuroparalytic disease in humans and animals. Efficient methods for the identification and isolation of C. botulinum are warranted for laboratory diagnostics of botulism and for food safety risk assessment. The cell wall binding domains (CBD) of phage lysins are recognized by their high specificity and affinity to distinct types of bacteria, which makes them promising for the development of diagnostic tools. We previously identified CBO1751, which is the first antibotulinal phage lysin showing a lytic activity against C. botulinum Group I. In this work, we assessed the host specificity of the CBD of CBO1751 and tested its feasibility as a probe for the specific isolation of C. botulinum Group I strains. We show that the CBO1751 CBD specifically binds to C. botulinum Group I sensu lato (including C. sporogenes) strains. We also demonstrate that some C. botulinum Group I strains possess an S-layer, the disruption of which by an acid glycine treatment is required for efficient binding of the CBO1751 CBD to the cells of these strains. We further developed CBO1751 CBD-based methods using flow cytometry and magnetic separation to specifically isolate viable cells of C. botulinum Group I. These methods present potential for applications in diagnostics and risk assessment in order to control the botulism hazard.

Strain Variability of Listeria monocytogenes under NaCl Stress Elucidated by a High-Throughput Microbial Growth Data Assembly and Analysis Protocol.

Listeria monocytogenes causes the severe foodborne illness listeriosis and survives in food-associated environments due to its high stress tolerance. A data assembly and analysis protocol for microbial growth experiments was compiled to elucidate the strain variability of L. monocytogenes stress tolerance. The protocol includes measurement of growth ability under stress (step 1), selection of a suitable method for growth parameter calculation (step 2), comparison of growth patterns between strains (step 3), and biological interpretation of the discovered differences (step 4). In step 1, L. monocytogenes strains (n = 388) of various serovars and origins grown on media with 9.0% NaCl were measured using a Bioscreen C microbiology reader. Technical variability of the growth measurements was assessed and eliminated. In step 2, the growth parameters determined by Gompertz, modified-Gompertz, logistic, and Richards models and model-free splines were compared, illustrating differences in the suitability of these methods to describe the experimental data. In step 3, hierarchical clustering was used to describe the NaCl tolerance of L. monocytogenes measured by strain-specific variation in growth ability; tolerant strains had higher growth rates and maximum optical densities and shorter lag phases than susceptible strains. The spline parameter area under the curve best classified "poor," "average," and "good" growers. In step 4, the tested L. monocytogenes lineage I strains (serovars 4b and 1/2b) proved to be significantly more tolerant toward 9.0% NaCl than lineage II strains (serovars 1/2a, 1/2c, and 3a). Our protocol provides systematic tools to gain comparable data for investigating strain-specific variation of bacterial growth under stress.IMPORTANCE The pathogen Listeria monocytogenes causes the foodborne disease listeriosis, which can be fatal in immunocompromised individuals. L. monocytogenes tolerates several environmental stressors and can persist in food-processing environments and grow in foodstuffs despite traditional control measures such as high salt content. Nonetheless, L. monocytogenes strains differ in their ability to withstand stressors. Elucidating the intraspecies strain variability of L. monocytogenes stress tolerance is crucial for the identification of particularly tolerant strains. To enhance reliable identification of variability in bacterial stress tolerance phenotypes, we compiled a large-scale protocol for the entire data assembly and analysis of microbial growth experiments, providing a systematic approach and checklist for experiments on strain-specific growth ability. Our study illustrated the diversity and strain-specific variation of L. monocytogenes stress tolerance with an unprecedented scope and discovered biologically relevant serovar- and lineage-dependent phenotypes of NaCl tolerance.

MouR controls the expression of the Listeria monocytogenes Agr system and mediates virulence.

The foodborne pathogen Listeria monocytogenes (Lm) causes invasive infection in susceptible animals and humans. To survive and proliferate within hosts, this facultative intracellular pathogen tightly coordinates the expression of a complex regulatory network that controls the expression of virulence factors. Here, we identified and characterized MouR, a novel virulence regulator of Lm. Through RNA-seq transcriptomic analysis, we determined the MouR regulon and demonstrated how MouR positively controls the expression of the Agr quorum sensing system (agrBDCA) of Lm. The MouR three-dimensional structure revealed a dimeric DNA-binding transcription factor belonging to the VanR class of the GntR superfamily of regulatory proteins. We also showed that by directly binding to the agr promoter region, MouR ultimately modulates chitinase activity and biofilm formation. Importantly, we demonstrated by in vitro cell invasion assays and in vivo mice infections the role of MouR in Lm virulence.

High prevalence of Clostridium botulinum in vegetarian sausages.

Clostridium botulinum is a significant food safety concern due to its ability to produce highly potent neurotoxin and resistant endospores. Vegetarian sausages have become a popular source of plant protein and alternative for meat products. While vegetarian sausages have not been linked to botulism, numerous outbreaks due to preserved vegetables suggest a frequent occurrence of C. botulinum spores in the raw material. The product formulation of vegetarian sausages involves limited NaCl and preservatives, and shelf-lives may be several months. The safety of vegetarian sausages thus relies mainly on heat treatment and chilled storage. The main food safety concern is C. botulinum Group II that can grow and produce toxin at refrigeration temperatures. Here we show a high overall prevalence (32%) of C. botulinum in 74 samples of vegetarian sausages from seven producers. Both Groups I and II strains and genes for neurotoxin types A, B, E and F were detected in the products. The highest cell counts (1200 spores/kg) were observed for C. botulinum Group II in products with remaining shelf-lives of 6 months at the time of purchase. We conclude that vacuum-packaged vegetarian sausage products frequently contain C. botulinum spores and may possess a high risk of C. botulinum growth and toxin production. Chilled storage below 3°C and thorough reheating before consumption are warranted.

MdrL, a major facilitator superfamily efflux pump of Listeria monocytogenes involved in tolerance to benzalkonium chloride.

Efflux pumps are recognized as an important mechanism for decreased susceptibility of benzalkonium chloride (BC) in Listeria monocytogenes. Previous studies showed that the efflux pump MdrL was overexpressed in L. monocytogenes exposed to BC. In the present work, we aimed to clarify the role of MdrL in tolerance to BC and environmental stresses including acid, alkali, osmotic, ethanol, and oxidative stresses, as well as resistance to other antimicrobial agents in L. monocytogenes EGD-e. In addition, regulation of the expression of mdrL by LadR was investigated. Gene deletion mutants were constructed by homologous recombination strategy. For the wild-type and mutant strains, minimum inhibitory concentrations (MICs) of antimicrobial agents were determined by the agar dilution, and the growth and survival analysis were also performed. LadR was expressed and the interaction between LadR and the mdrL promoter was investigated by electrophoretic mobility shift assay (EMSA). Compared to the wild-type strain, the growth of ΔmdrL deletion mutant strain was impaired in the presence of sublethal concentration of BC. Moreover, the mutant showed a lower level of survival than that of the wild-type strain in the presence of lethal concentration of BC. However, the deletion of mdrL had no impact on cefotaxime resistance and ethidium bromide efflux. BC could induce the expression of mdrL in L. monocytogenes and the mdrL expression was regulated by LadR instead of SigmaB. LadR was able to specifically bind to the mdrL promoter. The results showed that efflux pump MdrL was associated with BC tolerance in L. monocytogenes EGD-e. Moreover, our results also provided strong evidence that LadR negatively regulated the expression of mdrL. Since BC is commonly used in the food industry, efflux pump MdrL is beneficial for L. monocytogenes to survive this stress in food processing environments.

Processing plant and machinery sanitation and hygiene practices associate with Listeria monocytogenes occurrence in ready-to-eat fish products.

Listeria monocytogenes causes the foodborne illness listeriosis, which exhibits high fatality among people in risk groups. The incidence of listeriosis has increased in Europe, which raises concerns about L. monocytogenes occurrence in foodstuffs. Ready-to-eat seafood products are considered particularly risky vehicles. Poor hygiene at processing facilities predisposes them to L. monocytogenes contamination, which can be controlled by stringent self-checking system measures. We examined the association of fish-processing plant operational and hygiene practices with the occurrence of L. monocytogenes in vacuum-packaged gravad (cold-salted) and cold-smoked salmon and rainbow trout products. Product sampling of 21 fish-processing plants was carried out, and operational procedures relating to L. monocytogenes control were surveyed using an in-depth risk assessment questionnaire. L. monocytogenes occurred only in sliced and mainly in gravad products of seven fish-processing plants. Shortages in preventive measures were discovered predominantly among the L. monocytogenes positive fish-processing plants. Using generalized linear modeling, we identified the following features associated with L. monocytogenes product contamination: the number of processing machines, deficiencies in the processing environment and machinery sanitation, and staff movement from areas of low toward high hygiene. Furthermore, performing frequent periodic thorough sanitation alongside everyday sanitation practices associated with a decreased risk of product contamination.

Prevalence and Dynamics of Pathogenic Yersinia enterocolitica 4/O:3 Among Finnish Piglets, Fattening Pigs, and Sows.

Pigs are considered the main reservoir of Yersinia enterocolitica, and hence, understanding the ecology of this foodborne pathogen at the farm level is crucial. We calculated Bayesian estimates for the ability of a commercial enzyme-linked immunosorbent assay (ELISA) diagnostic test kit to detect antibodies against pathogenic Yersinia in pigs. The sensitivity and specificity of the test were 75.4% and 98.1%, respectively. We also studied the dynamics of Y. enterocolitica infection in 3 farrow-to-finish pig farms by following the same 30 pens of pigs through their lifetime from farrowing unit to slaughterhouse. Each farm was sampled 4 times, and 864 fecal and 730 serum samples were collected altogether. Pathogenic Y. enterocolitica 4/O:3 was isolated from 31.6% of the fecal samples by culturing, and Yersinia antibodies were detected in 38.2% of the serum samples with the commercial ELISA test. The pathogen was not isolated from farrowing units or all-in/all-out weaning units. However, in the weaning and fattening units using continuous management systems, the pathogen was isolated from every pen at some point of the study. After the pigs were transported into slaughterhouse, 150 tonsils were collected and 96.7% were positive by culturing. Among the strains isolated from feces and tonsils, 56 different genotypes of pathogenic Y. enterocolitica 4/O:3 were found by multilocus variable-number tandem-repeat analysis (MLVA). Finally, we collected tonsils of 266 sows from 115 farrowing farms, and Y. enterocolitica 4/O:3 was detected in 6.0% of the samples by the culture method, whereas 77.1% of the tonsils were serologically positive; the estimate for true seroprevalence was 95.8%. In conclusion, sows may not be the main source of Y. enterocolitica for piglets, although sows may still play a role in maintaining Y. enterocolitica in pig farms. Instead, pigs appear to get this foodborne pathogen mainly during the fattening period, especially if continuous management is applied.

Insights into the Phylogeny and Evolution of Cold Shock Proteins: From Enteropathogenic Yersinia and Escherichia coli to Eubacteria.

Psychrotrophic foodborne pathogens, such as enteropathogenic Yersinia, which are able to survive and multiply at low temperatures, require cold shock proteins (Csps). The Csp superfamily consists of a diverse group of homologous proteins, which have been found throughout the eubacteria. They are related to cold shock tolerance and other cellular processes. Csps are mainly named following the convention of those in Escherichia coli. However, the nomenclature of certain Csps reflects neither their sequences nor functions, which can be confusing. Here, we performed phylogenetic analyses on Csp sequences in psychrotrophic enteropathogenic Yersinia and E. coli. We found that representative Csps in enteropathogenic Yersinia and E. coli can be clustered into six phylogenetic groups. When we extended the analysis to cover Enterobacteriales, the same major groups formed. Moreover, we investigated the evolutionary and structural relationships and the origin time of Csp superfamily members in eubacteria using nucleotide-level comparisons. Csps in eubacteria were classified into five clades and 12 subclades. The most recent common ancestor of Csp genes was estimated to have existed 3585 million years ago, indicating that Csps have been important since the beginning of evolution and have enabled bacterial growth in unfavorable conditions.

Occurrence, Persistence, and Contamination Routes of Listeria monocytogenes Genotypes on Three Finnish Dairy Cattle Farms: a Longitudinal Study.

The molecular epidemiology of Listeria monocytogenes was investigated in a longitudinal study of three Finnish dairy farms during 2013 to 2016. A total of 186 bulk tank milk (BTM), 224 milk filter sock (MFS), and 1,702 barn environment samples were analyzed, and isolates of L. monocytogenes were genotyped using pulsed-field gel electrophoresis. L. monocytogenes occurred throughout the year in all sample types, and the prevalence in MFS increased significantly during the indoor season. L. monocytogenes was more prevalent in MFS (29%) than in BTM (13%) samples. However, the prevalence of L. monocytogenes varied more between farms in samples of MFS (13 to 48%) than in BTM (10 to 16%). For each farm, the L. monocytogenes genotypes detected were classified by persistence (defined as persistent if isolated from ≥3 samples during ≥6 months) and predominance (defined as predominant if >5% prevalence on at least one farm visit). The prevalence of sporadic genotypes was 4 to 5% on all three farms. In contrast, the prevalence of persistent predominant genotypes varied between farms by 4% to 16%. The highest prevalence of persistent predominant genotypes was observed on the farm with the poorest production hygiene. Persistent predominant genotypes were most prevalent on feeding surfaces, water troughs, and floors. Genotypes isolated from the milking system or from cow udders had a greater relative risk of occurring in BTM and MFS than genotypes that only occurred elsewhere in the farm, supporting the hypothesis that L. monocytogenes is transmitted to milk from contamination on the udder surface or in the milking equipment.IMPORTANCEListeria monocytogenes is a ubiquitous environmental bacterium and the causative agent of a serious foodborne illness, listeriosis. Dairy products are common vehicles of listeriosis, and dairy cattle farms harbor L. monocytogenes genotypes associated with human listeriosis outbreaks. Indeed, dairy cattle farms act as a reservoir of L. monocytogenes, and the organism is frequently detected in bulk tank milk (BTM) and in the feces of clinically healthy cows. The ecology of L. monocytogenes in the farm environment is complex and poorly understood. Isolates of the same L. monocytogenes genotype can occur in the farm for years, but the factors contributing to the persistence of genotypes on dairy farms are unknown. Knowledge of the persistence patterns and contamination routes of L. monocytogenes on dairy farms can improve management of the contamination pressure in the farm environment and aid in the development of focused control strategies to reduce BTM contamination.

Neurotoxin synthesis is positively regulated by the sporulation transcription factor Spo0A in Clostridium botulinum type E.

Clostridium botulinum produces the most potent natural toxin, the botulinum neurotoxin (BoNT), probably to create anaerobiosis and nutrients by killing the host, and forms endospores that facilitate survival in harsh conditions and transmission. Peak BoNT production coincides with initiation of sporulation in C. botulinum cultures, which suggests common regulation. Here, we show that Spo0A, the master regulator of sporulation, positively regulates BoNT production. Insertional inactivation of spo0A in C. botulinum type E strain Beluga resulted in significantly reduced BoNT production and in abolished or highly reduced sporulation in relation to wild-type controls. Complementation with spo0A restored BoNT production and sporulation. Recombinant DNA-binding domain of Spo0A directly bound to a putative Spo0A-binding box (CTTCGAA) within the BoNT/E operon promoter, demonstrating direct regulation. Spo0A is the first neurotoxin regulator reported in C. botulinum type E. Unlike other C. botulinum strains that are terrestrial and employ the alternative sigma factor BotR in directing BoNT expression, C. botulinum type E strains are adapted to aquatic ecosystems, possess distinct epidemiology and lack BotR. Our results provide fundamental new knowledge on the genetic control of BoNT production and demonstrate common regulation of BoNT production and sporulation, providing a key intervention point for control.

Screening of the two-component-system histidine kinases of Listeria monocytogenes EGD-e. LiaS is needed for growth under heat, acid, alkali, osmotic, ethanol and oxidative stresses.

To study the role of each two-component system (TCS) histidine kinase (HK) in stress tolerance of Listeria monocytogenes EGD-e, we monitored the growth of individual HK deletion mutant strains under heat (42.5 °C), acid (pH 5.6), alkali (pH 9.4), osmotic (6% NaCl), ethanol (3.5 vol%), and oxidative (5 mM H2O2) stresses. The growth of ΔliaS (Δlmo1021) strain was impaired under each stress, with the most notable decrease under heat and osmotic stresses. The ΔvirS (Δlmo1741) strain showed nearly completely restricted growth at high temperature and impaired growth in ethanol. The growth of ΔagrC (Δlmo0050) strain was impaired under osmotic stress and slightly under oxidative stress. We successfully complemented the HK mutations using a novel allelic exchange based approach. This approach avoided the copy-number problems associated with in trans complementation from a plasmid. The mutant phenotypes were restored to the wild-type level in the complemented strains. This study reveals novel knowledge on the HKs needed for growth of L. monocytogenes EGD-e under abovementioned stress conditions, with LiaS playing multiple roles in stress tolerance of L. monocytogenes EGD-e.

Evaluation of virulence genes in Yersinia enterocolitica strains using SYBR Green real-time PCR.

Yersinia enterocolitica comprises six biotypes 1A, 1B, 2, 3, 4, and 5. The virulence of the strains belonging to biotypes 1B and 2-5 depends on the presence of both chromosomal and plasmid-borne genes. Strains belonging to biotype 1A do not carry the virulence plasmid pYV. However, they carry other virulence genes, such as ystB and hreP. The aim of this study was to investigate the distribution of yadA, virF, inv, ystA, ystB, myfA, hreP and ymoA virulence genes in Y. enterocolitica strains in order to select the target genes that could be used for the development of a probe-specific real-time PCR to determine the presence of Y. enterocolitica in food samples. A total of 161 Y. enterocolitica strains isolated in eight countries and grouped into biotypes 1A, 2 (serotypes O3, O5 and O9), 3 (serotypes O3 and O9) and 4 (serotype O3) were examined for virulence genes. The most common virulence-associated gene in pathogenic Y. enterocolitica proved to be ystA, which can therefore be considered the best target gene to be amplified in order to evaluate the presence of pathogenic biotypes. By contrast, to identify Y. enterocolitica 1A strains, ystB, which codes for the enterotoxin YstB, can be proposed. This has been found in all non-pathogenic biotypes studied, but never in pathogenic biotypes.

Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia.

BACKGROUND: Clostridium botulinum is a diverse group of bacteria characterized by the production of botulinum neurotoxin. Botulinum neurotoxins are classified into serotypes (BoNT/A-G), which are produced by six species/Groups of Clostridia, but the genetic background of the bacteria remains poorly understood. The purpose of this study was to use comparative genomics to provide insights into the genetic diversity and evolutionary history of bacteria that produce the potent botulinum neurotoxin. RESULTS: Comparative genomic analyses of over 170 Clostridia genomes, including our draft genome assemblies for 59 newly sequenced Clostridia strains from six continents and publicly available genomic data, provided in-depth insights into the diversity and distribution of BoNT-producing bacteria. These newly sequenced strains included Group I and II strains that express BoNT/A,/B,/E, or/F as well as bivalent strains. BoNT-producing Clostridia and closely related Clostridia species were delineated with a variety of methods including 16S rRNA gene, concatenated marker genes, core genome and concatenated multi-locus sequencing typing (MLST) gene phylogenies that related whole genome sequenced strains to publicly available strains and sequence types. These analyses illustrated the phylogenetic diversity in each Group and the diversity of genomic backgrounds that express the same toxin type or subtype. Comparisons of the botulinum neurotoxin genes did not identify novel toxin types or variants. CONCLUSIONS: This study represents one of the most comprehensive analyses of whole genome sequence data for Group I and II BoNT-producing strains. Read data and draft genome assemblies generated for 59 isolates will be a resource to the research community. Core genome phylogenies proved to be a powerful tool for differentiating BoNT-producing strains and can provide a framework for the study of these bacteria. Comparative genomic analyses of Clostridia species illustrate the diversity of botulinum-neurotoxin-producing strains and the plasticity of the genomic backgrounds in which bont genes are found.

Large Diversity of Porcine Yersinia enterocolitica 4/O:3 in Eight European Countries Assessed by Multiple-Locus Variable-Number Tandem-Repeat Analysis.

A total of 253 multiple-locus variable-number tandem-repeat analysis (MLVA) types among 634 isolates were discovered while studying the genetic diversity of porcine Yersinia enterocolitica 4/O:3 isolates from eight different European countries. Six variable-number tandem-repeat (VNTR) loci V2A, V4, V5, V6, V7, and V9 were used to study the isolates from 82 farms in Belgium (n = 93, 7 farms), England (n = 41, 8 farms), Estonia (n = 106, 12 farms), Finland (n = 70, 13 farms), Italy (n = 111, 20 farms), Latvia (n = 66, 3 farms), Russia (n = 60, 10 farms), and Spain (n = 87, 9 farms). Cluster analysis revealed mainly country-specific clusters, and only one MLVA type consisting of two isolates was found from two countries: Russia and Italy. Also, farm-specific clusters were discovered, but same MLVA types could also be found from different farms. Analysis of multiple isolates originating either from the same tonsils (n = 4) or from the same farm, but 6 months apart, revealed both identical and different MLVA types. MLVA showed a very good discriminatory ability with a Simpson's discriminatory index (DI) of 0.989. DIs for VNTR loci V2A, V4, V5, V6, V7, and V9 were 0.916, 0.791, 0.901, 0.877, 0.912, and 0.785, respectively, when studying all isolates together, but variation was evident between isolates originating from different countries. Locus V4 in the Spanish isolates and locus V9 in the Latvian isolates did not differentiate (DI 0.000), and locus V9 in the English isolates showed very low discriminatory power (DI 0.049). The porcine Y. enterocolitica 4/O:3 isolates were diverse, but the variation in DI demonstrates that the well discriminating loci V2A, V5, V6, and V7 should be included in MLVA protocol when maximal discriminatory power is needed.

Two-component signal transduction system CBO0787/CBO0786 represses transcription from botulinum neurotoxin promoters in Clostridium botulinum ATCC 3502.

Blocking neurotransmission, botulinum neurotoxin is the most poisonous biological substance known to mankind. Despite its infamy as the scourge of the food industry, the neurotoxin is increasingly used as a pharmaceutical to treat an expanding range of muscle disorders. Whilst neurotoxin expression by the spore-forming bacterium Clostridium botulinum appears tightly regulated, to date only positive regulatory elements, such as the alternative sigma factor BotR, have been implicated in this control. The identification of negative regulators has proven to be elusive. Here, we show that the two-component signal transduction system CBO0787/CBO0786 negatively regulates botulinum neurotoxin expression. Single insertional inactivation of cbo0787 encoding a sensor histidine kinase, or of cbo0786 encoding a response regulator, resulted in significantly elevated neurotoxin gene expression levels and increased neurotoxin production. Recombinant CBO0786 regulator was shown to bind to the conserved -10 site of the core promoters of the ha and ntnh-botA operons, which encode the toxin structural and accessory proteins. Increasing concentration of CBO0786 inhibited BotR-directed transcription from the ha and ntnh-botA promoters, demonstrating direct transcriptional repression of the ha and ntnh-botA operons by CBO0786. Thus, we propose that CBO0786 represses neurotoxin gene expression by blocking BotR-directed transcription from the neurotoxin promoters. This is the first evidence of a negative regulator controlling botulinum neurotoxin production. Understanding the neurotoxin regulatory mechanisms is a major target of the food and pharmaceutical industries alike.

Two-Component-System Histidine Kinases Involved in Growth of Listeria monocytogenes EGD-e at Low Temperatures.

Two-component systems (TCSs) aid bacteria in adapting to a wide variety of stress conditions. While the role of TCS response regulators in the cold tolerance of the psychrotrophic foodborne pathogen Listeria monocytogenes has been demonstrated previously, no comprehensive studies showing the role of TCS histidine kinases of L. monocytogenes at low temperature have been performed. We compared the expression levels of each histidine kinase-encoding gene of L. monocytogenes EGD-e in logarithmic growth phase at 3°C and 37°C, as well as the expression levels 30 min, 3 h, and 7 h after cold shock at 5°C and preceding cold shock (at 37°C). We constructed a deletion mutation in each TCS histidine kinase gene, monitored the growth of the EGD-e wild-type and mutant strains at 3°C and 37°C, and measured the minimum growth temperature of each strain. Two genes, yycG and lisK, proved significant in regard to induced relative expression levels under cold conditions and cold-sensitive mutant phenotypes. Moreover, the ΔresE mutant showed a lower growth rate than that of wild-type EGD-e at 3°C. Eleven other genes showed upregulated gene expression but revealed no cold-sensitive phenotypes. The results show that the histidine kinases encoded by yycG and lisK are important for the growth and adaptation of L. monocytogenes EGD-e at low temperature.

Role of csp genes in NaCl, pH, and ethanol stress response and motility in Clostridium botulinum ATCC 3502.

Clostridium botulinum is a notable food pathogen and responsible for botulism due to production of botulinum neurotoxin. Strains of C. botulinum can adapt to and survive in stress conditions and food processing. The cold shock protein coding genes (csp) are involved in growth at low temperature, but they may also possess other functions. In this mutational analysis we show that cspB and cspC, but not cspA, are important for NaCl, pH and ethanol stress responses and for motility of C. botulinum ATCC 3502. In all NaCl concentrations tested, the cspB mutant had lower maximum growth rate and, together with the cspC mutant, a longer lag phase compared to the wild-type strain. At low pH, the cspB and cspC mutants showed either lower maximum growth rates or longer lag phases compared to the wild type. In all ethanol concentrations tested, the cspB mutant had lower maximum growth rates and the cspC mutant had a longer lag phase than the wild-type strain. Motility was reduced in cspA and cspC mutants, and flagella formation was affected. The results suggest that cspB plays a universal role in stress response and cspC aids C. botulinum in NaCl, pH and ethanol stress in C. botulinum ATCC 3502.

The ubiquitous nature of Listeria monocytogenes clones: a large-scale Multilocus Sequence Typing study.

Listeria monocytogenes is ubiquitously prevalent in natural environments and is transmitted via the food chain to animals and humans, in whom it can cause life-threatening diseases. We used Multilocus Sequence Typing (MLST) of ∼2000 isolates of L. monocytogenes to investigate whether specific associations existed between clonal complexes (CCs) and the environment versus diseased hosts. Most CCs (72%) were not specific for any single source, and many have been isolated from the environment, food products, animals as well as from humans. Our results confirm that the population structure of L. monocytogenes is largely clonal and consists of four lineages (I-IV), three of which contain multiple CCs. Most CCs have remained stable for decades, but one epidemic clone (CC101) was common in the mid-1950s and very rare until recently when it may have begun to re-emerge. The historical perspective used here indicates that the central sequence types of CCs were not ancestral founders but have rather simply increased in frequency over decades.

Positive regulation of botulinum neurotoxin gene expression by CodY in Clostridium botulinum ATCC 3502.

Botulinum neurotoxin, produced mainly by the spore-forming bacterium Clostridium botulinum, is the most poisonous biological substance known. Here, we show that CodY, a global regulator conserved in low-G+C Gram-positive bacteria, positively regulates the botulinum neurotoxin gene expression. Inactivation of codY resulted in decreased expression of botA, encoding the neurotoxin, as well as in reduced neurotoxin synthesis. Complementation of the codY mutation in trans rescued neurotoxin synthesis, and overexpression of codY in trans caused elevated neurotoxin production. Recombinant CodY was found to bind to a 30-bp region containing the botA transcription start site, suggesting regulation of the neurotoxin gene transcription through direct interaction. GTP enhanced the binding affinity of CodY to the botA promoter, suggesting that CodY-dependent neurotoxin regulation is associated with nutritional status.