Roles of fimB and fimE in site-specific DNA inversion associated with phase variation of type 1 fimbriae in Escherichia coli.

Evidence obtained with an improved in vivo assay of fimbrial phase variation in Escherichia coli supported a revised understanding of the roles of fimB and fimE in the site-specific DNA rearrangement with which they are associated. A previously proposed model argued that fimB and fimE play antagonistic, unidirectional roles in regulating the orientation of the invertible DNA element located immediately upstream of fimA, the gene encoding the major subunit of type 1 fimbriae. This conclusion, though, is based on an in vivo DNA inversion assay using recombinant plasmid substrates under conditions that, among other things, were incapable of detecting recombination of the fim invertible element from the on to the off orientation. Using a modified system that overcome this and several additional technical problems, we confirmed that fimB acts independently of fimE on the invertible element and that the additional presence of fimE results in the preferential rearrangement of the element to the off orientation. It is now demonstrated that fimE can act in the absence of fimB in this recombination to promote inversion primarily from on to off. In contrast to the previous studies, the effect of fimB on a substrate carrying the invertible element in the on orientation could be examined. It was found that fimB mediates DNA inversion from on to off, as well as from off to on, and that, contrary to prior interpretations, the fimB-associated inversion occurs with only minimal orientational preference to the on phase.     

Two regulatory fim genes, fimB and fimE, control the phase variation of type 1 fimbriae in Escherichia coli.

The expression of type 1 fimbriae in Escherichia coli is phase dependent, i.e. a cell is either completely fimbriated or bald. This phenomenon is due to the periodic inversion of a specific 300-bp DNA segment containing the promoter for the fimbrial subunit gene, fimA. The phase switch is controlled by the products of two regulatory genes, fimB and fimE, located upstream of fimA. The fimB and fimE proteins direct the phase switch into the 'on' and 'off' position, respectively. The DNA sequence of a 3000-bp region containing the two genes has been determined. The fimB and fimE proteins exhibit strong homology and have most likely originated by duplication of an ancestral gene. They are highly basic implying that they control the phase switch through interaction at the DNA level.     

Identification and characterization of a gene product that regulates type 1 piliation in Escherichia coli.

The recombinant plasmid pSH2 confers type 1 piliation (Pil+) on a nonpiliated (Pil-) strain of Escherichia coli K-12. At least four plasmid-encoded gene products are involved in pilus biosynthesis and expression. We present evidence which indicates that one gene encodes an inhibitor of piliation. Hyperpiliated (Hyp) mutants were isolated after Tn5 insertion mutagenesis of pSH2 and introduction of the plasmid DNA into a Pil- strain of E. coli as unique small, compact colonies. Also, Hyp mutants clumped during growth in static broth and were piliated under several cultural conditions that normally suppressed piliation. Electron microscopic examination of Hyp mutants associated an observed 40-fold increase in pilin antigen with an increase in the number and length of pili per cell. All Hyp mutants examined failed to produce a 23-kilodalton protein that was encoded by a gene adjacent to the structural (pilin) gene for type 1 pili, and all Tn5 insertion mutations that produced the Hyp phenotype mapped in this region (hyp). Piliation in Hyp mutants could be reduced to near parental levels by introducing a second plasmid containing a parental hyp gene. Thus the 23-kilodalton (hyp) protein appears to act in trans to regulate the level of piliation.     

Type 1 fimbriation and fimE mutants of Escherichia coli K-12.

We reexamined the influence of fimE, also referred to as hyp, on type 1 fimbriation in Escherichia coli K-12. We found that one strain used previously and extensively in the analysis of type 1 fimbriation, strain CSH50, is in fact a fimE mutant; the fimE gene of CSH50 contains a copy of the insertion sequence IS1. Using a recently described allelic exchange procedure, we transferred the fimE::IS1 allele from CSH50 to our present wild-type strain, MG1655. Characterization of this IS1-containing strain (AAEC137), together with another fimE mutant of MG1655 (AAEC143), led to two conclusions about the role of fimE. First, the formation of phase variant colony types, reported widely in strains of E. coli, depends on mutation of fimE, at least in K-12 strain MG1655. Here we showed that this phenomenon reflects the ability of fimE to stimulate the rapid inversion of the fim invertible element from on to off when the bacteria are grown on agar. Second, our analysis of fimE mutants, which is limited to chromosomal constructs, provided no evidence that they are hyperfimbriate. We believe that these results, which are at odds with a previous study using fim-containing multicopy plasmids, reflect differences in gene copy number.     

Isolation and characterization of Escherichia coli phase variants and mutants deficient in type 1 pilus production.

Type 1 pili of Escherichia coli are the prototype of the somatic class of pili found on many strains of bacteria. As a first step in the genetic analysis of type 1 piliation, an extensive series of nonpiliated derivatives of E. coli K-12 strain AW405, was characterized to produce attached or free pili when examined in the antiserum or appeared to produce attached or free pili when examined in the electron microscope. The derivatives fell into two classes; phase variants and mutants. Phase variants that formed colonies of two distinctive types, one associated with a predominantly piliated (P+), and the other associated with a nonpiliated (P-) phase, were obtained. Each phase could give rise to the other at a relatively high rate, which was greater in the P- to P+ direction during culture in unshaken liquid medium. In addition, 77 Pil- mutants were selected on the basis of a subtle difference in colonial morphology. The mutants reverted, if at all, at a much lower rate than that of the P- to P+ change. The stability of Pil- derivatives grown in unshaken liquid medium was used as a criterion for distinguishing between phase variants and mutants, Phase variation also effected colonial morphology and chemotactic swarming. These properties did not directly depend upon piliation since Pil- mutants were only slightly altered in colonial form and unaltered in chemotactic swarming. Piliation of Pil+ bacteria was quantitatively affected by growth conditions.     

Characterization of the fimA gene encoding bundle-forming fimbriae of the plant pathogen Xanthomonas campestris pv. vesicatoria.

The fimA gene of Xanthomonas campestris pv. vesicatoria was identified and characterized. A 20-mer degenerate oligonucleotide complementary to the N-terminal amino acid sequence of the purified 15.5-kDa fimbrillin was used to locate fimA on a 2.6-kb SalI fragment of the X. campestris pv. vesicatoria 3240 genome. The nucleotide sequence of a 1.4-kb fragment containing the fimA region revealed two open reading frames predicting highly homologous proteins FimA and FimB. FimA, which was composed of 136 amino acids and had a calculated molecular weight of 14,302, showed high sequence identity to the type IV fimbrillin precursors. fimB predicted a protein product of 135 amino acids and a molecular weight of 13,854. The open reading frame for fimB contained near the 5' end a palindromic sequence with a terminator loop potential, and the expression level of fimB in vitro and in Xanthomonas was considerably lower than that of fimA. We detected an efficiently transcribed fimA-specific mRNA of 600 bases as well as two weakly expressed, longer mRNA species that reacted with both fimA and fimB. A homolog of fimA but not of fimB was detected by Southern hybridization in strains of X. campestris pv. vesicatoria, campestris, begoniae, translucens, and graminis. A fimA::omega mutant of strain 3240 was not significantly reduced in virulence or adhesiveness to tomato leaves. However, the fimA mutant was dramatically reduced in cell aggregation in laboratory cultures and on infected tomato leaves. The fimA mutant strain also exhibited decreased tolerance to UV light.     

The molecular basis for the specificity of fimE in the phase variation of type 1 fimbriae of Escherichia coli K-12

The expression of type 1 fimbriae in Escherichia coli is phase variable, with cells switching between fimbriate (ON) and afimbriate (OFF) phases. The phase variation is dependent on the orientation of a 314 bp DNA element (the switch) that undergoes DNA inversion. DNA inversion requires either fimB or fimE, site-specific recombinases that differ in both specificity and activity. Whereas fimB promotes recombination with little orientational bias, fimE promotes recombination in the ON-to-OFF direction exclusively. In wild-type cells, fimE activity predominates and, hence, most bacteria are afimbriate. Here, it is shown that fimE specificity is caused by two different, but complementary, mechanisms. First, FimE shows a strong preference for the switch in the ON orientation as a substrate for recombination. Differences in the nucleotide sequence of the recombinase binding sites is a key factor in determining FimE specificity, although one or more additional cis-active sites that flank the fim switch also appear to be involved. Secondly, the orientation of the switch controls fimE in cis, most probably to control recombinase expression.     

The Type IV Fimbrial Subunit Gene (fimA) of Dichelobacter nodosus Is Essential for Virulence, Protease Secretion, and Natural Competence

Dichelobacter nodosus is the essential causative agent of footrot in sheep. The major D. nodosus-encoded virulence factors that have been implicated in the disease are type IV fimbriae and extracellular proteases. To examine the role of the fimbriae in virulence, allelic exchange was used to insertionally inactivate the fimA gene, which encodes the fimbrial subunit protein, from the virulent type G D. nodosus strain VCS1703A. Detailed analysis of two independently derived fimA mutants revealed that they no longer produced the fimbrial subunit protein or intact fimbriae and did not exhibit twitching motility. In addition, these mutants were no longer capable of undergoing natural transformation and did not secrete wild-type levels of extracellular proteases. These effects were not due to polar effects on the downstream fimB gene because insertionally inactivated fimB mutants were not defective in any of these phenotypic tests. Virulence testing of the mutants in a sheep pen trial conducted under controlled environmental conditions showed that the fimA mutants were avirulent, providing evidence that the fimA gene is an essential D. nodosus virulence gene. These studies represent the first time that molecular genetics has been used to determine the role of virulence genes in this slow growing anaerobic bacterium.     

Genetic analysis and morphological identification of pilus-like structures in members of the genus <Emphasis Type="Italic">Bifidobacterium</Emphasis>

BACKGROUND: Cell surface pili in Gram positive bacteria have been reported to orchestrate the colonization of host tissues, evasion of immunity and the development of biofilms. So far, little if any information is available on the presence of pilus-like structures in human gut commensals like bifidobacteria. RESULTS AND DISCUSSION: In this report, Atomic Force Microscopy (AFM) of various bifidobacterial strains belonging to Bifidobacterium bifidum, Bifidobacterium longum subsp. longum, Bifidobacterium dentium, Bifidobacterium adolescentis and Bifidobacterium animalis subsp. lactis revealed the existence of appendages resembling pilus-like structures. Interestingly, these microorganisms harbour two to six predicted pilus gene clusters in their genome, with each organized in an operon encompassing the major pilin subunit-encoding gene (designated fimA or fimP) together with one or two minor pilin subunit-encoding genes (designated as fimB and/or fimQ), and a gene encoding a sortase enzyme (strA). Quantitative Real Time (qRT)-PCR analysis and RT-PCR experiments revealed a polycistronic mRNA, encompassing the fimA/P and fimB/Q genes, which are differentially expressed upon cultivation of bifidobacteria on various glycans.