Synthesis of biologically active Shiga toxins in cell-free systems.

Shiga toxins (Stx) produced by pathogenic bacteria can cause mild to severe diseases in humans. Thus, the analysis of such toxins is of utmost importance. As an AB5 toxin, Stx consist of a catalytic A-subunit acting as a ribosome-inactivating protein (RIP) and a B-pentamer binding domain. In this study we synthesized the subunits and holotoxins from Stx and Stx2a using different cell-free systems, namely an E. coli- and CHO-based cell-free protein synthesis (CFPS) system. The functional activity of the protein toxins was analyzed in two ways. First, activity of the A-subunits was assessed using an in vitro protein inhibition assay. StxA produced in an E. coli cell-free system showed significant RIP activity at concentrations of 0.02 nM, whereas toxins synthesized in a CHO cell-free system revealed significant activity at concentrations of 0.2 nM. Cell-free synthesized StxA2a was compared to StxA2a expressed in E. coli cells. Cell-based StxA2a had to be added at concentrations of 20 to 200 nM to yield a significant RIP activity. Furthermore, holotoxin analysis on cultured HeLa cells using an O-propargyl-puromycin assay showed significant protein translation reduction at concentrations of 10 nM and 5 nM for cell-free synthesized toxins derived from E. coli and CHO systems, respectively. Overall, these results show that Stx can be synthesized using different cell-free systems while remaining functionally active. In addition, we were able to use CFPS to assess the activity of different Stx variants which can further be used for RIPs in general.

Rapid Acidification and Off-Flavor Reduction of Pea Protein by Fermentation with Lactic Acid Bacteria and Yeasts.

Pea protein is widely used as an alternative protein source in plant-based products. In the current study, we fermented pea protein to reduce off-flavor compounds, such as hexanal, and to produce a suitable fermentate for further processing. Laboratory fermentations using 5% (w/v) pea protein suspension were carried out using four selected lactic acid bacteria (LAB) strains, investigating their growth and acidification capabilities in pea protein. Rapid acidification of pea protein was achieved with Lactococcus lactis subsp. lactis strain LTH 7123. Next, this strain was co-inoculated together with either the yeasts Kluyveromyces lactis LTH 7165, Yarrowia lipolytica LTH 6056, or Kluyveromyces marxianus LTH 6039. Fermentation products of the mixed starter cultures and of the single strains were further analyzed by gas chromatography coupled with mass spectrometry to quantify selected volatile flavor compounds. Fermentation with L. lactis LTH 7123 led to an increase in compounds associated with the "beany" off-flavors of peas, including hexanal. However, significant reduction in those compounds was achieved after fermentation with Y. lipolytica LTH 6056 with or without L. lactis LTH 7123. Thus, fermentation using co-cultures of LAB and yeasts strains could prove to be a valuable method for enhancing quality attributes of pea protein-based products.

Sporadic Detection of Escherichia coli O104:H4 Strain C227/11Φcu in the Edible Parts of Lamb's Lettuce Cultured in Contaminated Agricultural Soil Samples.

In the current study, we demonstrate that E. coli O104:H4 strain C227/11Φcu, a derivative of the 2011 enterohemorrhagic/enteroaggregative (EHEC/EAEC) E. coli outbreak strain, migrated into the edible portion of lamb's lettuce plants upon contamination of the surrounding soil. Seeds were surface-sterilized and cultivated on Murashige-Skoog agar or in autoclaved agricultural soil. Migration into the edible portions was investigated by inoculating the agar or soil close to the plants with 108 colony-forming units (CFU). The edible parts, which did not come into contact with the contaminated medium or soil, were quantitatively analyzed for the presence of bacteria after 2, 4 and 8 weeks. Strain C227/11Φcu could colonize lamb's lettuce when contamination of medium or soil occurs. The highest recovery rate (27%) was found for lettuce cultivated in agar, and up to 1.6 × 103 CFU/g lettuce was detected. The recovery rate was lower for the soil samples (9% and 13.5%). Although the used contamination levels were high, migration of C227/11Φcu from the soil into the edible parts was demonstrated. This study further highlights the risk of crop plant contamination with pathogenic E. coli upon soil contamination.

Broad time-dependent transcriptional activity of metabolic genes of E. coli O104:H4 strain C227/11Φcu in a soil microenvironment at low temperature.

In the current study, metabolic genes and networks that influence the persistence of pathogenic Escherichia coli O104:H4 strain C227/11Φcu in agricultural soil microenvironments at low temperature were investigated. The strain was incubated in alluvial loam (AL) and total RNA was prepared from samples at time point 0, and after 1 and 4 weeks. Differential transcriptomic analysis was performed by RNA sequencing analysis and values obtained at weeks 1 and 4 were compared to those of time point 0. We found differential expression of more than 1500 genes for either time point comparison. The two lists of differentially expressed genes were then subjected to gene set enrichment of Gene Ontology terms. In total, 17 GO gene sets and 3 Pfam domains were found to be enriched after 1 week. After 4 weeks, 17 GO gene sets and 7 Pfam domains were statistically enriched. Especially stress response genes and genes of the primary metabolism were particularly affected at both time points. Genes and gene sets for uptake of carbohydrates, amino acids were strongly upregulated, indicating adjustment to a low nutrient environment. The results of this transcriptome analysis show that persistence of C227/11Φcu in soils is associated with a complex interplay of metabolic networks.

Genome-wide association reveals host-specific genomic traits in Escherichia coli.

BACKGROUND: Escherichia coli is an opportunistic pathogen which colonizes various host species. However, to what extent genetic lineages of E. coli are adapted or restricted to specific hosts and the genomic determinants of such adaptation or restriction is poorly understood. RESULTS: We randomly sampled E. coli isolates from four countries (Germany, UK, Spain, and Vietnam), obtained from five host species (human, pig, cattle, chicken, and wild boar) over 16 years, from both healthy and diseased hosts, to construct a collection of 1198 whole-genome sequenced E. coli isolates. We identified associations between specific E. coli lineages and the host from which they were isolated. A genome-wide association study (GWAS) identified several E. coli genes that were associated with human, cattle, or chicken hosts, whereas no genes associated with the pig host could be found. In silico characterization of nine contiguous genes (collectively designated as nan-9) associated with the human host indicated that these genes are involved in the metabolism of sialic acids (Sia). In contrast, the previously described sialic acid regulon known as sialoregulon (i.e. nanRATEK-yhcH, nanXY, and nanCMS) was not associated with any host species. In vitro growth experiments with a Δnan-9 E. coli mutant strain, using the sialic acids 5-N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) as sole carbon source, showed impaired growth behaviour compared to the wild-type. CONCLUSIONS: This study provides an extensive analysis of genetic determinants which may contribute to host specificity in E. coli. Our findings should inform risk analysis and epidemiological monitoring of (antimicrobial resistant) E. coli.

The Prophage and Us-Shiga Toxin Phages Revisited.

The authors first met in 1998 at the University of Würzburg, Germany, at the Institute of Hygiene and Microbiology, in Helge Karch's lab, where Herbert Schmidt worked as a PostDoc and Maite Muniesa visited the lab for a postdoctoral research stay to work on phages encoding Shiga toxin 2e (Stx2e) [...].

A Robust One-Step Recombineering System for Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) can cause severe diarrheic in humans. To improve therapy options, a better understanding of EHEC pathogenicity is essential. The genetic manipulation of EHEC with classical one-step methods, such as the transient overexpression of the phage lambda (λ) Red functions, is not very efficient. Here, we provide a robust and reliable method for increasing recombineering efficiency in EHEC based on the transient coexpression of recX together with gam, beta, and exo. We demonstrate that the genetic manipulation is 3-4 times more efficient in EHEC O157:H7 EDL933 Δstx1/2 with our method when compared to the overexpression of the λ Red functions alone. Both recombineering systems demonstrated similar efficiencies in Escherichia coli K-12 MG1655. Coexpression of recX did not enhance the Gam-mediated inhibition of sparfloxacin-mediated SOS response. Therefore, the additional inhibition of the RecFOR pathway rather than a stronger inhibition of the RecBCD pathway of SOS response induction might have resulted in the increased recombineering efficiency by indirectly blocking phage induction. Even though additional experiments are required to unravel the precise mechanistic details of the improved recombineering efficiency, we recommend the use of our method for the robust genetic manipulation of EHEC and other prophage-carrying E. coli isolates.

Novel Aspects of the SubA Subunit of the Subtilase Cytotoxin.

The subtilase cytotoxin (SubAB) belongs to the family of AB5 toxins and is produced together with Shiga toxin (Stx) by certain Stx-producing E. coli strains (STEC). For most AB-type toxins, it is assumed that cytotoxic effects can only be induced by a complete holotoxin complex consisting of SubA and SubB. However, it has been shown for SubAB that the enzymatically active subunit SubA, without its transport and binding domain SubB, induces cell death in different eukaryotic cell lines. Interestingly, the molecular structure of SubA resembles that of the SubAB complex. SubA alone is capable of binding to cells and then being taken up autonomously. Once inside the host cell, SubA is transported, similar to the SubAB holotoxin, via a retrograde transport into the endoplasmatic reticulum (ER). In the ER, it exhibits its enzymatic activity by cleaving the chaperone BiP/GRP78 and thereby triggering cell death. Therefore, the existence of toxic single SubA subunits that have not found a B-pentamer for holotoxin assembly might improve the pathogenic potential of subtilase-producing strains. Moreover, from a pharmacological aspect, SubA might be an interesting molecule for the targeted transport of therapeutic molecules into the ER, in order to investigate and specifically modulate processes in the context of ER stress-associated diseases. Since recent studies on bacterial AB5 toxins contributed mainly to the understanding of the biology of AB-type holotoxins, this mini-review specifically focus on that recently observed single A-effect of the subtilase cytotoxin and addresses whether a fundamental shift of the traditional AB5 paradigm might be required.

Survival of Enterohemorrhagic Escherichia coli O104:H4 Strain C227/11Φcu in Agricultural Soils Depends on rpoS and Environmental Factors.

The consumption of contaminated fresh produce caused outbreaks of enterohemorrhagic (EHEC) Escherichia coli. Agricultural soil might be a reservoir for EHEC strains and represent a contamination source for edible plants. Furthermore, the application of manure as fertilizer is an important contamination route. Thus, the German fertilizer ordinance prohibits the use of manure 12 weeks before crop harvest to avoid pathogen transmission into the food chain. In this study, the survival of E. coli O104:H4 strain C227/11Φcu in soil microenvironments with either diluvial sand or alluvial loam at two temperatures was investigated for more than 12 weeks. It was analyzed whether the addition of cattle manure extends EHEC survival in these microenvironments. The experiments were additionally performed with isogenic ΔrpoS and ΔfliC deletion mutants of C227/11Φcu. The survival of C227/11Φcu was highest at 4 °C, whereas the soil type had a minor influence. The addition of cattle manure increased the survival at 22 °C. Deletion of rpoS significantly decreased the survival period under all cultivation conditions, whereas fliC deletion did not have any influence. The results of our study demonstrate that EHEC C227/11Φcu is able to survive for more than 12 weeks in soil microenvironments and that RpoS is an important determinant for survival.

Effects of Protein, Calcium, and pH on Gene Transcription, Cell-Envelope Peptidase Activity of Lactococcus lactis Strains, and the Formation of Bitter Peptides.

Calcium- and protein-rich fermented milk products, such as concentrated yoghurts and fresh cheeses, may contain undesired bitter peptides, which are generated by the proteolytic cleavage of casein. Up to now, it is not clear whether this process is caused by endogenous milk enzymes, such as plasmin and cathepsin D, or whether proteolytic enzymes from applied starter cultures, such as the lactococcal cell-envelope peptidase PrtP, are involved. A sensory analysis of fresh cheese products made from milk concentrates fermented with prtP-negative and -positive Lactococcus lactis strains revealed bitterness in the products fermented with prtP-positive L. lactis strains. Two prtP-positive strains, LTH 7122 and LTH 7123, were selected to investigate the effect of increased calcium concentrations (additional 5 mM and 50 mM CaCl2) at neutral (pH 6.6) and acidic (pH 5.5) pH-values on the transcription of the prtP gene and its corresponding PrtP peptidase activity in milk citrate broth (MCB). For both strains, it was shown that prtP transcription was upregulated only under slightly elevated calcium conditions (5 mM CaCl2) after 5 h of growth. In concordance with these findings, PrtP peptidase activity also increased. When higher concentrations of calcium were used (50 mM), prtP expression of both strains decreased strongly by more than 50%. Moreover, PrtP peptidase activity of strain LTH 7123 decreased by 15%, but enzymatic activity of strain LTH 7122 increased slightly during growth under elevated calcium concentrations (50 mM CaCl2). Fermentations of reconstituted casein medium with 3.4% (w/v) and 8.5% (w/v) protein and different calcium concentrations using strain LTH 7122 revealed no clear relationship between prtP transcription and calcium or protein concentration. However, an increase in PrtP peptidase activity under elevated protein and calcium conditions was observed. The activity increase was accompanied by increased levels of bitter peptides derived from different casein fractions. These findings could be a possible explanation for the bitterness in fermented milk concentrates that was detected by a trained bitter panel.

Cytotoxic Effects of Recombinant StxA2-His in the Absence of Its Corresponding B-Subunit.

AB5 protein toxins are produced by certain bacterial pathogens and are composed of an enzymatically active A-subunit and a B-subunit pentamer, the latter being responsible for cell receptor recognition, cellular uptake, and transport of the A-subunit into the cytosol of eukaryotic target cells. Two members of the AB5 toxin family were described in Shiga toxin-producing Escherichia coli (STEC), namely Shiga toxin (Stx) and subtilase cytotoxin (SubAB). The functional paradigm of AB toxins includes the B-subunit being mandatory for the uptake of the toxin into its target cells. Recent studies have shown that this paradigm cannot be maintained for SubAB, since SubA alone was demonstrated to intoxicate human epithelial cells in vitro. In the current study, we raised the hypothesis that this may also be true for the A-subunit of the most clinically relevant Stx-variant, Stx2a. After separate expression and purification, the recombinant Stx2a subunits StxA2a-His and StxB2a-His were applied either alone or in combination in a 1:5 molar ratio to Vero B4, HeLa, and HCT-116 cells. For all cell lines, a cytotoxic effect of StxA2a-His alone was detected. Competition experiments with Stx and SubAB subunits in combination revealed that the intoxication of StxA2a-His was reduced by addition of SubB1-His. This study showed that the enzymatic subunit StxA2a alone was active on different cells and might therefore play a yet unknown role in STEC disease development.

Bacteriophages of Shiga Toxin-Producing Escherichia coli and Their Contribution to Pathogenicity.

Shiga toxins (Stx) of Shiga toxin-producing Escherichia coli (STEC) are generally encoded in the genome of lambdoid bacteriophages, which spend the most time of their life cycle integrated as prophages in specific sites of the bacterial chromosome. Upon spontaneous induction or induction by chemical or physical stimuli, the stx genes are co-transcribed together with the late phase genes of the prophages. After being assembled in the cytoplasm, and after host cell lysis, mature bacteriophage particles are released into the environment, together with Stx. As members of the group of lambdoid phages, Stx phages share many genetic features with the archetypical temperate phage Lambda, but are heterogeneous in their DNA sequences due to frequent recombination events. In addition to Stx phages, the genome of pathogenic STEC bacteria may contain numerous prophages, which are either cryptic or functional. These prophages may carry foreign genes, some of them related to virulence, besides those necessary for the phage life cycle. Since the production of one or more Stx is considered the major pathogenicity factor of STEC, we aim to highlight the new insights on the contribution of Stx phages and other STEC phages to pathogenicity.

The enzyme subunit SubA of Shiga toxin-producing E. coli strains demonstrates comparable intracellular transport and cytotoxic activity as the holotoxin SubAB in HeLa and HCT116 cells in vitro.

The subtilase cytotoxin (SubAB) is secreted by certain Shiga toxin-producing Escherichia coli (STEC) strains and is composed of the enzymatically active subunit SubA and the pentameric binding/transport subunit SubB. We previously demonstrated that SubA (10 µg/ml), in the absence of SubB, binds and intoxicates the human cervix cancer-derived epithelial cell line HeLa. However, the cellular and molecular mechanisms underlying the cytotoxic activity of SubA in the absence of SubB remained unclear. In the present study, the cytotoxic effects mediated by SubA alone were investigated in more detail in HeLa cells and the human colon cancer cell line HCT116. We found that in the absence of SubB, SubA (10 µg/ml) is internalized into the endoplasmic reticulum (ER), where it cleaves the chaperone GRP78, an already known substrate for SubA after its canonical uptake into cells via SubB. The autonomous cellular uptake of SubA and subsequent cleavage of GRP78 in cells is prevented by treatment of cells with 10 µM brefeldin A, which inhibits the transport of protein toxins into the ER. In addition, by analyzing the SubA mutant SubAΔC344, we identified the C-terminal SEEL motif as an ER-targeting signal. Conclusively, our results strongly suggest that SubA alone shares the same intracellular transport route and cytotoxic activity as the SubAB holotoxin.

Removal of the innocuity test from The International Pharmacopoeia and WHO recommendations for vaccines and biological products.

The innocuity test was indicated as a quality control test to release pharmaceutical and biological products to the market. The test was intended to detect possible extraneous toxic contaminants derived from the manufacturing processes of the product. The test was included in WHO Recommendations and Guidelines for vaccines, biotherapeutics and blood products and in some monographs on antibiotics in The International Pharmacopoeia. Over the past years, the requirements in WHO Recommendations/Guidelines for conducting the test evolved such that it could be waived for routine release of product once consistency of production was established to the satisfaction of the NRA, or that the need for this test should be discussed and agreed with the NRA. However, some users of WHO written standards for biologicals (i.e., Recommendations, Guidelines) and WHO specifications for pharmaceuticals (i.e., The International Pharmacopoeia) requested that the innocuity test be deleted from WHO written standards based on its lack of specificity and scientific relevance. In response to that request, we studied the history of this test and its use by the member states of WHO, and the recommendations in WHO written standards. The outcomes of the study were reviewed by the relevant WHO Expert Committee on Biological Standardization and Expert Committee on Specifications for Pharmaceutical Products who then decided to discontinue this test in WHO Recommendations for vaccines and biologicals and to omit the test from The International Pharmacopoeia.

How bacterial pathogens of the gastrointestinal tract use the mucosal glyco-code to harness mucus and microbiota: New ways to study an ancient bag of tricks.

During the last decades, the flourishing scientific field of molecular pathogenesis brought groundbreaking knowledge of the mechanisms of pathogenicity and the underlying bacterial virulence factors to cause infectious diseases. However, a major paradigm shift is currently occurring after it became increasingly evident that bacterial-host and host-host cell interactions including immune responses orchestrated by defined virulence factors are not the sole drivers of infectious disease development. Strong evidence has been collected that information and nutrient flow within complex microbial communities, as well as to and from host cells and matrices are equally important for successful infection. This particularly holds true for gastrointestinal (GI) pathogens and the GI microbiota interacting and communicating with each other as well as with the host GI mucus and mucosa. Gut-adapted pathogens appear to have developed powerful and specific strategies to interact with human GI mucus including the microbiota for nutrient acquisition, mucosal adhesion, inter-species communication and traversing the mucus barrier. This review covers the existing evidence on these topics and explores the mutual dynamics of host GI mucus, the mucosal habitat and incoming acute and chronic pathogens during GI infections. A particular focus is placed on the role of carbohydrates in diverse mucosal interaction, communication and competition processes. Novel techniques to analyze and synthesize mucus-derived carbohydrates and to generate mucus mimetics are introduced. Finally, open questions and future objectives for pathogen - host GI mucus research will be discussed.

Plant variety and soil type influence Escherichia coli O104:H4 strain C227/11ϕcu adherence to and internalization into the roots of lettuce plants.

Human disease outbreaks caused by pathogenic Escherichia coli are increasingly associated with the consumption of contaminated fresh produce. Internalization of enteroaggregative/enterohemorrhagic E. coli (EAEC/EHEC) strains into plant tissues may present a serious threat to public health. In the current study, the ability of the fluorescing Shiga toxin-negative E. coli O104:H4 strain C227/11ϕcu/pKEC2 to adhere to and to internalize into the roots of Lactuca sativa and Valerianella locusta grown in diluvial sand (DS) and alluvial loam (AL) was investigated. In parallel, the soil microbiota was analyzed by partial 16S rRNA gene sequencing. The experiments were performed in a safety level 3 greenhouse to simulate agricultural practice. The adherence of C227/11ϕcu/pKEC2 to the roots of both plant varieties was increased by at least a factor three after incubation in DS compared to AL. Compared to V. locusta, internalization into the roots of L. sativa was increased 12-fold in DS and 108-fold in AL. This demonstrates that the plant variety had an impact on the internalization ability, whereas for a given plant variety the soil type also affected bacterial internalization. In addition, microbiota analysis detected the inoculated strain and showed large differences in the bacterial composition between the soil types.

Variants of Escherichia coli Subtilase Cytotoxin Subunits Show Differences in Complex Formation In Vitro.

The subtilase cytotoxin (SubAB) of Shiga toxin-producing Escherichia coli (STEC) is a member of the AB5 toxin family. In the current study, we analyzed the formation of active homo- and hetero-complexes of SubAB variants in vitro to characterize the mode of assembly of the subunits. Recombinant SubA1-His, SubB1-His, SubA2-2-His, and SubB2-2-His subunits, and His-tag-free SubA2-2 were separately expressed, purified, and biochemically characterized by circular dichroism (CD) spectroscopy, size-exclusion chromatography (SEC), and analytical ultracentrifugation (aUC). To confirm their biological activity, cytotoxicity assays were performed with HeLa cells. The formation of AB5 complexes was investigated with aUC and isothermal titration calorimetry (ITC). Binding of SubAB2-2-His to HeLa cells was characterized with flow cytometry (FACS). Cytotoxicity experiments revealed that the analyzed recombinant subtilase subunits were biochemically functional and capable of intoxicating HeLa cells. Inhibition of cytotoxicity by Brefeldin A demonstrated that the cleavage is specific. All His-tagged subunits, as well as the non-tagged SubA2-2 subunit, showed the expected secondary structural compositions and oligomerization. Whereas SubAB1-His complexes could be reconstituted in solution, and revealed a Kd value of 3.9 ± 0.8 µmol/L in the lower micromolar range, only transient interactions were observed for the subunits of SubAB2-2-His in solution, which did not result in any binding constant when analyzed with ITC. Additional studies on the binding characteristics of SubAB2-2-His on HeLa cells revealed that the formation of transient complexes improved binding to the target cells. Conclusively, we hypothesize that SubAB variants exhibit different characteristics in their binding behavior to their target cells.

Genetics, Toxicity, and Distribution of Enterohemorrhagic Escherichia coli Hemolysin.

The ability to produce enterohemolysin is regarded as a potential virulence factor for enterohemorrhagic Escherichia coli (EHEC) and is frequently associated with severe human diseases such as hemorrhagic colitis (HC) and the hemolytic uremic syndrome (HUS). The responsible toxin, which has also been termed EHEC-hemolysin (EHEC-Hly, syn. Ehx), belongs to the Repeats in Toxin (RTX)-family of pore-forming cytolysins and is characterized by the formation of incomplete turbid lysis zones on blood agar plates containing defibrinated sheep erythrocytes. Besides the expression of Shiga toxins (Stx) and the locus of enterocyte effacement (LEE), EHEC-Hly is a commonly used marker for the detection of potential pathogenic E. coli strains, although its exact role in pathogenesis is not completely understood. Based on the current knowledge of EHEC-Hly, this review describes the influence of various regulator proteins, explains the different mechanisms leading to damage of target cells, discusses the diagnostic role, and gives an insight of the prevalence and genetic evolution of the toxin.

Differential transcriptome analysis of enterohemorrhagic Escherichia coli strains reveals differences in response to plant-derived compounds.

BACKGROUND: Several serious vegetable-associated outbreaks of enterohemorrhagic Escherichia coli (EHEC) infections have occurred during the last decades. In this context, vegetables have been suggested to function as secondary reservoirs for EHEC strains. Increased knowledge about the interaction of EHEC with plants including gene expression patterns in response to plant-derived compounds is required. In the current study, EHEC O157:H7 strain Sakai, EHEC O157:H- strain 3072/96, and the EHEC/enteroaggregative E. coli (EAEC) hybrid O104:H4 strain C227-11φcu were grown in lamb's lettuce medium and in M9 minimal medium to study the differential transcriptional response of these strains to plant-derived compounds with RNA-Seq technology. RESULTS: Many genes involved in carbohydrate degradation and peptide utilization were similarly upregulated in all three strains, suggesting that the lamb's lettuce medium provides sufficient nutrients for proliferation. In particular, the genes galET and rbsAC involved in galactose metabolism and D-ribose catabolism, respectively, were uniformly upregulated in the investigated strains. The most prominent differences in shared genome transcript levels were observed for genes involved in the expression of flagella. Transcripts of all three classes of the flagellar hierarchy were highly abundant in strain C227-11φcu. Strain Sakai expressed only genes encoding the basal flagellar structure. In addition, both strains showed increased motility in presence of lamb's lettuce extract. Moreover, strain 3072/96 showed increased transcription activity for genes encoding the type III secretion system (T3SS) including effectors, and was identified as a powerful biofilm-producer in M9 minimal medium. CONCLUSION: The current study provides clear evidence that EHEC and EHEC/EAEC strains are able to adjust their gene expression patterns towards metabolization of plant-derived compounds, demonstrating that they may proliferate well in a plant-associated environment. Moreover, we propose that flagella and other surface structures play a fundamental role in the interaction of EHEC and EHEC/EAEC with plants.

Transcription of the Subtilase Cytotoxin Gene subAB1 in Shiga Toxin-Producing Escherichia coli Is Dependent on hfq and hns.

Certain foodborne Shiga toxin-producing Escherichia coli (STEC) strains carry genes encoding the subtilase cytotoxin (SubAB). Although the mode of action of SubAB is under intensive investigation, information about the regulation of subAB gene expression is currently not available. In this study, we investigated the regulation of the chromosomal subAB1 gene in laboratory E. coli strain DH5α and STEC O113:H21 strain TS18/08 using a luciferase reporter gene assay. Special emphasis was given to the role of the global regulatory protein genes hfq and hns in subAB1 promoter activity. Subsequently, quantitative real-time PCR was performed to analyze the expression of Shiga toxin 2a (Stx2a), SubAB1, and cytolethal distending toxin V (Cdt-V) genes in STEC strain TS18/08 and its isogenic hfq and hns deletion mutants. The deletion of hfq led to a significant increase of up to 2-fold in subAB1 expression, especially in the late growth phase, in both strains. However, deletion of hns showed different effects on the promoter activity during the early and late exponential growth phases in both strains. Furthermore, upregulation of stx2a and cdt-V was demonstrated in hfq and hns deletion mutants in TS18/08. These data showed that the expression of subAB1, stx2a, and cdt-V is integrated in the regulatory network of global regulators Hfq and H-NS in Escherichia coliIMPORTANCE Shiga toxin-producing Escherichia coli (STEC) strains are responsible for outbreaks of foodborne diseases, such as hemorrhagic colitis and the hemolytic uremic syndrome. The pathogenicity of those strains can be attributed to, among other factors, the production of toxins. Recently, the subtilase cytotoxin was detected in locus of enterocyte effacement (LEE)-negative STEC, and it was confirmed that it contributes to the cytotoxicity of those STEC strains. Although the mode of action of SubAB1 is under intensive investigation, the regulation of gene expression is currently not known. The global regulatory proteins H-NS and Hfq have impact on many cellular processes and have been described to regulate virulence factors as well. Here, we investigate the role of hns and hfq in expression of subAB1 as well as stx2a and cdt-V in an E. coli laboratory strain as well as in wild-type STEC strain TS18/08.