Intergenic sequence comparison of Escherichia coli isolates reveals lifestyle adaptations but not host specificity.

Establishing the risk of human infection is one of the goals of public health. For bacterial pathogens, the virulence and zoonotic potential can often be related to their host source. Escherichia coli bacteria are common contaminants of water associated with human recreation and consumption, and many strains are pathogenic. In this study, we analyzed three promoter-containing intergenic regions from 284 diverse E. coli isolates in an attempt to identify molecular signatures associated with specific host types. Promoter sequences controlling production of curli fimbriae, flagella, and nutrient import yielded a phylogenetic tree with isolates clustered by established phylogenetic grouping (A, B1, B2, and D) but not by host source. Virulence genes were more prevalent in groups B2 and D isolates and in human isolates. Group B1 isolates, primarily from nonhuman sources, were the most genetically similar, indicating that they lacked molecular adaptations to specific host environments and were likely host generalists. Conversely, B2 isolates, primarily from human sources, displayed greater genetic distances and were more likely to be host adapted. In agreement with these hypotheses, prevalence of σ(S) activity and the rdar morphotype, phenotypes associated with environmental survival, were significantly higher in B1 isolates than in B2 isolates. Based on our findings, we speculate that E. coli host specificity is not defined by genome-wide sequence changes but, rather, by the presence or absence of specific genes and associated promoter elements. Furthermore, the requirements for colonization of the human gastrointestinal tract may lead to E. coli lifestyle changes along with selection for increased virulence.

Sleeping beauty mutase (sbm) is expressed and interacts with ygfd in Escherichia coli.

In Escherichia coli, a four-gene operon, sbm-ygfD-ygfG-ygfH, has been shown to encode a putative cobalamin-dependent pathway with the ability to produce propionate from succinate in vitro [Haller T, Buckel T, Retey J, Gerlt JA. Discovering new enzymes and metabolic pathways: conversion of succinate to propionate by Escherichia coli. Biochemistry 2000;39:4622-4629]. However, the operon was thought to be silent in vivo, illustrated by the eponym describing its first gene, "sleeping beauty mutase" (methylmalonyl-CoA mutase, MCM). Of the four genes described, only ygfD could not be assigned a function. In this study, we have evaluated the functional integrity of YgfD and Sbm and show that, indeed, both proteins are expressed in E. coli and that YgfD has GTPase activity. We show that YgfD and Sbm can be co-immunoprecipitated from E. coli extracts using antibody to either protein, demonstrating in vivo interaction, a result confirmed using a strain deleted for ygfD. We show further that, in vitro, purified His-tagged YgfD and Sbm behave as a monomer and dimer, respectively, and that they form a multi-subunit complex that is dependent on pre-incubation of YgfD with non-hydrolysable GTP, an outcome that was not affected by the state of Sbm, as holo- or apoenzyme. These studies reinforce a role for the in vivo interaction of YgfD and Sbm.

Aggregation via the red, dry, and rough morphotype is not a virulence adaptation in Salmonella enterica serovar Typhimurium.

The Salmonella rdar (red, dry, and rough) morphotype is an aggregative and resistant physiology that has been linked to survival in nutrient-limited environments. Growth of Salmonella enterica serovar Typhimurium was analyzed in a variety of nutrient-limiting conditions to determine whether aggregation would occur at low cell densities and whether the rdar morphotype was involved in this process. The resulting cultures consisted of two populations of cells, aggregated and nonaggregated, with the aggregated cells preferentially displaying rdar morphotype gene expression. The two groups of cells could be separated based on the principle that aggregated cells were producing greater amounts of thin aggregative fimbriae (Tafi or curli). In addition, the aggregated cells retained some physiological characteristics of the rdar morphotype, such as increased resistance to sodium hypochlorite. Competitive infection experiments in mice showed that nonaggregative DeltaagfA cells outcompeted rdar-positive wild-type cells in all tissues analyzed, indicating that aggregation via the rdar morphotype was not a virulence adaptation in Salmonella enterica serovar Typhimurium. Furthermore, in vivo imaging experiments showed that Tafi genes were not expressed during infection but were expressed once Salmonella was passed out of the mice into the feces. We hypothesize that the primary role of the rdar morphotype is to enhance Salmonella survival outside the host, thereby aiding in transmission.

Persistence of Salmonella on egg conveyor belts is dependent on the belt type but not on the rdar morphotype.

Commercial caged layer flocks in Alberta, Canada, are commonly monitored for Salmonella enterica serovar Enteritidis (SE) and S. enterica serovar Typhimurium (ST) by environmental sampling. In one recent case, a SE strain isolated from the egg conveyor belt was a source of persistent infection for the flock. This study was undertaken to examine Salmonella colonization on egg conveyor belts and to determine whether the rdar morphotype, a conserved physiology associated with aggregation and long-term survival, contributed to persistence. Four woven belts constructed of natural or nonnatural fibers and a 1-piece belt made of vinyl were tested with rdar-positive ST and SE strains and a rdar-negative ST DeltaagfD reference strain. The type of egg belt was the most important factor influencing Salmonella colonization and persistence. The vinyl belt, with the least surface area available for colonization, had the fewest Salmonella remaining after washing and disinfection, whereas the hemp-plastic belt, with the greatest surface area, had the most Salmonella remaining. Real-time gene expression indicated that the rdar morphotype was involved in colonizing the egg belt pieces; however, it was not essential for persistence. In addition, rdar-positive and rdar-negative strains were equally similarly to disinfection on the egg belt pieces. The results indicate that Salmonella can persist on a variety of egg belts by mechanisms other than the rdar morphotype, and that using egg conveyer belts with reduced surface area for bacterial colonization can lessen contamination problems.

An efficient system for markerless gene replacement applicable in a wide variety of enterobacterial species.

We describe an improved allelic-exchange method for generating unmarked mutations and chromosomal DNA alterations in enterobacterial species. Initially developed for use in Salmonella enterica, we have refined the method in terms of time, simplicity, and efficiency. We have extended its use into related bacterial species that are more recalcitrant to genetic manipulations, including enterohemorrhagic and enteropathogenic Escherichia coli and Vibrio parahaemolyticus. Data from over 50 experiments are presented including gene inactivations, site-directed mutagenesis, and promoter exchanges. In each case, desired mutations were identified by polymerase chain reaction screening typically from as few as 10-20 colonies up to a maximum of 300 colonies. The method does not require antibiotic nor nutritional markers in target genes and works efficiently in wild-type strains, obviating the need for specialized hosts or genetic systems. The use is simple, requiring basic laboratory materials, and represents an alternative to existing methods for gene manipulation in the Enterobacteriaceae.

AgfC and AgfE facilitate extracellular thin aggregative fimbriae synthesis in Salmonella enteritidis.

Salmonella thin aggregative fimbriae (Tafi; curli) are important in pathogenesis and biofilm formation; however, less is known of their structure and morphogenesis. In the Salmonella agfBAC Tafi operon, the transcription and role of agfC have been elusive. In this study, agfBAC transcripts were detected using a sensitive reverse transcriptase technique. Native AgfC was not detected using polyclonal antibodies generated against purified hexahistidine-tagged AgfC; however, in trans expression revealed that AgfC was localized to the periplasm as a mature form. An isogenic DeltaagfC mutant displayed an abundance of 20 nm fibres, in addition to native Tafi (5-7 nm), and had an increase in cell surface hydrophobicity. Purified 20 nm fibres were depolymerized under exceptionally stringent conditions to release what proved to be AgfA subunits. This revealed that the 20 nm fibres represented a different form of Tafi. The role of AgfC in Tafi assembly was investigated further using an antibody-capture assay of isogenic Deltaagf mutants. A soluble antibody-accessible form of AgfA was captured in wild-type (wt), DeltaagfB and DeltaagfF strains, in support of the extracellular nucleation-precipitation pathway of Tafi assembly, but not in DeltaagfC or DeltaagfE mutants. This indicates that AgfC and AgfE are important for AgfA extracellular assembly, facilitating the synthesis of Tafi.

Salmonella produces an O-antigen capsule regulated by AgfD and important for environmental persistence.

In this study, we show that Salmonella produces an O-antigen capsule coregulated with the fimbria- and cellulose-associated extracellular matrix. Structural analysis of purified Salmonella extracellular polysaccharides yielded predominantly a repeating oligosaccharide unit similar to that of Salmonella enterica serovar Enteritidis lipopolysaccharide O antigen with some modifications. Putative carbohydrate transport and regulatory operons important for capsule assembly and translocation, designated yihU-yshA and yihVW, were identified by screening a random transposon library with immune serum generated to the capsule. The absence of capsule was confirmed by generating various isogenic Deltayih mutants, where yihQ and yihO were shown to be important in capsule assembly and translocation. Luciferase-based expression studies showed that AgfD regulates the yih operons in coordination with extracellular matrix genes coding for thin aggregative fimbriae and cellulose. Although the capsule did not appear to be important for multicellular behavior, we demonstrate that it was important for survival during desiccation stress. Since the yih genes are conserved in salmonellae and the O-antigen capsule was important for environmental persistence, the formation of this surface structure may represent a conserved survival strategy.

Comparative genetics of the rdar morphotype in Salmonella.

The Salmonella rdar morphotype is a distinct, rough and dry colony morphology formed by the extracellular interaction of thin aggregative fimbriae (Tafi or curli), cellulose, and other polysaccharides. Cells in rdar colonies are more resistant to desiccation and exogenous stresses, which is hypothesized to aid in the passage of pathogenic Salmonella spp. between hosts. Here we analyzed the genetic and phenotypic conservation of the rdar morphotype throughout the entire Salmonella genus. The rdar morphotype was conserved in 90% of 80 isolates representing all 7 Salmonella groups; however, the frequency was only 31% in a reference set of 16 strains (Salmonella reference collection C [SARC]). Comparative gene expression analysis was used to separate cis- and trans-acting effects on promoter activity for the 16 SARC strains, focusing on the 780-bp intergenic region containing divergent promoters for the master regulator of the rdar morphotype (agfD) and the Tafi structural genes (agfB). Surprisingly, promoter functionality was conserved in most isolates, and loss of the phenotype was due primarily to defects in trans-acting regulatory factors. We hypothesize that trans differences have been caused by domestication, whereas cis differences, detected for Salmonella enterica subsp. arizonae isolates, may reflect an evolutionary change in lifestyle. Our results demonstrate that the rdar morphotype is conserved throughout the salmonellae, but they also emphasize that regulation is an important source of variability among isolates.

Thin aggregative fimbriae and cellulose enhance long-term survival and persistence of Salmonella.

Salmonella spp. are environmentally persistent pathogens that have served as one of the important models for understanding how bacteria adapt to stressful conditions. However, it remains poorly understood how they survive extreme conditions encountered outside their hosts. Here we show that the rdar morphotype, a multicellular phenotype characterized by fimbria- and cellulose-mediated colony pattern formation, enhances the resistance of Salmonella to desiccation. When colonies were stored on plastic for several months in the absence of exogenous nutrients, survival of wild-type cells was increased compared to mutants deficient in fimbriae and/or cellulose production. Differences between strains were further highlighted upon exposure to sodium hypochlorite, as cellulose-deficient strains were 1,000-fold more susceptible. Measurements of gene expression using luciferase reporters indicated that production of thin aggregative fimbriae (Tafi) may initiate formation of colony surface patterns characteristic of the rdar morphotype. We hypothesize that Tafi play a role in the organization of different components of the extracellular matrix. Conservation of the rdar morphotype among pathogenic S. enterica isolates and the survival advantages that it provides collectively suggest that this phenotype could play a role in the transmission of Salmonella between hosts.

Extracellular polysaccharides associated with thin aggregative fimbriae of Salmonella enterica serovar enteritidis.

Lipopolysaccharide (LPS) O polysaccharide was identified as the principle factor impeding intercellular formation of intact thin aggregative fimbriae (Tafi) in Salmonella enterica serovar Enteritidis. The extracellular nucleation-precipitation assembly pathway for these organelles was investigated by quantifying fimbrial formation between deltaagfA (AgfA recipient) and deltaagfB (AgfA donor) cells harboring mutations in LPS (galE::Tn10) and/or cellulose (deltabcsA) synthesis. Intercellular complementation could be detected between deltaagfA and deltaagfB strains only when both possessed the galE mutation. LPS O polysaccharide appears to be an impenetrable barrier to AgfA assembly between cells but not within individual cells. The presence of cellulose did not restrict Tafi formation between cells. Transmission electron microscopy of w+ S. enterica serovar Enteritidis 3b cells revealed diffuse Tafi networks without discernible fine structure. In the absence of cellulose, however, individual Tafi fibers were clearly visible, appeared to be occasionally branched, and showed the generally distinctive appearance described for Escherichia coli K-12 curli. A third extracellular matrix component closely associated with cellulose and Tafi was detected on Western blots by using immune serum raised to whole, purified Tafi aggregates. Cellulose was required to tightly link this material to cells. Antigenically similar material was also detected in S. enterica serovar Typhimurium and one diarrheagenic E. coli isolate. Preliminary analysis indicated that this material represented an anionic, extracellular polysaccharide that was distinct from colanic acid. Therefore, Tafi in their native state appear to exist as a complex with cellulose and at least one other component.

Structure and characterization of AgfB from Salmonella enteritidis thin aggregative fimbriae.

The agfBAC operon of Salmonella enteritidis encodes thin aggregative fimbriae, fibrous, polymeric structures primarily composed of AgfA fimbrins. Although uncharacterized, AgfB shows a 51 % overall amino acid sequence similarity to AgfA. Using AgfB epitope-specific antiserum, AgfB was detected as a minor component of whole, purified fimbriae. Like AgfA, AgfB was released from purified fimbriae by >70 % formic acid, whereupon both AgfA-AgfA and AgfA-AgfB dimers as well as monomers were detected. This suggested that AgfB may form specific, highly stable, structural associations with AgfA in native fimbrial filaments, associations that were weakened in structurally unstable fibers derived from AgfA chimeric fimbrial mutants. Detailed sequence comparisons between AgfA and AgfB showed that AgfB harbored a similar fivefold repeated sequence pattern (x(6)QxGx(2)NxAx(3)Q), and contained structural motifs similar to the parallel beta helix model proposed for AgfA. Molecular modeling of AgfB revealed a 3D structure remarkably similar to that of AgfA, the structures differing principally in the surface disposition of non-conserved, basic, acidic and non-polar residues. Thus AgfB is a fimbrin-like structural homologue of AgfA and an integral, minor component of native thin aggregative fimbrial fibers. AgfB from an agfA deletion strain was detected as a non-fimbrial, SDS-insoluble form in the supernatant and was purified. AgfA from an agfB deletion strain was found in both SDS-soluble and insoluble, non-fimbrial forms. No AgfA-AgfA dimers were detected in the absence of AgfB. Fimbriae formation by intercellular complementation between agfB and agfA deletion strains could not be shown under a variety of conditions, indicating that AgfA and AgfB are not freely diffusible in S. enteritidis. This has important implications on the current assembly hypothesis for thin aggregative fimbriae.

The efficacy of topical anaesthesia in flexible nasendoscopy: a double-blind randomised controlled trial.

The routine use of topical anaesthesia during flexible nasendoscopy has been questioned, and the degree to which topical vasoconstrictors can affect patient discomfort has yet to be elucidated. Patients' experiences with Lignocaineand phenylephrine, Lignocaine alone, xylometazoline and no preparation were compared. One hundred patients were recruited in this double-blind, randomised control trial and put into these four groups. Each patient completed a visual analogue scoring chart to determine the severity of unpleasantness and other undesirable effects (pain, bad taste, burning, choking, numbness and difficulty in swallowing). The results confirmed that vasoconstriction is a major contributing factor towards reducing overall unpleasantness (P = 0.022), topical anaesthesia can produce a bad taste (P = 0.022), and that none of the preparations have any effect on the pain during nasendoscopy. In conclusion, xylometazoline is recommended for nasendoscopy as it is effective and is significantly cheaper than the other preparations. Not using any preparations leads to the experience of severe symptoms.

Salmonella enteritidis fimbriae displaying a heterologous epitope reveal a uniquely flexible structure and assembly mechanism.

Two distinct Salmonella fimbrins, AgfA and SefA, comprising thin aggregative fimbriae SEF17 and SEF14, respectively, were each genetically engineered to carry PT3, an alpha-helical 16-amino acid Leishmania T-cell epitope derived from the metalloprotease gp63. To identify regions within AgfA and SefA fimbrins amenable to replacement with this epitope, PCR-generated chimeric fimbrin genes were constructed and used to replace the native chromosomal agfA and sefA genes in Salmonella enteritidis. Immunoblot analysis using anti-SEF17 and anti-PT3 sera demonstrated that all ten AgfA chimeric fimbrin proteins were expressed by S. enteritidis under normal growth conditions. Immunoelectron microscopy confirmed that eight of the AgfA::PT3 proteins were effectively assembled into cell surface-exposed fimbriae. The PT3 replacements in AgfA altered Congo red (CR) binding, cell-cell adhesion and cell surface properties of S. enteritidis to varying degrees. However, these chimeric fimbriae were still highly stable, being resistant to proteinase K digestion and requiring harsh formic acid treatment for depolymerization. In marked contrast to AgfA, none of the chimeric SefA proteins were expressed or assembled into fimbriae. Since each PT3 replacement constituted over 10% of the AgfA amino acid sequence and all ten replacements collectively represented greater than 75% of the entire AgfA primary sequence, the ability of AgfA to accept large sequence substitutions and still assemble into fibers is unique among fimbriae and other structural proteins. This structural flexibility may be related to the novel fivefold repeating sequence of AgfA and its recently proposed structure Proper formation of chimeric fimbrial fibers suggests an unusual assembly mechanism for thin aggregative fimbriae which tolerates aberrant structures. This study opens a range of possibilities for Salmonella thin aggregative fimbriae as a carrier of heterologous epitopes and as an experimental model for studies of protein structure.

High efficiency gene replacement in Salmonella enteritidis: chimeric fimbrins containing a T-cell epitope from Leishmania major.

A simple, high frequency chromosomal gene replacement method of general utility was developed for Salmonella enteritidis. This system uses an unstable, imperfectly segregating, temperature-sensitive replicon, pHSG415, as a carrier of the recombinant gene of interest. It also allows for site-specific replacement of chromosomal genes without the need for antibiotic resistance markers in the recombinant genes or the use of specific bacterial strains. This strategy was used to replace the chromosomal sefA and agfA fimbrin genes of S. enteritidis 3b with recombinant genes containing a 48 bp DNA fragment encoding PT3, an immunoprotective T-cell epitope from GP63 of Leishmania major. The fidelity of chimeric fimbrial replacements were confirmed by DNA sequence analysis. Nearly 30% of the S. enteritidis clones selected in the final stage of sefA mutagenesis contained the sefA::PT3 recombinant gene, whereas for agfA the efficiency was as high as 10%. To our knowledge, this is the first report of fimbrial epitope replacement in the Salmonellae and the first chimeric fimbrin genes that have been reconstituted into a wild-type genetic background for any organism. As such, this model represents a promising 'organelle' expression system for epitope display in vaccinology.

Periplasmic and fimbrial SefA from Salmonella enteritidis.

Salmonella enteritidis produces thin, filamentous fimbriae composed of the fimbrin subunit SefA. Although insoluble in most detergents and chaotropic agents, these fimbriae were soluble at pH 10.5. Furthermore, in sodium dodecyl sulfate, these fibers depolymerized into monomers, dimers and other multimers of SefA, which precipitated on removal of the detergent. In contrast, unassembled periplasmic SefA fimbrins purified from Escherichia coli expressing cloned sefA and sefB were readily soluble in aqueous solution. Fimbrial and periplasmic SefA also differed in their reaction with an anti-SEF14 monoclonal antibody and in their surface hydrophobicity, indicating that the two forms had different properties. Precise mass measurements of periplasmic and fimbrial SefA by mass spectroscopy showed that these variations were not due to post-translational modifications. Periplasmic SefA consisted primarily of intact as well as some N-terminally truncated forms. The main 24 amino acid, N-terminally truncated form of periplasmic SefA was present as a 12.2 kDa monomer which had a low tendency to dimerize whereas intact periplasmic SefA was present as a 34.1 kDa homodimer. Intact periplasmic SefA also formed stable multimers at low concentrations of chemical cross-linker but multimerization of the truncated form required high concentrations of protein or cross-linker. Thus, SefA fimbrins appear to multimerize through their N-termini and undergo a conformational change prior to assembly into fibers. Within these fibers, subunit-subunit contact is maintained through strong hydrophobic interactions.

tRNA(Arg) (fimU) and expression of SEF14 and SEF21 in Salmonella enteritidis.

A Tn10 insertion affecting SEF14 fimbrial synthesis in Salmonella enteritidis was located 13 bp upstream of a gene designated fimU. The 77-bp DNA sequence of fimU from S. enteritidis was identical to that of fimU encoding tRNA(Arg) (UCU) from Salmonella typhimurium and 96% identical to that of the Escherichia coli argU homolog. Furthermore, the open reading frame adjacent to and overlapping the 3' end of fimU was similar to the prophage DLP12 integrase gene. The fimU-encoded transcript comigrated with total cellular tRNA and was predicted to form a tRNA-like cloverleaf structure containing the arginine anticodon UCU. Thus, fimU encoded a tRNA(Arg) specific for the rare codon AGA. fimU mapped to the SEF21 fim operon located 15 C's from the sef14 gene cluster. Although fimU was located within the SEF21 fim gene cluster, the fimU Tn10 insertion mutant of S. enteritidis was found to be defective in SEF14 as well as SEF21 (type 1) fimbria production. SEF17 and SEF18 fimbria production was not affected. Complementation of this mutant with plasmid-borne fimU restored normal production of the fimbrins SefA and FimA as well as their respective fimbriae SEF14 and SEF21. This is the first description of tRNA simultaneously controlling the production of two distinct fimbriae.

Machine learning techniques in early screening for gastric and oesophageal cancer.

A database on 2692 dyspeptic patients over the age of 40 was established, consisting of 73 epidemiological and clinical variables. A tree-based machine learning algorithm (PREDICTOR) was applied to this database, in order to attempt to find rules which would classify patients into 2 groups, i.e., those suffering from gastric or oesophageal cancer, and the remainder. The results were encouraging. The cross-validated classification performance figure showed that by classifying 61.3% of the patients as high risk, a sensitivity of 94.9% and a specificity of 39.8% could be achieved. It is planned to construct an expert system based on the rules produced by the machine learning algorithm, in order to provide preliminary screening for cancer in dyspeptic patients.

Hydration of single-stranded phosphodiester and phosphorothioate oligodeoxyribonucleotides.

Infrared spectroscopy was used to identify hydration-sensitive structural differences between single- stranded phosphorothioate (PS) and phosphodiester (PO) oligodeoxyribonucleotides. Spectra were recorded in the mid-infrared region, 500-1800 cm-1, at relative humidities between 0 and 98%; the PS and PO spectra are substantially different. The hydration effects on spectral bands in these single-stranded oligodeoxyribonucleotides is markedly different from such behavior in double- and triple-stranded oligodeoxyribonucleotides. A strong absorption occurs at 656 cm-1 in the phosphorothioate sample which is completely absent from the PO spectra. Gravimetric measurements were carried out on one PS and one PO sample to monitor and confirm hydration. The calculated BET adsorption constants [Brunauer, S., Emmett, RH. and Teller, E. (1938) J. Am. Chem. Soc., 60, 309-319] are 1.2 and 1.4 water molecules per nucleotide in the first hydration layer of PS and PO respectively. While the gravimetric data indicate that the single-stranded oligodeoxyribonucleotides hydrate very similarly to duplex DNA, the mid-infrared conformational marker bands are strikingly different from those observed for duplex DNA. In particular, the Vas of the phosphate group (PO2) at 1222 cm-1 in the single-stranded PO spectra is independent of relative humidity.