Alternatives to antibiotics in a One Health context and the role genomics can play in reducing antimicrobial use.

BACKGROUND: This review follows on from the International Conference on One Health Antimicrobial Resistance (ICOHAR 2019), where strategies to improve the fundamental understanding and management of antimicrobial resistance at the interface between humans, animals and the environment were discussed. OBJECTIVE: This review identifies alternatives to antimicrobials in a One Health context, noting how advances in genomic technologies are assisting their development and enabling more targeted use of antimicrobials. SOURCES: Key articles on the use of microbiota modulation, livestock breeding and gene editing, vaccination, antivirulence strategies and bacteriophage therapy are discussed. CONTENT: Antimicrobials are central for disease control, but reducing their use is paramount as a result of the rise of transmissible antimicrobial resistance. This review discusses antimicrobial alternatives in the context of improved understanding of fundamental host-pathogen and microbiota interactions using genomic tools. IMPLICATIONS: Host and microbial genomics and other novel technologies play an important role in devising disease control strategies for healthier animals and humans that in turn reduce our reliance on antimicrobials.

British Escherichia coli O157 in Cattle Study (BECS): to determine the prevalence of E. coli O157 in herds with cattle destined for the food chain.

Escherichia coli O157 are zoonotic bacteria for which cattle are an important reservoir. Prevalence estimates for E. coli O157 in British cattle for human consumption are over 10 years old. A new baseline is needed to inform current human health risk. The British E. coli O157 in Cattle Study (BECS) ran between September 2014 and November 2015 on 270 farms across Scotland and England & Wales. This is the first study to be conducted contemporaneously across Great Britain, thus enabling comparison between Scotland and England & Wales. Herd-level prevalence estimates for E. coli O157 did not differ significantly for Scotland (0·236, 95% CI 0·166-0·325) and England & Wales (0·213, 95% CI 0·156-0·283) (P = 0·65). The majority of isolates were verocytotoxin positive. A higher proportion of samples from Scotland were in the super-shedder category, though there was no difference between the surveys in the likelihood of a positive farm having at least one super-shedder sample. E. coli O157 continues to be common in British beef cattle, reaffirming public health policy that contact with cattle and their environments is a potential infection source.

Bacteriophages as pathogens and immune modulators?

While Shiga toxins (Stx) are key determinants of enterohemorrhagic Escherichia coli (EHEC) pathophysiology in humans, their dissemination to target organs following gastrointestinal EHEC infection is still poorly understood. Most types of Stx target cells with globotriaosylceramide (Gb3) receptors, which are expressed on endothelial cells. According to current theory, Stx is trafficked on the surface of peripheral blood cells, and transfer of toxin from these trafficking cells to endothelial cells results in microvascular damage to target organs, including the kidneys and brain. Inside the cell, Stx inhibits protein synthesis, resulting in cell death. Host "repair" responses can lead to microthrombus formation, erythrocyte damage, and reduced oxygen supply, potentially resulting in organ failure. A recent study [L. V. Bentancor et al., mBio 4(5):e00501-13, 2013, doi:10.1128/mBio.00501-13] indicates that another mechanism for Stx "dissemination" needs to be considered. Bentancor et al. demonstrated that high-pressure injection of a plasmid encoding the "prokaryotic" Stx2 sequence into mice can lead to mortality, with pathology indicative of Stx activity and antibody responses to Stx. While the plasmid levels and injection methodology were extreme, the study indicates that these sequences are potentially taken up into eukaryotic cells, transcribed, and translated, producing active Stx. Stx genes are present on integrated bacteriophage genomes in EHEC, and Stx-encoding phages are released following bacterial lysis in the gastrointestinal tract. We therefore need to consider whether bacteriophage sequences can be expressed in eukaryotic cells, what the wider implications are for our understanding of many "bacterial" diseases, and the possibility of developing novel interventions that target bacteriophages.

Associations between the presence of virulence determinants and the epidemiology and ecology of zoonotic Escherichia coli.

The severity of human infection with pathogenic Escherichia coli depends on two major virulence determinants (eae and stx) that, respectively, produce intimin and Shiga toxin. In cattle, both may enhance colonization, but whether this increases fitness by enhancing cattle-to-cattle transmission in the field is unknown. In E. coli O157, the almost uniform presence of the virulence determinants in cattle isolates prevents comparative analysis. The availability to this study of extensive non-O157 E. coli data, with much greater diversity in carriage of virulence determinants, provides the opportunity to gain insight into their potential impact on transmission. Dynamic models were used to simulate expected prevalence distributions for serogroups O26 and O103. Transmission parameters were estimated by fitting model outputs to prevalence data from Scottish cattle using a Bayesian Markov chain Monte Carlo (MCMC) approach. Despite similar prevalence distributions for O26 and O103, their transmission dynamics were distinct. Serogroup O26 strains appear well adapted to the cattle host. The dynamics are characterized by a basic reproduction ratio (R(0)) of >1 (allowing sustained cattle-to-cattle transmission), a relatively low transmission rate from environmental reservoirs, and substantial association with eae on transmission. The presence of stx(2) was associated with reduced transmission. In contrast, serogroup O103 appears better adapted to the noncattle environment, characterized by an R(0) value of <1 for plausible test sensitivities, a significantly higher transmission rate from noncattle sources than serogroup O26, and an absence of fitness benefits associated with the carriage of eae. Thus, the association of eae with enhanced transmission depends on the E. coli serogroup. Our results suggest that the capacity of E. coli strains to derive fitness benefits from virulence determinants influences the prevalence in the cattle population and the ecology and epidemiology of the host organism.

Heterogeneous shedding of Escherichia coli O157 in cattle and its implications for control.

Identification of the relative importance of within- and between-host variability in infectiousness and the impact of these heterogeneities on the transmission dynamics of infectious agents can enable efficient targeting of control measures. Cattle, a major reservoir host for the zoonotic pathogen Escherichia coli O157, are known to exhibit a high degree of heterogeneity in bacterial shedding densities. By relating bacterial count to infectiousness and fitting dynamic epidemiological models to prevalence data from a cross-sectional survey of cattle farms in Scotland, we identify a robust pattern: approximately 80% of the transmission arises from the 20% most infectious individuals. We examine potential control options under a range of assumptions about within- and between-host variability in infection dynamics. Our results show that the within-herd basic reproduction ratio, R(0), could be reduced to <1 with targeted measures aimed at preventing infection in the 5% of individuals with the highest overall infectiousness. Alternatively, interventions such as vaccination or the use of probiotics that aim to reduce bacterial carriage could produce dramatic reductions in R(0) by preventing carriage at concentrations corresponding to the top few percent of the observed range of counts. We conclude that a greater understanding of the cause of the heterogeneity in bacterial carriage could lead to highly efficient control measures to reduce the prevalence of E. coli O157.

Phenotypic and functional characterisation of follicle-associated epithelium of rectal lymphoid tissue.

Lymphoid follicles cluster in the terminal rectum of various animal species and of man and hence this site may be important in the development of immune responses to pathogens. For the induction of immune responses at mucosal sites, interplay is required between various cell types performing functions ranging from antigen-sampling cells via antigen-presenting cells to antigen-specific lymphocytes. Therefore, we have characterised the cell populations and relevant functioning of follicle-associated epithelium (FAE) and associated follicles in the terminal portion of rectum in cattle as a representative mammal. Immunohistochemical studies of this region identified immune cell subsets (CD4+, CD8+, WC 1+gammadelta, CD2+, CD 21+ and CD 40+ cells) characteristic of an immune-inductive site. Examination of FAE identified a subset of cells with structural and functional features of antigen-sampling M-cells. Cells of the FAE and adjacent follicle-associated crypts expressed vimentin and a subset of these cells internalised microparticles, a further attribute of M-cells. The FAE cells were phenotypically heterogeneous and therefore the function and phenotype of these cell subsets requires further characterisation, particularly with respect to their potentially important role in the interaction of hosts with pathogens and the development of immune responses.

Type III secretion of the Salmonella effector protein SopE is mediated via an N-terminal amino acid signal and not an mRNA sequence.

Type III secretion systems (TTSS) are virulence-associated components of many gram-negative bacteria that translocate bacterial proteins directly from the bacterial cytoplasm into the host cell. The Salmonella translocated effector protein SopE has no consensus cleavable amino-terminal secretion sequence, and the mechanism leading to its secretion through the Salmonella pathogenicity island 1 (SPI-1) TTSS is still not fully understood. There is evidence from other bacteria which suggests that the TTSS signal may reside within the 5' untranslated region (UTR) of the mRNA of secreted effectors. We investigated the role of the 5' UTR in the SPI-1 TTSS-mediated secretion of SopE using promoter fusions and obtained data indicating that the mRNA sequence is not involved in the secretion process. To clarify the proteinaceous versus RNA nature of the signal, we constructed frameshift mutations in the amino-terminal region of SopE of Salmonella enterica serovar Typhimurium SL1344. Only constructs with the native amino acid sequence were secreted, highlighting the importance of the amino acid sequence versus the mRNA sequence for secretion. Additionally, we obtained frameshift mutation data suggesting that the first 15 amino acids are important for secretion of SopE independent of the presence of the chaperone binding site. These data shed light on the nature of the signal for SopE secretion and highlight the importance of the amino-terminal amino acids for correct targeting and secretion of SopE via the SPI-1-encoded TTSS during host cell invasion.

Characterization of Escherichia coli strains causing urinary tract infections in patients with transposed intestinal segments.

PURPOSE: Transposition of intestinal segments into the urinary tract predisposes to urinary tract infections. We characterized bacterial infections in these patients and examined the virulence genotype and persistence of Escherichia coli isolates. MATERIALS AND METHODS: We followed 26 patients who underwent bladder reconstructive surgery using transposed intestinal segments. E. coli strains isolated from the urine of these patients were genotyped for established virulence determinants and the frequency of carriage was compared with E. coli strains isolated from community acquired urinary infections and the fecal flora of anonymous volunteers. A longitudinal study of E. coli strains in 9 patients was also done using pulsed field gel electrophoresis. RESULTS: E. coli was the most frequently isolated organism, responsible for 59% (62 of 105) of monobacterial infections. Other bacteria isolated included Klebsiella species, Proteus species and Enterococcus faecalis. Community acquired E. coli strains were more likely to carry multiple determinants for particular adhesins (P and S fimbriae) and toxins (alpha-hemolysin and cytotoxic necrotizing factor) than fecal strains. Carriage frequency for bladder reconstruction strains was intermediary and not significantly different. The key finding was that E. coli strains persisted for prolonged periods, including 2 years in certain patients, often despite various antimicrobial treatments. CONCLUSIONS: This study highlights that further steps must be taken to prevent and treat urinary tract infections in this susceptible group. Particular attention should be given to the treatment of persistent infections.

Regulation, secretion and activity of type III-secreted proteins of enterohaemorrhagic Escherichia coli O157.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 causes gastrointestinal disease with the potential for life-threatening sequelae. Although Shiga-like toxins are responsible for much of the serious pathology in humans, the bacterium also possesses a type III protein secretion system that is responsible for intimate attachment to host intestinal mucosa. This sophisticated interaction requires co-ordination that is governed by environmental and genetic factors. Ongoing research supports the following model for how EHEC enables and controls this process: (i) specific environmental cues that are present in the host result in the expression of a number of adhesins, including fimbriae, which allow the initial binding to the mucosal surface. The same conditions support the expression of the basal type III secretion apparatus; (ii) targeting and assembly of the translocon requires both an mRNA signal and chaperones, with coupled translation and secretion of translocon proteins, EspA, B and D; (iii) opening up of a conduit between the bacterium and host cell releases a cytoplasmic pool of effector proteins. A consequence of this is increased expression of particular effector proteins. Potentially, different proteins could be released into the cell at different times or have activities modulated with time; (iv) intimate contact between the translocated intimin receptor (Tir) and the bacterial surface factor intimin requires translocon expression to be down-regulated and translocon filaments to be lost. Fluorescent protein fusions allow contact-mediated regulation and protein targeting through the type III secretion system to be studied in detail.

PapB paralogues and their effect on the phase variation of type 1 fimbriae in Escherichia coli.

Recent work has demonstrated that expression of type 1 fimbriae is repressed by PapB, a regulator of pyelonephritis-associated pili (P-pili). PapB belongs to family of related adhesin regulators, for which consensus residues required for DNA binding and oligomerization have been identified. Of the regulators tested in this study, PapB, SfaB (S-fimbriae) and PefB (Salmonella enterica serovar Typhimurium--plasmid-encoded fimbriae) repressed FimB-promoted off-to-on inversion of the fim switch, although complete repression was only demonstrated by PapB. DaaA, FaeB, FanA, FanB and ClpB had no effect on fim switching. In addition, only PapB stimulated FimE-promoted on-to-off inversion. Deletion analysis demonstrated that this specificity resides in the carboxy terminal of the protein, and not the amino terminal, with the central region being homologous among the family members. Exchange of Leu(82) and Ile(83) of PapB for the equivalent residues from the DaaA protein (Phe and Gln) within the carboxy terminal virtually abolished cross-talk activity. Whereas PapB can bind to a region around the left inverted repeat of the fim switch, DaaA and the PapB double mutant were effectively unable to bind this region. A previously characterized PapB DNA binding mutant also failed to bind to this region and failed to inhibit FimB activity at the fim switch. Thus, repression of fim expression appears unique to PapB and SfaB within E. coli and requires DNA binding involving amino acid residues located both within the homologous core and in the heterogeneous carboxy terminus. The variation in the carboxy terminus between the PapB family members explains their differential effects on fim. This mechanism of cross-talk seems restricted to the P and S family adhesins with type 1 fimbriae and may ensure variable and sequential expression of adhesins during urinary tract infections.

Differences in levels of secreted locus of enterocyte effacement proteins between human disease-associated and bovine Escherichia coli O157.

Ongoing extensive epidemiological studies of verotoxin-carrying Escherichia coli O157 (stx(+) eae(+)) have shown this bacterial pathogen to be common in cattle herds in the United States and the United Kingdom. However, the incidence of disease in humans due to this pathogen is still very low. This study set out to investigate if there is a difference between strains isolated from human disease cases and those isolated from asymptomatic cattle which would account for the low disease incidence of such a ubiquitous organism. The work presented here has compared human disease strains from both sporadic and outbreak cases with a cross-section, as defined by pulsed-field gel electrophoresis, of E. coli O157 strains from cattle. Human (n = 22) and bovine (n = 31) strains were genotyped for carriage of the genes for Shiga-like toxin types 1, 2, and 2c; E. coli secreted protein genes espA, espB, and espP; the enterohemolysin gene; eae (intimin); ast (enteroaggregative E. coli stable toxin [EAST]); and genes for common E. coli adhesins. Strains were also phenotyped for hemolysin, EspP, Tir, and EspD expression as well as production of actin and cytoskeletal rearrangement associated with attaching and effacing (A/E) lesions on HeLa cells. The genotyping confirmed that there was little difference between the two groups, including carriage of stx(2) and stx(2c), which was similar in both sets. ast alleles were confirmed to all contain mutations that would prevent EAST expression. espP mutations were found only in cattle strains (5 of 30). Clear differences were observed in the expression of locus of enterocyte effacement (LEE)-encoded factors between strains and in different media. EspD, as an indicator of LEE4 (espA, -B, and -D) expression, and Tir levels in supernatants were measured. Virtually all strains from both sources could produce EspD in Luria-Bertani broth, although at very different levels. Standard trichloroacetic acid precipitation of secreted proteins from tissue culture medium produced detectable levels of EspD from the majority of strains of human origin (15 of 20) compared with only a few (4 of 20) bovine strains (P < 0.001), which is indicative of much higher levels of protein secretion from the human strains. Addition of bovine serum albumin carrier protein before precipitation and enhanced detection techniques confirmed that EspD could be detected after growth in tissue culture medium for all strains, but levels from strains of human origin were on average 90-fold higher than those from strains of bovine origin. In general, levels of secretion also correlated with ability to form A/E lesions on HeLa cells, with only the high-level protein secretors in tissue culture medium exhibiting a localized adherence phenotype. This research shows significant differences between human- and bovine-derived E. coli O157 (stx(+) eae(+)) strains and their production of certain LEE-encoded virulence factors. These data support the recent finding of Kim et al. (J. Kim, J. Nietfeldt, and A. K. Benson, Proc. Natl. Acad. Sci. USA 96:13288-13293, 1999) proposing different E. coli O157 lineages in cattle and humans and extend the differential to the regulation of virulence factors. Potentially only a subset of E. coli O157 isolates (stx(+) eae(+)) in cattle may be capable of causing severe disease in humans.

Analysis of Escherichia coli strains causing bacteriuria during pregnancy: selection for strains that do not express type 1 fimbriae.

Escherichia coli isolates from patients with bacteriuria of pregnancy were compared by PCR with isolates from patients with community-acquired cystitis for the presence of established virulence determinants. The strains from patients with bacteriuria of pregnancy were less likely to carry genes for P-family, S-family, and F1C adhesins, cytotoxic necrotizing factor 1, and aerobactin, but virtually all of the strains carried the genes for type 1 fimbriae. Standard mannose-sensitive agglutination of yeast cells showed that only 15 of 42 bacteriuria strains (36%) expressed type 1 fimbriae compared with 32 of 42 strains from community-acquired symptomatic infections (76%) (P < 0.01). This difference was confirmed by analysis of all isolates for an allele of the type 1 fimbrial regulatory region (fim switch), which negates type 1 fimbrial expression by preventing the fim switch from being inverted to the on phase. This allele, fimS49, was found in 8 of 47 bacteriuria strains from pregnant women (17.0%) compared with 2 of 60 strains isolated from patients with cystitis (3.3%) (P < 0.05). Determination of the phase switch orientation in vivo by analysis of freshly collected infected urine from patients with bacteriuria showed that the fim switch was detectable in the off orientation in 17 of 23 urine samples analyzed (74%). These data indicate that type 1 fimbriae are not necessary to maintain the majority of E. coli bacteriurias in pregnant women since there appears to be selection against their expression in this particular group. This is in contrast to the considered role of this adhesin in community-acquired symptomatic infections. The lack of type 1 fimbria expression is likely to contribute to the asymptomatic nature of bacteriuria in pregnant women, although approximately one-third of the bacteriuria isolates do possess key virulence determinants. If left untreated, this subset of isolates pose the greatest threat to the health of the mother and unborn child.

Analysis of type 1 fimbriae expression in verotoxigenic Escherichia coli: a comparison between serotypes O157 and O26.

Previous research has shown that verotoxin-producing Escherichia coli (VTEC) O157 strains appear unable to express type 1 fimbriae although other serotypes such as O26 and O118 can. This study has investigated the molecular basis of this difference. The study confirmed the presence of a 16 bp deletion within the regulatory region of fimA (fim switch) in 63 VTEC O157 strains but not in other VTEC serotypes tested. The fim switch was shown to be detectable only in the phase off orientation in VTEC O157, but detection of the switch in the phase on orientation correlated with the degree of mannose-sensitive yeast agglutination in VTEC O26. Repair of the 16 bp deletion in the VTEC O157 fim switch region restored phase-variable expression of fimA in a permissive background. Non-O157 VTEC, especially O26 and O118, can be pathogenic in cattle; the role of type 1 fimbriae in this and colonization is discussed.

The biology of cystitis: host and bacterial factors.

Cystitis is caused by a relatively small number of bacterial species. To colonize and grow in the urinary tract, these organisms have developed and acquired special properties (virulence factors) that allow them to overcome the defences of the urinary tract, particularly clearance by urine flow. These virulence factors are unlikely to be required during transmission from host to host, and sometimes their constitutive expression may actually be disadvantageous. Such factors are therefore regulated by the environment and in a coordinate manner to ensure their most appropriate expression for the conditions encountered. This review focuses on the biology of the urinary tract and the bacterial properties necessary to cause cystitis. The regulation of virulence factors at the different stages of the infection is considered, and a general model for the pathogenesis of urinary tract infection is proposed.

Regulation of type 1 fimbrial expression in uropathogenic Escherichia coli: heterogeneity of expression through sequence changes in the fim switch region.

Over 80% of uropathogenic Escherichia coli express type 1 fimbriae. Expression is phase variable, and regulation of phase switching can differ between isolates. Previously, this was explained by differences in the expression of the fim recombinases, FimB and FimE. Our study of 50 uropathogenic E. coli isolates confirms variation in the regulation of type 1 fimbriae but, in many cases, the variation could be accounted for by sequence changes within and adjacent to the fim switch, rather than by differences in recombinase expression. This was demonstrated by moving the switch from the isolates into an isogenic background and comparing the switching behaviour with that of the original isolate. Isolates could be arranged into groups based on fim switch regulation and sequence similarity. In certain cases, the altered regulation was located to specific basepair changes within the fim switch. Sequence changes were found that had a marked effect on the activity of either FimB or FimE switching, while others affected FimB switching in only one direction. These results emphasize the value of using naturally selected sequence variation to further the understanding of gene regulation.

Differential temperature modulation by H-NS of the fimB and fimE recombinase genes which control the orientation of the type 1 fimbrial phase switch.

Phase variation of type 1 fimbriation in Escherichia coli is associated with the inversion of a 314-bp DNA element. This DNA switch directs transcription of fimA, encoding the major type 1 fimbrial subunit, in the on orientation but not in the off orientation. Inversion of the DNA element requires either FimB (confers both on and off orientations) or FimE (confers off orientation). Here we show, by chromosomally located fimB- and fimE-lacZ cassettes in isogenic strain sets differing only in the hns locus, how the global regulator H-NS affects the expression of type 1 fimbriae. H-NS was found to downregulate fimB and fimE in a temperature-dependent manner which affected the genes inversely at 30 degrees C and 37 degrees C. By gel-retardation assays H-NS binding was demonstrated to the regions containing the fimB promoter and the fimE promoter, respectively. In vitro recombination analysis suggested no direct involvement of H-NS in the inversion of the phase switch. Rather than directly affecting the switching process per se, it appeared that the orientation of this element was affected by the differential temperature modulation of H-NS of the fimB and fimE genes. Taken together the results suggest that H-NS modulates expression of type 1 fimbriae in a way which seems to favor a fimbriate state at the mammalian body temperature.

The Pai-associated leuX specific tRNA5(Leu) affects type 1 fimbriation in pathogenic Escherichia coli by control of FimB recombinase expression.

The uropathogenic Escherichia coli strain 536 (06:K15:H31) carries two large chromosomal pathogenicity islands (Pais). Both Pais are flanked by tRNA genes. Spontaneous deletion of Pai II results in truncation of the leuX tRNA5Leu gene. This tRNA is required for the expression of type 1 fimbriae (Fim) and other virulence factors. Transcription of fimA, encoding the major type 1 fimbrial subunit is controlled by an invertable DNA switch. The inversion is catalysed by two recombinases, FimB and FimE. FimB is able to turn the switch on, FimE only off. The fimB gene of strain 536 contains five TTG codons recognized by tRNA5Leu, fimE contains only two. It was proposed that turning on the fim switch requires efficient translation of FimB, in turn requiring tRNA5Leu. Strains in which the TTG codons in fimB were replaced with CTG codons at the wild-type locus were able to produce type 1 fimbriae in the absence of leuX. fimB transcription was influenced by the presence of leuX, but only slightly affected by the presence or absence of leuX codons in fimB. FimB translation was significantly higher from codon-replaced fimB genes than that of wild-type fimB genes in various strain backgrounds. The fim switch was shown to be switched off in leuX-derivatives of E. coli 536, but could be found in the on position when the codon-altered fimB gene was exchanged into the chromosome of these strains. From these data, it is apparent that tRNA5Leu is required for efficient translation of FimB, in turn, leading to type 1 fimbrial expression.

Interaction of FimB and FimE with the fim switch that controls the phase variation of type 1 fimbriae in Escherichia coli K-12.

The phase variation of type 1 fimbriae in Escherichia coli is associated with the site-specific inversion of a short DNA element. Recombination at fim requires fimB and fimE, and their products are considered to be the fim recombinases. In this study, FimB and FimE were overproduced and extracts containing the proteins were shown to (i) bind to and (ii) invert the fim switch in vitro. Phenanthroline-copper protection of DNA-protein complexes showed that both FimB and FimE bind to half-sites that flank, and overlap with, the left and right inverted repeats (IRL and IRR, respectively) of the fim switch. Alignment of the four half-sites identified a conserved 5'-CA doublet; mutation of these two bases lowers the affinity of binding of both FimB and FimE to the inverted repeats, and greatly diminishes inversion of the fim switch in vivo. The specificity of the fim recombinases observed in vivo (FimB switching in both directions; FimE switching from on-to-off only) was maintained in vitro. Furthermore, the different binding affinities of FimB and FimE for the various half-site combinations suggests that the specificity of FimE could arise, in part, from the low affinity of FimE for IRL (off).

The leucine-responsive regulatory protein binds to the fim switch to control phase variation of type 1 fimbrial expression in Escherichia coli K-12.

Phase variation of type 1 fimbriation in Escherichia coli is associated with the site-specific recombination of a 314-bp DNA invertible element. The fim switch directs transcription of fimA, the major fimbrial subunit gene, in one orientation (on) but not the other (off). Switching requires either fimB (on-to-off or off-to-on inversion) or fimE (on-to-off inversion only) and is reduced sharply in strains containing lrp::Tn10 mutations. Both fimE-promoted switching and fimB-promoted switching are stimulated by the amino acids alanine, isoleucine, leucine, and valine, and this regulation requires lrp. Here it is shown that the leucine-responsive regulatory protein (Lrp) binds in and adjacent to the fim switch. Mutations in fim that lower Lrp binding in vitro have corresponding effects on both fimB-promoted switching and fimE-promoted switching in vivo. Lrp initiates binding at one of two sites within the fim switch. Additional cooperative binding results in an extensive region of protection from both DNase I and 1,10-phenanthroline-copper complex-activated DNA cleavage. The region of protection can extend to within 12 bp of the right inverted repeat (switch off) and occupies over one-third of the switch. It is proposed that wrapping of fim DNA around an Lrp complex is required to form a recombination-proficient structure.