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
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
References (56)
- et al.
Genome-wide transcriptional profile of Escherichia coli in response to high levels of the second messenger 3′,5′-cyclic diguanylic acid
J Biol Chem
(2006) - et al.
Mutational analysis of the PapB transcriptional regulator in Escherichia coli. Regions important for DNA binding and oligomerization
J Biol Chem
(1999) - et al.
The Yersinia high-pathogenicity island is present in different members of the family Enterobacteriaceae
FEMS Microbiol Lett
(2000) - et al.
Radioimmunological screening method for specific membrane proteins
Anal Biochem
(1979) - et al.
A complementation analysis of the restriction and modification of DNA in Escherichia coli
J Mol Biol
(1969) - et al.
Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors
Gene
(1985) - et al.
Construction and characterization of new cloning vehicles. II. A multipurpose cloning system
Gene
(1977) - et al.
Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector
Gene
(1986) - et al.
Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria
Gene
(1997) - et al.
Extraintestinal pathogenic Escherichia coli
Foodborne Pathog Dis
(2007)
Cloning and characterization of genes involved in production of mannose-resistant, neuraminidase-susceptible (X) fimbriae from a uropathogenic O6:K15:H31 Escherichia coli strain
Infect Immun
Functional analysis of the sialic acid-binding adhesin SfaS of pathogenic Escherichia coli by site-specific mutagenesis
Infect Immun
Cloning and characterization of the S fimbrial adhesin II complex of an Escherichia coli O18:K1 meningitis isolate
Infect Immun
S-Fimbria-encoding determinant sfa(I) is located on pathogenicity island III(536) of uropathogenic Escherichia coli strain 536
Infect Immun
Analysis of the S-fimbriae-minor-subunit-proteins mediating adherence of pathogenic Escherichia coli
Mutations in E coli cistrons affecting adhesion to human cells do not abolish Pap pili fiber formation
EMBO J
Genes of pyelonephritogenic E. coli required for digalactoside-specific agglutination of human cells
EMBO J
Horizontal gene transfer of the Escherichia coli pap and prs pili operons as a mechanism for the development of tissue-specific adhesive properties
Mol Microbiol
GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility
Mol Microbiol
Cyclic diguanylate is a ubiquitous signaling molecule in bacteria: insights into biochemistry of the GGDEF protein domain
J Bacteriol
The ubiquitous protein domain EAL is a cyclic diguanylate-specific phosphodiesterase: enzymatically active and inactive EAL domains
J Bacteriol
Serotypes, hemolysin production, and receptor recognition of Escherichia coli strains associated with neonatal sepsis and meningitis
Infect Immun
matB, a common fimbrillin gene of Escherichia coli, expressed in a genetically conserved, virulent clonal group
J Bacteriol
Genetic relationships and clonal structure of strains of Escherichia coli causing neonatal septicemia and meningitis
Infect Immun
H-NS: a modulator of environmentally regulated gene expression
Mol Microbiol
Role of histone-like proteins H-NS and StpA in expression of virulence determinants of uropathogenic Escherichia coli
J Bacteriol
Use of a wild-type gene fusion to determine the influence of environmental conditions on expression of the S fimbrial adhesin in an Escherichia coli pathogen
J Bacteriol
Transcriptional activation of a Pap pilus virulence operon from uropathogenic Escherichia coli
EMBO J
Cited by (24)
Uropathogenic Escherichia coli
2013, Escherichia coli: Pathotypes and Principles of Pathogenesis: Second EditionThe c-di-GMP phosphodiesterase VmpA absent in Escherichia coli K12 strains affects motility and biofilm formation in the enterohemorrhagic O157: H7 serotype
2013, Veterinary Immunology and ImmunopathologyCitation Excerpt :Claret et al. (2007) showed that in the Crohn disease-associated adherent-invasive E. coli, c-di-GMP signaling pathways affect the fine tuning of flagellar and type-1 pili synthesis. Sjostrom et al. (2008) showed that in a newborn meningitis E. coli isolate, a putative PDE-encoding gene is present in the main S fimbrial operon. In non-pathogenic E. coli, c-di-GMP signaling has been shown to play a role in synthesis of curli, an important pathogenesis factor (Weber et al., 2006; Pesavento et al., 2008), however the role of c-di-GMP in curli synthesis in pathogenic E. coli has not been elucidated.
Expression and purification of SfaX<inf>II</inf>, a protein involved in regulating adhesion and motility genes in extraintestinal pathogenic Escherichia coli
2012, Protein Expression and PurificationCitation Excerpt :In keeping with the localization of these genes, the proteins have been named PapX, FocX, PrsX and PrfX, respectively, after the corresponding upstream operon [11,19]. The sequence identity between SfaXII and these 17-kDa proteins varies between 87–96% [9,11]. A target DNA sequence for the PapX protein was recently determined in the flhD regulatory region [20], but otherwise little is known about the properties of this family of proteins.
The SfaX<inf>II</inf> protein from newborn meningitis E. coli is involved in regulation of motility and type 1 fimbriae expression
2009, Microbial PathogenesisCitation Excerpt :In this study we showed that the sfaXII gene, present in strains with the ability to express S fimbriae, negatively affects the expression of type 1 fimbriae and flagella production. The sfaXII gene was recently shown to be part of the major sfaII fimbrial operon [15]. Here we demonstrate that sfaXII is a new component also in the regulatory cross-talk between fimbrial genetic determinants.
Metabolic and Morphotypic Trade-Offs within the Eco-Evolutionary Dynamics of Escherichia coli
2022, Microbiology Spectrum