Gabriele Blum-Oehler

Genetic Structure and Distribution of Four Pathogenicity Islands (PAI I536 to PAI IV536) of Uropathogenic Escherichia coli Strain 536

ABSTRACT For the uropathogenic Escherichia coli strain 536 (O6:K15:H31), the DNA sequences of three pathogenicity islands (PAIs) (PAI I536 to PAI III536) and their flanking regions (about 270 kb) were determined to further characterize the virulence potential of this strain. PAI I536 to PAI III536 exhibit features typical of PAIs, such as (i) association with tRNA-encoding genes; (ii) G+C content differing from that of the host genome; (iii) flanking repeat structures; (iv) a mosaic-like structure comprising a multitude of functional, truncated, and nonfunctional putative open reading frames (ORFs) with known or unknown functions; and (v) the presence of many fragments of mobile genetic elements. PAI I536 to PAI III536 range between 68 and 102 kb in size. Although these islands contain several ORFs and known virulence determinants described for PAIs of other extraintestinal pathogenic E. coli (ExPEC) isolates, they also consist of as-yet-unidentified ORFs encoding putative virulence factors. The genetic structure of PAI IV536, which represents the core element of the so-called high-pathogenicity island encoding a siderophore system initially identified in pathogenic yersiniae, was further characterized by sample sequencing. For the first time, multiple PAI sequences (PAI I536 to PAI IV536) in uropathogenic E. coli were studied and their presence in several wild-type E. coli isolates was extensively investigated. The results obtained suggest that these PAIs or at least large fragments thereof are detectable in other pathogenic E. coli isolates. These results support our view that the acquisition of large DNA regions, such as PAIs, by horizontal gene transfer is an important factor for the evolution of bacterial pathogens.

A Single Nucleotide Exchange in the wzy Gene Is Responsible for the Semirough O6 Lipopolysaccharide Phenotype and Serum Sensitivity of Escherichia coli Strain Nissle 1917

Structural analysis of lipopolysaccharide (LPS) isolated from semirough, serum-sensitive Escherichia coli strain Nissle 1917 (DSM 6601, serotype O6:K5:H1) revealed that this strains LPS contains a bisphosphorylated hexaacyl lipid A and a tetradecasaccharide consisting of one E. coli O6 antigen repeating unit attached to the R1-type core. Configuration of the GlcNAc glycosidic linkage between O-antigen oligosaccharide and core (beta) differs from that interlinking the repeating units in the E. coli O6 antigen polysaccharide (alpha). The wa(*) and wb(*) gene clusters of strain Nissle 1917, required for LPS core and O6 repeating unit biosyntheses, were subcloned and sequenced. The DNA sequence of the wa(*) determinant (11.8 kb) shows 97% identity to other R1 core type-specific wa(*) gene clusters. The DNA sequence of the wb(*) gene cluster (11 kb) exhibits no homology to known DNA sequences except manC and manB. Comparison of the genetic structures of the wb(*)(O6) (wb(*) from serotype O6) determinants of strain Nissle 1917 and of smooth and serum-resistant uropathogenic E. coli O6 strain 536 demonstrated that the putative open reading frame encoding the O-antigen polymerase Wzy of strain Nissle 1917 was truncated due to a point mutation. Complementation with a functional wzy copy of E. coli strain 536 confirmed that the semirough phenotype of strain Nissle 1917 is due to the nonfunctional wzy gene. Expression of a functional wzy gene in E. coli strain Nissle 1917 increased its ability to withstand antibacterial defense mechanisms of blood serum. These results underline the importance of LPS for serum resistance or sensitivity of E. coli.

Instability of Pathogenicity Islands in Uropathogenic Escherichia coli 536

The uropathogenic Escherichia coli strain 536 carries at least five genetic elements on its chromosome that meet all criteria characteristic of pathogenicity islands (PAIs). One main feature of these distinct DNA regions is their instability. We applied the so-called island-probing approach and individually labeled all five PAIs of E. coli 536 with the counterselectable marker sacB to evaluate the frequency of PAI-negative colonies under the influence of different environmental conditions. Furthermore, we investigated the boundaries of these PAIs. According to our experiments, PAI II536 and PAI III536 were the most unstable islands followed by PAI I536 and PAI V536, whereas PAI IV536 was stable. In addition, we found that deletion of PAI II536 and PAI III536 was induced by several environmental stimuli. Whereas excision of PAI I536, PAI II536, and PAI V536 was based on site-specific recombination between short direct repeat sequences at their boundaries, PAI III536 was deleted either by site-specific recombination or by homologous recombination between two IS100-specific sequences. In all cases, deletion is thought to lead to the formation of nonreplicative circular intermediates. Such extrachromosomal derivatives of PAI II536 and PAI III536 were detected by a specific PCR assay. Our data indicate that the genome content of uropathogenic E. coli can be modulated by deletion of PAIs.

S-Fimbria-Encoding Determinant sfaI Is Located on Pathogenicity Island III536 of Uropathogenic Escherichia coli Strain 536

ABSTRACT The sfaI determinant encoding the S-fimbrial adhesin of uropathogenic Escherichia colistrains was found to be located on a pathogenicity island of uropathogenic E. coli strain 536. This pathogenicity island, designated PAI III536, is located at 5.6 min of theE. coli chromosome and covers a region of at least 37 kb between the tRNA locus thrW and yagU. As far as it has been determined, PAI III536 also contains genes which code for components of a putative enterochelin siderophore system of E. coli and Salmonella spp. as well as for colicin V immunity. Several intact or nonfunctional mobility genes of bacteriophages and insertion sequence elements such as transposases and integrases are present on PAI III536. The presence of known PAI III536 sequences has been investigated in several wild-type E. coli isolates. The results demonstrate that the determinants of the members of the S-family of fimbrial adhesins may be located on a common pathogenicity island which, in E. coli strain 536, replaces a 40-kb DNA region which represents anE. coli K-12-specific genomic island.

The Pathogenicity Island-Associated K15 Capsule Determinant Exhibits a Novel Genetic Structure and Correlates with Virulence in Uropathogenic Escherichia coli Strain 536

ABSTRACT The K15 capsule determinant of uropathogenic Escherichia coli strain 536 (O6:K15:H31) is part of a novel 79.6-kb pathogenicity island (PAI) designated PAI V536 that is absent from the genome of nonpathogenic E. coli K-12 strain MG1655. PAI V536 shows typical characteristics of a composite PAI that is associated with the pheV tRNA gene and contains the pix fimbriae determinant as well as genes coding for a putative phosphoglycerate transport system, an autotransporter protein, and hypothetical open reading frames. A gene cluster coding for a putative general secretion pathway system, together with a kpsK15 determinant, is localized downstream of a truncated pheV gene (′pheV) also present in this chromosomal region. The distribution of genes present on PAI V536 was studied by PCR in different pathogenic and nonpathogenic E. coli isolates of various sources. Analysis of the 20-kb kps locus revealed a so far unknown genetic organization. Generally, the kpsK15 gene cluster resembles that of group 2 and 3 capsules, where two conserved regions (regions 1 and 3) are located up- or downstream of a highly variable serotype-specific region (region 2). Interestingly, recombination of a group 2 and 3 determinant may have been involved in the evolution of the K15 capsule-encoding gene cluster. Expression of the K15 capsule is important for virulence in a murine model of ascending urinary tract infection but not for serum resistance of E. coli strain 536.

Development of strain-specific PCR reactions for the detection of the probiotic Escherichia coli strain Nissle 1917 in fecal samples

PCR was used to establish a specific detection system for the non-pathogenic Escherichia coli strain Nissle 1917 (DSM6601), which is used as a probiotic drug against intestinal disorders and diseases. Five PCR assays have been developed which are based on the chromosomally encoded major fimbrial subunit genes fimA (type 1 fimbriae) and focA (F1C fimbriae), and the two small cryptic plasmids pMUT1 and pMUT2. The assays were validated by testing a collection of 354 different pathogenic and non-pathogenic E. coli strains from various origins, including E. coli K-12, fecal and environmental as well as pathogenic extraintestinal and intestinal E. coli strains. The most specific results were obtained with primers based on DNA sequences from plasmid pMUT2. The plasmid-based PCR assays described can be used to detect E. coli strain Nissle 1917 in feces from patients without prior cultivation.

The Pathogenicity Islands (PAIs) of the Uropathogenic Escherichia coli Strain 536: Island Probing of PAI II536

Pathogenicity islands (PAIs) represent distinct pieces of DNA that are present in the genomes of pathogenic bacteria but absent from the genomes of related nonpathogenic strains. They carry (often more than one) virulence genes and are linked to transfer RNA (tRNA) loci. In addition, they carry mobility genes and direct repeats at their ends and are often unstable [1]. The first PAIs have been identified in the genomes of uropathogenic Escherichia coli (UPEC) [2–4], and PAIs of 120 species of pathogens have been described [5]. UPECs can produce various virulence factors, such as specific adhesins (P, S, type I, F1C), toxins (a-hemolysin, cytotoxic necrotizing factor I), capsules (K1, K5, K12), specific O antigens (O1, O2, O4, O6, O18), iron-uptake systems (aerobactin, yersiniabactin), and factors contributing to serum resistance. Most genes, coding for these factors are located on PAIs [3, 4]. The uropathogenic strain E. coli 536, which was isolated from a patient with pyelonephritis, represents a model organism for the analysis of the genetic basis of urovirulence [2, 6]. The strain exhibits the serotype O6:K15:H31 and carries four PAIs in its chromosome. PAI I536 and PAI II536, which are 70 and 120 kb in size, carry the genes for hemolysin and, in the case of PAI II536, P fimbriae. PAI III536 carries the S fimbrial gene cluster, and PAI IV536 is almost identical to the functional core region of the “high pathogenicity island” (HPI) of pathogenic Yersinia species, encoding the yersiniabactin iron-uptake system [4, 7]. In addition, the genes responsible for the capsule synthesis seem also to be located on another PAI (Janke B, et al., unpublished data). As shown recently, PAI I536 and PAI II536, which are located next to the tRNA genes selC and leuX at map positions 82 and 97 in the E. coli chromosome, respectively, are unstable [2]. With relatively high frequencies, these PAIs can be deleted from the chromosome of strain 536. In previous studies [2], the deletion processes of PAI I536 and PAI II536 have been analyzed on the basis of the loss of hemolysin production. In this report, however, we use the new method of “island probing,” as described by Rajakumar et al. [8], for the first time

Toxin genes on pathogenicity islands: impact for microbial evolution.

Toxin-specific genes are often located on mobile genetic elements such as phages, plasmids and pathogenicity islands (PAIs). The uropathogenic E. coli strain 536 carries two alpha-hemolysin gene clusters, which are part of the pathogenicity islands I536 and II536, respectively. Using different genetic techniques, two additional PAIs were identified in the genome of the E. coli strain 536, and it is likely that further PAIs are located on the genome of this strain. Pathogenicity islands are often associated with tRNA genes. In the case of the E. coli strain 536, the PAI-associated tRNA gene leuX, which encodes a minor leucyl-tRNA, affects the expression of various virulence traits including alpha-hemolysin production. The exact mode of action of the tRNA5Leu-dependent gene expression has to be identified in the future.

DNA sequence analysis of the composite plasmid pTC conferring virulence and antimicrobial resistance for porcine enterotoxigenic Escherichia coli.

In this study the plasmid pTC, a 90 kb self-conjugative virulence plasmid of the porcine enterotoxigenic Escherichia coli (ETEC) strain EC2173 encoding the STa and STb heat-stable enterotoxins and tetracycline resistance, has been sequenced in two steps. As a result we identified five main distinct regions of pTC: (i) the maintenance region responsible for the extreme stability of the plasmid, (ii) the TSL (toxin-specific locus comprising the estA and estB genes) which is unique and characteristic for pTC, (iii) a Tn10 transposon, encoding tetracycline resistance, (iv) the tra (plasmid transfer) region, and (v) the colE1-like origin of replication. It is concluded that pTC is a self-transmissible composite plasmid harbouring antibiotic resistance and virulence genes. pTC belongs to a group of large conjugative E. coli plasmids represented by NR1 with a widespread tra backbone which might have evolved from a common ancestor. This is the first report of a completely sequenced animal ETEC virulence plasmid containing an antimicrobial resistance locus, thereby representing a selection advantage for spread of pathogenicity in the presence of antimicrobials leading to increased disease potential.

Pathogenicity Islands of Uropathogence E. Coli and Evolution of Virulence

E. coli bacteria are able to cause a large range of infectious diseases in humans. Among these are infections of the gastrointestinal tract as well as extraintestinal infections of great importance. Intestinal E. coli can be grouped in at least six different pathotypes including enterotoxigenic (ETEC), enteropathogenic (EPEC), enterohemorrhagic (EHEC) and enteroaggregative (EaggEC) E. coli. Extraintestinal E. coli fall into three groups: meningitis (MENEC), septicemia (SEPEC) and uropathogenic (UPEC) E. coli. UPECs are by far the most common cause of uncomplicated bacterial urinary tract infections (UTIs). About 80 % of all UTIs are due to E. coli. UPECs differ from non-pathogenic E. coli variants by the presence of certain virulence factors which contribute to their ability to cause disease. These are two different types of toxins, α -hemolysin (Hly) and the cytotoxic necrotizing factor 1 (CNF1), fimbrial adhesins e. g. P-, type 1 and S-fimbriae and iron-uptake systems like aerobactin, enterobactin or yersiniabactin. Furthermore, UPECs belong to certain serotypes and have the capacity to survive in human serum. About ten years ago it was detected that UPECs contain distinct blocks of DNA carrying closely linked virulence genes. Later on these structures were