Umeå University's logo

The genomes of pathogenic E. coli may contain several different fimbrial operons. How bacteria regulate and coordinate the choice of fimbrial expression under different circumstances remains largely unanswered. In this report we have investigated the role of the sfaX_II gene associated to the Sfa_II fimbrial determinant in the E. coli isolate IHE3034. sfaX_II belongs to a subfamily of genes, the 17kDa genes, located near different fimbrial operons in uropathogenic and newborn meningitis E. coli (NMEC) strains. Using the NMEC isolate IHE3034 and non-pathogenic E. coli strains we found that the sfaX_II gene had an inhibitory effect on type 1 fimbriae expression. Down regulation of type 1 fimbriae was exerted at transcriptional level both by inhibiting expression from the fimA promoter and by reducing the frequency of OFF-to-ON switching. The effect of sfaX_II on expression of the recombinase FimB that catalyzes OFF to ON switching might explain the described reduction in percentage of ON cells. Moreover, expression of the sfaX_II gene strongly influenced motility and flagella production of the NMEC isolate IHE3034. We propose that the sfaX_II gene, and presumably other members in the 17kDa gene family, may play a role in the control of virulence related gene expression in pathogenic E. coli.

S pili are members of the chaperone-usher-pathway-assembled pili family that are predominantly associated with neonatal meningitis (S(II)) and believed to play a role in ascending urinary tract infections (S(I)). We used force-measuring optical tweezers to characterize the intrinsic biomechanical properties and kinetics of S(II) and S(I) pili. Under steady-state conditions, a sequential unfolding of the layers in the helix-like rod occurred at somewhat different forces, 26 pN for S(II) pili and 21 pN for S(I) pili, and there was an apparent difference in the kinetics, 1.3 and 8.8 Hz. Tests with bacteria defective in a newly recognized sfa gene (sfaX (II)) indicated that absence of the sfaX (II) gene weakens the interactions of the fimbrium slightly and decreases the kinetics. Data of S(I) are compared with those of previously assessed pili primary associated with urinary tract infections, the P and type 1 pili. S pili have weaker layer-to-layer bonds than both P and type 1 pili, 21, 28 and 30 pN, respectively. In addition, the S pili kinetics are ~10 times faster than the kinetics of P pili and ~550 times faster than the kinetics of type 1 pili. Our results also show that the biomechanical properties of pili expressed ectopically from a plasmid in a laboratory strain (HB101) and pili expressed from the chromosome of a clinical isolate (IHE3034) are identical. Moreover, we demonstrate that it is possible to distinguish, by analyzing force-extension data, the different types of pili expressed by an individual cell of a clinical bacterial isolate.

We describe the expression and regulation of the gene sfaX_II located near the Sfa_II fimbrial determinant in the newborn meningitis Escherichia coli (NMEC) isolate IHE3034. sfaX_II belongs to a gene family, the 17kDa genes, typically located downstream (300 – 3000 bp) of different fimbrial operons found in E. coli isolates of uropathogenic and newborn meningitis origin. Using transcriptional sfaX_II reporter gene fusions we found that different environmental conditions commonly affecting expression of fimbrial genes also affected sfaX_II expression. Analysis of the sfaX_II 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 sfaX_II 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 sfaX_II mutant derivative of IHE3034 did not reveal effects on Sfa_II fimbrial biogenesis as monitored by e.g. immunofluorescence microscopy. Nevertheless, a mutation in sfaX_II resulted in altered expression of other surface components. Moreover, we define a new gene, sfaY_II, coding for a putative phosphodiesterase that is located in between the sfaX_II gene and the fimbrial biogenesis genes. Our studies by ectopic expression of sfaY_II in Vibrio cholerae showed that the gene product caused reduced biofilm formation and it is proposed that sfaY_II 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.

The regulatory gene sfaX_II in the S fimbrial gene cluster of the newborn meningitis E. coli isolate IHE3034 is expressed in a growth phase dependent fashion with the highest levels at the onset of the stationary phase. It is then mainly transcribed from a promoter proximal to the gene. We have assessed the potential influence by the stationary phase sigma factor, σ^S, in the sfaX_II regulation. In contrast to the stimulatory role commonly seen with stationary phase induced genes, we found that σ^S exerted a strong repressive effect on sfaX_II transcription. Tests with an hfq mutant strain suggested that also the RNA chaperon Hfq caused negative regulation of sfaX_II transcription. In both cases the effects were considered indirect via some other regulatory factor(s). Results from a transposon insertion mutagenesis experiment indicated that it may be possible to identify additional genes involved in sfaX_II regulation.