BACKGROUND
enterohemorrhagic escherichia coli , a subgroup of shiga toxin  producing e. coli , are enteric pathogens frequently causing severe illness in humans.
ehec infection may lead to non-bloody and bloody diarrhea and most dangerously, the extraintestinal complication hemolytic uremic syndrome   <cit> .
in order to cause gastrointestinal disease, bacteria must pass through the acidic gastric barrier.
when taking into account the apparent low infectious dose of enterohemorrhagic e. coli, which may be as little as  <dig> viable organisms  <cit> , it becomes obvious that acid resistance is an important virulence trait of ehec.
investigation of the complex mechanisms conferring acid tolerance onto e. coli has revealed many new insights in the last years.
currently, four different acid stress protection systems are being discussed.
at least three of these are controlled by σs, an alternative sigma subunit of rna polymerase encoded by the rpos gene  <cit> .
σs, which is seen as a master regulator of general stress response, confers promoter specificity to the core rna polymerase and is activated under a variety of stress conditions, as well as during stationary phase  <cit> .
the glutamate-dependent acid resistance  system is believed to provide best protection for bacterial cells below ph  <dig>  <cit> .
bhagwat et al.
have recently reported, that in natural populations of pathogenic e. coli mutations in gade exist which, in addition to mutant alleles of rpos, may contribute to varying acid resistance phenotypes of ehec  <cit> .
gade encodes the gade protein, a regulatory molecule of the gdar system  <cit> .
in  <dig> small et al.
already reported that the growth ph is important for expression of acid resistance in e. coli  <cit> .
waterman et al.
who have investigated a large set of stec for acid resistance  <cit> , identified defective rpos genes as cause for loss of acid resistance in individual e. coli isolates.
recently it was demonstrated by king et al., that modulation of genome usage enables regulatory diversity which contributes to strain variation in e. coli  <cit> .
a similar observation was reported by bhagwat et al., who investigated stress tolerance of ehec and could observe a functional heterogeneity of rpos  <cit> .
our study was initially focused on analyzing the role of σs in acid stress behavior of two different ehec isolates used as model organisms in our laboratory: the sequenced prototype o157:h <dig> ehec edl <dig>   <cit>  and a very well characterized ehec o26:h <dig> isolate from a hus patient  <cit> .
we expected these results to improve the interpretation of data obtained from in vitro and in vivo virulence experiments investigating pathogen-host interactions.
using homologous recombination and suicide vector technique we constructed unmarked isogenic rpos deletion mutants of both strains.
resistance of wild type organisms and their mutants towards acidic conditions at ph  <dig>  and  <dig>  were tested as described by lange et al.
<cit>  with slight modifications.
while the ehec o26:h <dig> patient isolate was highly dependent on a functional rpos gene for an adequate response to acid stress, surprisingly, deletion of rpos in ehec edl <dig> had no measurable effect when compared to its wild type strain.
further investigation revealed a point mutation in the ehec edl <dig> rpos gene which leads to a taa stop codon being responsible for this phenotype.
we then evaluated another  <dig> isolates of human and porcine origin and functionally characterized a subgroup of five selected rpos genes.
surprisingly, σs activity of stec ed- <dig>  <cit>  appeared to be dependent on a yet unknown regulatory mechanism that modulated its activity.
in the following, we could also observe a similar phenomenon with the well characterized probiotic e. coli nissle  <dig>  <cit> .
RESULTS
construction and confirmation of unmarked isogenic rpos deletion mutants
after pcr screening of potential mutants one rpos negative isolate of each ehec wild type strain, edl933a and  <dig> , was subjected to further testing by southern blotting.
they both showed dna fragments of the expected size, after restriction digest of their genomic dna with either xmni or stui  thereby indicating the correct insertion of the rpos deletion mutation into the genome of the two ehec strains.
the mutants were termed e. coli mhh933- <dig> and mhh126- <dig> respectively.
additionally, the mutation was confirmed by dna sequencing of a pcr product generated with primers rpos  <dig> and rpos  <dig>  antibiotic resistance patterns of the mutants did not differ from their parental strains.
biochemical reaction profiles of all strains, as determined with api  <dig> e strips, were quite similar .
ehec edl933a and its mutant, which both could not ferment sorbitol, were identified as  <dig>  % e. coli according to their api profile index "5144172".
sorbitol positive ehec  <dig> and mutant strain e. coli mhh126- <dig> generated the profile index "5144562" which was  <dig>  % specific for e. coli.
additionally, all e. coli o157:h <dig> isolates were able to ferment rhamnose, while the o26:h <dig> strains were not.
after  <dig> h treatment with  <dig> mm hydrogen peroxide no viable bacteria could be recovered from cultures of e. coli mhh933- <dig>  mhh126- <dig> and ehec edl933a, while ehec  <dig> and edl933b  still produced more than  <dig> % colony forming units  compared to the blank value .
acid resistance assays
the strains became acid resistant from od <dig>  <dig>  for ehec  <dig> and from od <dig>  <dig>  for ehec edl933a, respectively.
ehec  <dig> very effectively responded to acid stress  and showed resistance rates up to  <dig> % at ph  <dig> , indicating bacterial growth at this low ph, and  <dig> % at ph  <dig> .
in contrast, ehec edl933a was only moderately acid resistant at ph  <dig>  as was its isogenic Δrpos mutant .
the survival of these strains was about  <dig> % at most, which appeared to be independent of the rpos genotype.
the behavior of a second clone of ehec edl933a, obtained from atcc, was identical .
ehec edl933b however, was nearly as acid resistant as ehec  <dig> .
furthermore, e. coli mhh126- <dig> was completely unable to survive acidic growth conditions at ph  <dig>  and ph  <dig>  .
by complementation of e. coli mhh126- <dig> with psc <dig> bearing its own rpos gene, a wild type like phenotype could be restored.
at ph  <dig>  it was even more resistant than the parental organism.
at ph  <dig>  its survival was still about  <dig> % .
when complemented with psc <dig>  harboring rpos*, the rpos gene from ehec edl933a, e. coli mhh126- <dig> was completely sensitive to acidic growth conditions further on .
however, when e. coli mhh126- <dig> was transformed with plasmid pmh <dig>  containing an rpos* allele cured by site directed mutagenesis from the taa stop codon at position  <dig>  its acid resistance increased to more than  <dig> % .
thus, we could unequivocally prove that the point mutation g721t in rpos* was solely responsible for the acid sensitive phenotype of ehec edl933a.
homology comparison of rpos genes
homology of all sequenced rpos genes was compared with the software package bioedit .
in the following all nucleotide positions are enumerated as described in materials and methods.
we again sequenced the rpos gene of ehec edl933b and used it as reference for all comparisons since the respective nucleotide data from the genome sequence nc_ <dig> had two sequencing errors, g57c and g61c.
the main rpos promoter rposp is located within the nlpd gene at position - <dig> to - <dig>  the  <dig> and the - <dig> region are positioned at - <dig> to - <dig> and - <dig> to - <dig> respectively.
both, the highly acid resistant ehec strain  <dig> as well as the stec isolate e-d <dig> had the point mutation g-570a, located in between the  <dig> region and rposp.
furthermore, these strains had point mutation a-521g leading to an amino acid exchange from threonine to alanine in nlpd.
ehec  <dig> and  <dig>  stec e-d <dig> and e-d <dig> and ecn also showed mutations t-306c in the nlpd gene, as well as a543c in rpos.
with the exception of ehec  <dig>  these strains were mutated at position t387c as well.
ehec  <dig> and the two stec strains e-d <dig> and e-d <dig> also carried mutation a819g.
furthermore, ehec  <dig> and stec e-d <dig> had the additional mutation g-465a in nlpd.
stec e-d <dig> showed four more point mutations: c-472t, which leads to an amino acid exchange from threonine to isoleucine in nlpd, c-293t, g-183t and a-162g.
ecn carried the nucleotide exchange c-463t resulting in isoleucine instead of threonine in nlpd.
furthermore, the rpos gene of ecn had point mutations t171c, c272t, t365g, t470c, t581c, and c995t.
except for the three amino acid exchanges in nlpd described above, all other point mutations observed in either rpos or nlpd of all e. coli strains investigated were silent.
in contrast, ehec 86- <dig> had an  <dig> bp duplicate sequence  in rpos beginning at position  <dig> which caused a shift in the open reading frame and a stop codon  <dig> bp later.
functional analysis of further rpos genes
all experiments are illustrated in figure  <dig>  plasmids pud <dig>  pud <dig> and pud <dig> conferred a highly acid resistant phenotype on to e. coli mhh126- <dig>  comparable to the respective wild type organisms ehec edl933b, ehec  <dig> and stec e-d <dig>  as expected, complementation with pud <dig> did not mediate acid resistance to e. coli mhh126- <dig>  this behavior was comparable to the parental strain ehec 86- <dig>  however, when the test strain e. coli mhh126- <dig> was transformed with pud <dig>  surprisingly it became strongly acid resistant.
this was in sharp contrast to the manner of the corresponding wild type strain stec e-d <dig>  which only showed an acid resistance ≤  <dig>  %.
this response becomes comprehensible considering the unaffected open reading frame in the rpos sequence of pud <dig>  to confirm this effect we constructed pud <dig>  a second independent plasmid containing rpos from stec e-d <dig>  with this plasmid the acid resistance of complemented e. coli mhh126- <dig> was identical to pud <dig>  interestingly, we observed a similar phenomenon when we investigated acid tolerance of probiotic ecn.
while survival of the wild type organism after ph  <dig>  treatment was only around  <dig> %, test strain e. coli mhh126- <dig> became fully acid resistant, when complemented with plasmid pds <dig> containing rpos of ecn .
discussion
gastric acid is a natural barrier that all bacteria entering the lower intestine have to pass.
with ph values ranging between  <dig>  and  <dig> , the stomach is one of the most inhospitable areas in the human body.
a correlation between an infectious dose of enterobacteriaceae and their capacity to withstand acidic conditions is well known.
with respect to the very low infectious dose of ehec, of about  <dig> to  <dig> organisms  <cit> , it becomes obvious, that acid resistance is a key factor for virulence of these bacteria.
e. coli have developed elegant regulatory systems, that enable their survival under such conditions  <cit> .
the alternative sigma factor σs is instrumental in the regulation of acid protection mechanisms in this species of enterobacteriacae  <cit> .
in this study we have investigated the role of the rpos genotype on the acid stress response of a set of shiga toxin producing e. coli as well as the widely used probiotic e. coli strain nissle  <dig>  <cit> .
we first constructed unmarked isogenic rpos deletion mutants of the highly virulent ehec patient isolate  <dig>  <cit>  and of the completely sequenced prototype ehec edl <dig>  <cit> , labeled ehec edl933a in this study.
the patient isolate was highly dependent on a functional rpos gene for an adequate response to acid stress.
by complementation of its rpos deletion mutant e. coli mhh126- <dig> with the rpos gene from ehec  <dig> a wild type like phenotype could be restored.
surprisingly, ehec edl933a and its mutant did not exhibit major differences regarding their acid tolerance, but both appeared to be σs defective.
indeed, when we sequenced rpos* from ehec edl933a we could identify a point mutation that caused a premature stop codon which was in conflict with the published database sequence  <cit> .
in order to rule out that this mutation was an artifact generated in our laboratory, we purchased a second isolate of sequenced ehec edl <dig>  it carried the identical point mutation, which may have occurred during passage prior to storage of the isolate at the strain collection.
allelic variations in the rpos gene are not uncommon since it is localized in a highly mutable region of the e. coli genome  <cit> .
in a large study waterman et al.
identified ehec strains that were defective in their response to low ph  <cit> .
they attributed this phenotype to a non functional σs as a consequence of rpos mutations.
phenotypical characterization of ehec edl933b, a further clone of edl <dig>  and complementation experiments clearly showed that the stop codon in rpos* causally determined the stress phenotype of atcc strain  <dig>  when considering the significant impact of σs in the regulatory network of e. coli, which controls up to  <dig> % of the e. coli genes directly or indirectly  <cit> , this observation is of importance for those working in the field of ehec and planning to do both, in vitro or in vivo studies with this particular sequenced isolate.
further investigation of six rpos genes from shiga toxin producing e. coli and a probiotic e. coli strain revealed that σs activity is not always dependent on the rpos genotype.
despite having a functional rpos gene, as shown by complementation experiments in a Δrpos background, stec e-d <dig> as well as ecn behaved σs defective regarding their acid resistance.
while stec e-d <dig> was completely sensitive to acid stress, probiotic ecn exhibited about  <dig> % survival in the same test environment.
with respect to ecn, this observation adds to an actual study of bhagwat et al.
where the authors observed functional heterogeneity of σs in food-borne and clinical ehec isolates  <cit> .
however, in stec e-d <dig> as well as in ecn, mutations in the gene encoding the gdar system regulator gade  <cit>  have to be ruled out.
such mutations have been reported by bhagwat et al.
as another reason for attenuated acid resistance in e. coli wild type isolates  <cit> .
with regard to the varying σs activities in individual e. coli strains observed in our study, it should be mentioned that all isolates analyzed here carried glutamate at codon  <dig>  resulting in σs .
this seems to account for a higher variability in rpos related phenotypes as recently described by atlung et al.
<cit> .
price et al.
have shown recently, that ehec make use of their different acid resistance systems depending on the type of acid stress they are exposed to  <cit> .
in either case σs was important for a sufficient acid stress response of ehec edl <dig>  in vitro as well as in vivo  <cit> .
it should be investigated though, whether this impact of σs on the in vivo acid resistance of ehec also allows assumptions about the virulence of a particular strain.
in our laboratory environment for instance wild type ehec 86- <dig> was highly virulent in an oral infection model with gnotobiotic piglets  <cit> , although it has a non functional rpos gene and is only weakly acid resistant as shown above.
krogfelt et al.
have published an accomplished experiment which clearly demonstrates that rpos gene function may be a disadvantage for e. coli colonizing the intestine  <cit> .
the authors therefore conclude that the benefit of a functional rpos regulon for e. coli depends on the actual growth phase of a particular strain.
the apparent down regulation of σs activity in stec e-d <dig> and ecn in our study seems to be another way of adapting the rpos regulon to specific growth conditions.
we have performed further experiments to investigate the impact of rpos in ecn on regulation of potential host probiotic marker genes which we have recently identified  <cit> .
our long-term objective is to establish to what extent e. coli is able to modulate pathogenic but also beneficial properties using its rpos regulatory network.
CONCLUSIONS
the results of our study clearly confirm the central role of σs as a key regulator for acid resistance in stec and ecn.
when interpreting in vitro or in vivo data generated with ehec edl933a it is important to realize, that this prototype ehec has an attenuated σs phenotype.
we could also show that rpos gene function is modified in singular e. coli isolates by regulatory mechanisms that lead to an altered σs activity, as exemplified by stec strain e-d <dig> and probiotic ecn.
this is in line with the observations regarding functional heterogeneity of rpos in stress tolerance of k- <dig> e. coli strains and ehec isolates, king et al.
and bhagwat et al.
have reported  <cit> , and expands them to a commensal e. coli with beneficial traits.

 enterohemorrhagic escherichia coli, a subgroup of shiga toxin producing e. coli, are enteric pathogens frequently causing severe illness in humans. ehec infection may lead to non-bloody and bloody diarrhea and most dangerously, the extraintestinal complication hemolytic uremic syndrome. in order to cause gastrointestinal disease, bacteria must pass through the acidic gastric barrier. when taking into account the apparent low infectious dose of enterohemorrhagic e. coli, which may be as little as viable organisms, it becomes obvious that acid resistance is an important virulence trait of ehec. investigation of the complex mechanisms conferring acid tolerance onto e. coli has revealed many new insights in the last years. currently, four different acid stress protection systems are being discussed. at least three of these are controlled by σs, an alternative sigma subunit of rna polymerase encoded by the rpos gene. σs, which is seen as a master regulator of general stress response, confers promoter specificity to the core rna polymerase and is activated under a variety of stress conditions, as well as during stationary phase. the glutamate-dependent acid resistance system is believed to provide best protection for bacterial cells below ph. bhagwat et al. have recently reported, that in natural populations of pathogenic e. coli mutations in gade exist which, in addition to mutant alleles of rpos, may contribute to varying acid resistance phenotypes of ehec. gade encodes the gade protein, a regulatory molecule of the gdar system. in small et al. already reported that the growth ph is important for expression of acid resistance in e. coli. waterman et al. who have investigated a large set of stec for acid resistance, identified defective rpos genes as cause for loss of acid resistance in individual e. coli isolates. recently it was demonstrated by king et al., that modulation of genome usage enables regulatory diversity which contributes to strain variation in e. coli. a similar observation was reported by bhagwat et al., who investigated stress tolerance of ehec and could observe a functional heterogeneity of rpos. our study was initially focused on analyzing the role of σs in acid stress behavior of two different ehec isolates used as model organisms in our laboratory: the sequenced prototype o157:h ehec edl and a very well characterized ehec o26:h isolate from a hus patient. we expected these results to improve the interpretation of data obtained from in vitro and in vivo virulence experiments investigating pathogen-host interactions. using homologous recombination and suicide vector technique we constructed unmarked isogenic rpos deletion mutants of both strains. resistance of wild type organisms and their mutants towards acidic conditions at ph and were tested as described by lange et al. with slight modifications. while the ehec o26:h patient isolate was highly dependent on a functional rpos gene for an adequate response to acid stress, surprisingly, deletion of rpos in ehec edl had no measurable effect when compared to its wild type strain. further investigation revealed a point mutation in the ehec edl rpos gene which leads to a taa stop codon being responsible for this phenotype. we then evaluated another isolates of human and porcine origin and functionally characterized a subgroup of five selected rpos genes. surprisingly, σs activity of stec ed- appeared to be dependent on a yet unknown regulatory mechanism that modulated its activity. in the following, we could also observe a similar phenomenon with the well characterized probiotic e. coli nissle.  construction and confirmation of unmarked isogenic rpos deletion mutants after pcr screening of potential mutants one rpos negative isolate of each ehec wild type strain, edl933a and, was subjected to further testing by southern blotting. they both showed dna fragments of the expected size, after restriction digest of their genomic dna with either xmni or stui thereby indicating the correct insertion of the rpos deletion mutation into the genome of the two ehec strains. the mutants were termed e. coli mhh933- and mhh126- respectively. additionally, the mutation was confirmed by dna sequencing of a pcr product generated with primers rpos and rpos antibiotic resistance patterns of the mutants did not differ from their parental strains. biochemical reaction profiles of all strains, as determined with api e strips, were quite similar. ehec edl933a and its mutant, which both could not ferment sorbitol, were identified as % e. coli according to their api profile index "5144172". sorbitol positive ehec and mutant strain e. coli mhh126- generated the profile index "5144562" which was % specific for e. coli. additionally, all e. coli o157:h isolates were able to ferment rhamnose, while the o26:h strains were not. after h treatment with mm hydrogen peroxide no viable bacteria could be recovered from cultures of e. coli mhh933- mhh126- and ehec edl933a, while ehec and edl933b still produced more than % colony forming units compared to the blank value. acid resistance assays the strains became acid resistant from od for ehec and from od for ehec edl933a, respectively. ehec very effectively responded to acid stress and showed resistance rates up to % at ph, indicating bacterial growth at this low ph, and % at ph. in contrast, ehec edl933a was only moderately acid resistant at ph as was its isogenic Δrpos mutant. the survival of these strains was about % at most, which appeared to be independent of the rpos genotype. the behavior of a second clone of ehec edl933a, obtained from atcc, was identical. ehec edl933b however, was nearly as acid resistant as ehec. furthermore, e. coli mhh126- was completely unable to survive acidic growth conditions at ph and ph. by complementation of e. coli mhh126- with psc bearing its own rpos gene, a wild type like phenotype could be restored. at ph it was even more resistant than the parental organism. at ph its survival was still about %. when complemented with psc harboring rpos*, the rpos gene from ehec edl933a, e. coli mhh126- was completely sensitive to acidic growth conditions further on. however, when e. coli mhh126- was transformed with plasmid pmh containing an rpos* allele cured by site directed mutagenesis from the taa stop codon at position its acid resistance increased to more than %. thus, we could unequivocally prove that the point mutation g721t in rpos* was solely responsible for the acid sensitive phenotype of ehec edl933a. homology comparison of rpos genes homology of all sequenced rpos genes was compared with the software package bioedit. in the following all nucleotide positions are enumerated as described in materials and methods. we again sequenced the rpos gene of ehec edl933b and used it as reference for all comparisons since the respective nucleotide data from the genome sequence nc_ had two sequencing errors, g57c and g61c. the main rpos promoter rposp is located within the nlpd gene at position - to - the and the - region are positioned at - to - and - to - respectively. both, the highly acid resistant ehec strain as well as the stec isolate e-d had the point mutation g-570a, located in between the region and rposp. furthermore, these strains had point mutation a-521g leading to an amino acid exchange from threonine to alanine in nlpd. ehec and stec e-d and e-d and ecn also showed mutations t-306c in the nlpd gene, as well as a543c in rpos. with the exception of ehec these strains were mutated at position t387c as well. ehec and the two stec strains e-d and e-d also carried mutation a819g. furthermore, ehec and stec e-d had the additional mutation g-465a in nlpd. stec e-d showed four more point mutations: c-472t, which leads to an amino acid exchange from threonine to isoleucine in nlpd, c-293t, g-183t and a-162g. ecn carried the nucleotide exchange c-463t resulting in isoleucine instead of threonine in nlpd. furthermore, the rpos gene of ecn had point mutations t171c, c272t, t365g, t470c, t581c, and c995t. except for the three amino acid exchanges in nlpd described above, all other point mutations observed in either rpos or nlpd of all e. coli strains investigated were silent. in contrast, ehec 86- had an bp duplicate sequence in rpos beginning at position which caused a shift in the open reading frame and a stop codon bp later. functional analysis of further rpos genes all experiments are illustrated in figure plasmids pud pud and pud conferred a highly acid resistant phenotype on to e. coli mhh126- comparable to the respective wild type organisms ehec edl933b, ehec and stec e-d as expected, complementation with pud did not mediate acid resistance to e. coli mhh126- this behavior was comparable to the parental strain ehec 86- however, when the test strain e. coli mhh126- was transformed with pud surprisingly it became strongly acid resistant. this was in sharp contrast to the manner of the corresponding wild type strain stec e-d which only showed an acid resistance ≤ %. this response becomes comprehensible considering the unaffected open reading frame in the rpos sequence of pud to confirm this effect we constructed pud a second independent plasmid containing rpos from stec e-d with this plasmid the acid resistance of complemented e. coli mhh126- was identical to pud interestingly, we observed a similar phenomenon when we investigated acid tolerance of probiotic ecn. while survival of the wild type organism after ph treatment was only around %, test strain e. coli mhh126- became fully acid resistant, when complemented with plasmid pds containing rpos of ecn. discussion gastric acid is a natural barrier that all bacteria entering the lower intestine have to pass. with ph values ranging between and, the stomach is one of the most inhospitable areas in the human body. a correlation between an infectious dose of enterobacteriaceae and their capacity to withstand acidic conditions is well known. with respect to the very low infectious dose of ehec, of about to organisms, it becomes obvious, that acid resistance is a key factor for virulence of these bacteria. e. coli have developed elegant regulatory systems, that enable their survival under such conditions. the alternative sigma factor σs is instrumental in the regulation of acid protection mechanisms in this species of enterobacteriacae. in this study we have investigated the role of the rpos genotype on the acid stress response of a set of shiga toxin producing e. coli as well as the widely used probiotic e. coli strain nissle. we first constructed unmarked isogenic rpos deletion mutants of the highly virulent ehec patient isolate and of the completely sequenced prototype ehec edl, labeled ehec edl933a in this study. the patient isolate was highly dependent on a functional rpos gene for an adequate response to acid stress. by complementation of its rpos deletion mutant e. coli mhh126- with the rpos gene from ehec a wild type like phenotype could be restored. surprisingly, ehec edl933a and its mutant did not exhibit major differences regarding their acid tolerance, but both appeared to be σs defective. indeed, when we sequenced rpos* from ehec edl933a we could identify a point mutation that caused a premature stop codon which was in conflict with the published database sequence. in order to rule out that this mutation was an artifact generated in our laboratory, we purchased a second isolate of sequenced ehec edl it carried the identical point mutation, which may have occurred during passage prior to storage of the isolate at the strain collection. allelic variations in the rpos gene are not uncommon since it is localized in a highly mutable region of the e. coli genome. in a large study waterman et al. identified ehec strains that were defective in their response to low ph. they attributed this phenotype to a non functional σs as a consequence of rpos mutations. phenotypical characterization of ehec edl933b, a further clone of edl and complementation experiments clearly showed that the stop codon in rpos* causally determined the stress phenotype of atcc strain when considering the significant impact of σs in the regulatory network of e. coli, which controls up to % of the e. coli genes directly or indirectly, this observation is of importance for those working in the field of ehec and planning to do both, in vitro or in vivo studies with this particular sequenced isolate. further investigation of six rpos genes from shiga toxin producing e. coli and a probiotic e. coli strain revealed that σs activity is not always dependent on the rpos genotype. despite having a functional rpos gene, as shown by complementation experiments in a Δrpos background, stec e-d as well as ecn behaved σs defective regarding their acid resistance. while stec e-d was completely sensitive to acid stress, probiotic ecn exhibited about % survival in the same test environment. with respect to ecn, this observation adds to an actual study of bhagwat et al. where the authors observed functional heterogeneity of σs in food-borne and clinical ehec isolates. however, in stec e-d as well as in ecn, mutations in the gene encoding the gdar system regulator gade have to be ruled out. such mutations have been reported by bhagwat et al. as another reason for attenuated acid resistance in e. coli wild type isolates. with regard to the varying σs activities in individual e. coli strains observed in our study, it should be mentioned that all isolates analyzed here carried glutamate at codon resulting in σs. this seems to account for a higher variability in rpos related phenotypes as recently described by atlung et al.. price et al. have shown recently, that ehec make use of their different acid resistance systems depending on the type of acid stress they are exposed to. in either case σs was important for a sufficient acid stress response of ehec edl in vitro as well as in vivo. it should be investigated though, whether this impact of σs on the in vivo acid resistance of ehec also allows assumptions about the virulence of a particular strain. in our laboratory environment for instance wild type ehec 86- was highly virulent in an oral infection model with gnotobiotic piglets, although it has a non functional rpos gene and is only weakly acid resistant as shown above. krogfelt et al. have published an accomplished experiment which clearly demonstrates that rpos gene function may be a disadvantage for e. coli colonizing the intestine. the authors therefore conclude that the benefit of a functional rpos regulon for e. coli depends on the actual growth phase of a particular strain. the apparent down regulation of σs activity in stec e-d and ecn in our study seems to be another way of adapting the rpos regulon to specific growth conditions. we have performed further experiments to investigate the impact of rpos in ecn on regulation of potential host probiotic marker genes which we have recently identified. our long-term objective is to establish to what extent e. coli is able to modulate pathogenic but also beneficial properties using its rpos regulatory network.  the results of our study clearly confirm the central role of σs as a key regulator for acid resistance in stec and ecn. when interpreting in vitro or in vivo data generated with ehec edl933a it is important to realize, that this prototype ehec has an attenuated σs phenotype. we could also show that rpos gene function is modified in singular e. coli isolates by regulatory mechanisms that lead to an altered σs activity, as exemplified by stec strain e-d and probiotic ecn. this is in line with the observations regarding functional heterogeneity of rpos in stress tolerance of k- e. coli strains and ehec isolates, king et al. and bhagwat et al. have reported, and expands them to a commensal e. coli with beneficial traits. 
it should also be a relevant characteristic of e. coli strains used for therapeutic purposes such as the probiotic e. coli nissle. in e. coli and related enteric bacteria it has been extensively demonstrated, that the alternative sigma factor σs, encoded by the rpos gene, acts as a master regulator mediating resistance to various environmental stress factors. using rpos deletion mutants of a highly virulent ehec o26:h patient isolate and the sequenced prototype ehec edl of serotype o157:h we investigated the impact of a functional rpos gene for orchestrating a satisfactory response to acid stress in these strains. we then functionally characterized rpos of probiotic ecn and five rpos genes selected from stec isolates pre-investigated for acid resistance. moreover, to our surprise, one stec strain as well as ecn was acid sensitive in our test environment, although their cloned rpos genes could effectively complement acid sensitivity of an rpos deletion mutant. enterohemorrhagic e. coli, a subgroup of shiga toxin producing e. coli, may cause severe enteritis and hemolytic uremic syndrome and is transmitted orally via contaminated foods or from person to person. furthermore our data supports recently published observations, that individual e. coli isolates are able to significantly modulate their acid resistance phenotype independent of their rpos genotype. the attenuation of sequenced ehec edl might be of importance for anyone planning to do either in vitro or in vivo studies with this prototype strain. acid resistance therefore is an important mechanism of ehec virulence. first, we found out that atcc isolate of ehec edl has a point mutation in rpos, not present in the published sequence, leading to a premature stop codon. the infectious dose is known to be very low, which requires most of the bacteria to survive the gastric acid barrier.
 enterohemorrhagic e. coli, a subgroup of shiga toxin producing e. coli, may cause severe enteritis and hemolytic uremic syndrome and is transmitted orally via contaminated foods or from person to person. the infectious dose is known to be very low, which requires most of the bacteria to survive the gastric acid barrier. acid resistance therefore is an important mechanism of ehec virulence. it should also be a relevant characteristic of e. coli strains used for therapeutic purposes such as the probiotic e. coli nissle. in e. coli and related enteric bacteria it has been extensively demonstrated, that the alternative sigma factor σs, encoded by the rpos gene, acts as a master regulator mediating resistance to various environmental stress factors. methods using rpos deletion mutants of a highly virulent ehec o26:h patient isolate and the sequenced prototype ehec edl of serotype o157:h we investigated the impact of a functional rpos gene for orchestrating a satisfactory response to acid stress in these strains. we then functionally characterized rpos of probiotic ecn and five rpos genes selected from stec isolates pre-investigated for acid resistance.  first, we found out that atcc isolate of ehec edl has a point mutation in rpos, not present in the published sequence, leading to a premature stop codon. moreover, to our surprise, one stec strain as well as ecn was acid sensitive in our test environment, although their cloned rpos genes could effectively complement acid sensitivity of an rpos deletion mutant.  the attenuation of sequenced ehec edl might be of importance for anyone planning to do either in vitro or in vivo studies with this prototype strain. furthermore our data supports recently published observations, that individual e. coli isolates are able to significantly modulate their acid resistance phenotype independent of their rpos genotype. 