Elsevier

Microbial Pathogenesis

Volume 46, Issue 3, March 2009, Pages 150-158
Microbial Pathogenesis

Analysis of the sfaXII locus in the Escherichia coli meningitis isolate IHE3034 reveals two novel regulatory genes within the promoter-distal region of the main S fimbrial operon

https://doi.org/10.1016/j.micpath.2008.12.001Get rights and content

Abstract

We describe the expression and regulation of the gene sfaXII located near the SfaII fimbrial determinant in the newborn meningitis Escherichia coli (NMEC) isolate IHE3034. sfaXII belongs to a gene family, the 17-kDa genes, typically located downstream (300–3000 bp) of different fimbrial operons found in E. coli isolates of uropathogenic and newborn meningitis origin. Using transcriptional sfaXII reporter gene fusions we found that different environmental conditions commonly affecting expression of fimbrial genes also affected sfaXII expression. Analysis of the sfaXII transcripts showed that the gene is part of the main fimbrial operon as it is transcribed together with the rest of the fimbrial genes. In addition, the sfaXII gene can be expressed from a more proximal promoter and is found to be subject to strong down-regulation by the nucleoid protein H-NS. Studies with an sfaXII mutant derivative of IHE3034 did not reveal effects on SfaII fimbrial biogenesis as monitored by e.g. immunofluorescence microscopy. Nevertheless, a mutation in sfaXII resulted in altered expression of other surface components. Moreover, we define a new gene, sfaYII, coding for a putative phosphodiesterase that is located in between the sfaXII gene and the fimbrial biogenesis genes. Our studies by ectopic expression of sfaYII in Vibrio cholerae showed that the gene product caused reduced biofilm formation and it is proposed that sfaYII can influence cyclic-di-GMP turnover in the bacteria. Our findings demonstrate that the operons typical for S-fimbriae of extraintestinal pathogenic E. coli include previously unrecognized novel regulatory genes.

Introduction

Extraintestinal pathogenic Escherichia coli (ExPEC) commonly cause urinary tract infections but they also occur as infectious agents in other human diseases such as meningitis, sepsis, pneumonia, and surgical site infections [1]. ExPEC strains possess several virulence traits that facilitate colonization, invasion and pathogenesis in particular bodily sites. For example, the ability to adhere to tissue surfaces is commonly mediated by different types of fimbriae such as the S and P fimbrial adhesins that in a specific manner recognize receptors on the host cell surfaces. The genetic determinants for fimbrial biosynthesis are gene clusters that typically are organized in a major operon for the structural components and a promoter proximal regulatory region including a minor, often divergently oriented operon for some regulatory gene(s). Several environmental cues e.g. changes in temperature, pH, carbon source, and osmolarity are influencing ExPEC fimbrial expression. The S fimbrial determinants (type I and II) present in ExPEC causing urinary tract infections or meningitis are well studied examples of such an operon organization and the genes, designated sfaA-H, have been characterized genetically and functionally [2], [3], [4].

By genome sequence analyses, an increasing number of genes coding for tentative 17-kDa proteins has been detected in close association with fimbrial operons (Fig. 1). In the sequenced uropathogenic E. coli (UPEC) isolate 536 (accession number CP000247), such 17-kDa genes are found 1.2 kb downstream of the sfaHI gene (S fimbrial adhesin) of the sfaI determinant [5] and 0.3 kb downstream of the prfG gene (P fimbrial adhesin) of the prf fimbrial operon. In the NMEC isolate IHE3034, a 17-kDa gene is located 1.2 kb downstream of the sfaHII gene (S fimbrial adhesin) of the sfaII determinant [6] (U. Dobrindt, in preparation). Similarly, in the UPEC strain J96, the open reading frame (ORF) coding for the 17-kDa protein is found 2.3 kb downstream of the papG gene (P fimbrial adhesin) of the well characterized pap gene cluster. This ORF is however not included in the cloned pap gene determinant that has been used for most of the studies and is evidently not required for P fimbriae biogenesis per se in E. coli K-12 [7], [8]. In the same strain, a highly homologous ORF (also termed the 17-kDa gene) is found 0.3 kb downstream of the prsG gene (P fimbrial adhesin) of the prs (pap related sequences) determinant [9].

All 17-kDa genes are approximately 0.5 kb in size and their GC-content is less than 40% compared to approximately 50% in other E. coli chromosomal genes [9]. According to predictions, the 17-kDa genes encode for putative cytoplasmatic proteins that share homology with regulatory proteins belonging to the MarR family. However, the biological role and mode of action of the 17-kDa protein family have remained unknown. Since the 17-kDa genes are found nearby different fimbrial operons, the genes have nevertheless been given designations in accordance to their respective closely situated genetic loci, e.g. papX, prsX, sfaXI and sfaXII, but in neither case has any direct functional relationship to fimbrial expression been demonstrated. Here, the sfaXII gene, a 17-kDa gene encoded in close proximity to the sfaII determinant of the NMEC strain IHE3034 was further characterized.

Moreover, our analysis of the promoter-distal region of the sfaII fimbrial operon showed that, in addition to the sfaXII locus, it includes a gene (denoted sfaYII) coding for a protein with an EAL domain, which might be involved in 3′, 5′-cyclic-diguanylic acid (c-di-GMP) turnover. The intracellular levels of this modified nucleotide are depending on expression of diguanylate cyclases, containing a GGDEF domain, and c-di-GMP-specific phosphodiesterases, containing an EAL domain. The activity of the first class of enzymes increases the c-di-GMP levels while the latter decreases the c-di-GMP pool [10], [11], [12]. Recently it has been shown that c-di-GMP is acting as an intracellular signal molecule in many bacterial species and that it affects expression of many different classes of genes. Several genes encoding cell surface and membrane-bound proteins as well as genes for transcriptional regulators respond to altered c-di-GMP levels [13]. Our studies on the expression and putative function of the two so far uncharacterized genes downstream of the sfaII fimbrial operon, sfaXII and sfaYII, provide evidence that the corresponding gene products might act in trans as regulators of several surface structures and related functions.

Section snippets

Expression profile of the sfaXII gene

The sfaXII gene was cloned from the clinical NMEC isolate IHE3034 and sequenced (accession number AJ492821). This strain was isolated from a meningitis patient and shares the common characteristics of this highly conserved E. coli serogroup (O18:K1:H7): expression of S, type 1, and Mat fimbriae and lack of the determinants encoding for α-haemolysin, P, and type 1C fimbriae [14], [15], [16]. It is thereby considered to carry only one copy of the 17-kDa gene family in contrast to many typical

Bacterial strains, plasmids, and growth conditions

The E. coli strains and plasmids used in the present work are described in Table 1 and the primers used are described in Table S1, supplementary data. Unless otherwise stated, the strains were grown at 37 °C in either Luria-Bertani (LB) broth with vigorous shaking or on tryptone yeast (TYS) agar. When necessary, antibiotics were added at following concentrations: Carbenicillin (Cb) - 50 μg ml−1, Kanamycin (Km) - 50 μg ml−1 and Chloramphenicol (Cm) - 12 μg ml−1.

Cloning and recombinant DNA techniques

Molecular genetic manipulations were

Acknowledgements

This work was carried out in the frame of the European Virtual Institute for Functional Genomics of Bacterial Pathogens (CEE LSHB-CT-2005-512061) and the ERA-NET project “Deciphering the intersection of commensal and extraintestinal pathogenic E. coli” and was supported by grants from the Swedish Research Council, the Swedish Foundation for International Cooperation in Research and Higher Education (STINT), the Faculty of Medicine, Umeå University, and it was performed within the Umeå Centre

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