Bart J. A. M. Jordi

The positive regulator CfaD overcomes the repression mediated by histone-like protein H-NS (H1) in the CFA/I fimbrial operon of Escherichia coli.

CFA/I fimbriae of enterotoxigenic Escherichia coli are expressed at 37 degrees C and not at 20 degrees C. Expression of CFA/I fimbriae requires two DNA regions (regions 1 and 2) which are separated by 40 kb on the wild type plasmid. Region 2 encodes a protein (CfaD) which activates the promoter in region 1. We investigated whether the histone‐like protein H‐NS (H1) of E.coli is involved in the temperature regulated expression of CFA/I fimbriae. As demonstrated recently with other temperature regulated genes, a mutation in the gene coding for this nucleoid‐associated H‐NS (H1) protein resulted in derepression of CFA/I expression. CFA/I fimbriae were now expressed both at 20 degrees C and 37 degrees C. More strinkingly, the positive regulator CfaD was not needed for CFA/I expression in an H‐NS‐ strain. This indicates that CfaD diminishes an inhibitory effect of the H‐NS nucleoid‐associated protein. We also showed that in the H‐NS‐ strain the CfaD protein still has a positive effect on the transcription of CFA/I.

H-NS and Lrp serve as positive modulators of traJ expression from the Escherichia coli plasmid pRK100

Conjugative transfer of F-like plasmids is a tightly regulated process. The TraJ protein is the main positive activator of the tra operon which encodes products required for conjugative transfer of F-like plasmids. Nucleotide sequence analysis revealed potential Lrp and H-NS binding sites in the traJ regulatory region. Expression of a traJ-lacZ fusion in hns and lrp mutant strains showed that both are positive modulators of traJ expression. Competitive RT-PCR demonstrated that H-NS and Lrp exert their effect at the transcriptional level. Electrophoretic mobility-shift assays showed that H-NS and Lrp proteins bind to the traJ promoter. Conjugative transfer of pRK100 was decreased in hns but not in lrp mutant strains. Together, the results indicate H-NS and Lrp function as activators of traJ transcription.

The Cyclic AMP-Cyclic AMP Receptor Protein Complex Regulates Activity of the traJ Promoter of the Escherichia coli Conjugative Plasmid pRK100

The TraJ protein is a central activator of F-like plasmid conjugal transfer. In a search for regulators of traJ expression, we studied the possible regulatory role of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex in traJ transcription using a traJ-lacZ reporter system. A comparison of the enzyme activities in the wild-type Escherichia coli strain MC4100 with those in cya and crp mutants indicated that disruption of the formation of the cAMP-CRP complex negatively influenced the activity of the traJ promoter of the F-like plasmid pRK100. The defect in the cya mutant was partially restored by addition of exogenous cAMP. Competitive reverse transcription-PCR performed with RNA isolated from the wild-type and mutant strains showed that the cAMP-CRP complex exerted its effect at the level of transcription. Electrophoretic mobility shift assays with purified CRP demonstrated that there was direct binding of CRP to the traJ promoter region. DNase I footprint experiments mapped the CRP binding site around position -67.5 upstream of the putative traJ promoter. Targeted mutagenesis of the traJ promoter region confirmed the location of the CRP binding site. Consistent with the demonstrated regulation of TraJ by the cAMP-CRP complex, mutants with defects in cya or crp exhibited reduced conjugal transfer from pRK100.

Regions of the CFA/I promoter involved in the activation by the transcriptional activator CfaD and repression by the histone-like protein H-NS

Expression of CFA/I fimbriae of Escherichia coli requires the transcriptional activator CfaD. The mechanism by which CfaD activates the CFA/I promoter is to overcome the repression by H-NS, one of the histone-like proteins in E coli. This study addresses the question of which sequences in the promoter region of CFA/I interact with CfaD and H-NS. In order to determine this, deletion mutants of the CFA/I promoter were constructed and cloned upstream of the promoterless lacZ gene. The effect of CfaD and H-NS on the expression of these constructs was determined.

Codon-usage based regulation of colicin K synthesis by the stress alarmone ppGpp: Translational regulation of colicin K by ppGpp

The molecular mechanism of the upregulation of Escherichia coli colicin K (Cka) synthesis during stress conditions was studied. Nutrient starvation experiments and the use of relA spoT mutant strains, IPTG‐regulated overproduction of ppGpp and lacZ fusions revealed that the stringent response alarmone guanosine 3′,5′‐bispyrophosphate (ppGpp) is the main positive effector of Cka synthesis. Comparison of the amounts of protein produced (Western blotting) and specific mRNA (Northern blotting) before and after nutrient starvation demonstrated increases in Cka protein with unaltered specific mRNA levels, suggesting a post‐transcriptional regulatory mechanism. Reporter (β‐galactosidase) assays using truncated cka of variable length fused to lacZ located the key regulatory region close to the 5′ end of the cka mRNA. Closer analysis of this region indicated the presence of several rare codons, including the leucine‐encoding codon CUA. Synonymous exchange of the rare codons with more frequently used ones abolished the regulatory effect of ppGpp. Supplementation of the strain with the plasmid CodonPlus carrying several rare tRNA genes yielded similar results, indicating that codon usage (in particular, the fifth codon for the amino acid leucine) and tRNA availability (i.e. tRNAleu) are the key elements of the regulatory function of ppGpp. We conclude that ppGpp regulates Cka synthesis via a novel post‐transcriptional mechanism that is based on rare codon usage and variable cognate tRNA availability.

Regulation of Expression of Fimbriae of Human Enterotoxigenic Escherichia Coli

Adhesion of bacteria to epithelial cells of the host is the first and probably the most important step in the pathogenesis of bacterial infections. In humans, adhesion of enterotoxigenic E.coli (ETEC) to the mucosa of the small intestines is mediated by serologically distinct and host-specific fimbrial antigens on the bacterial surface. The first fimbrial antigen to be identified in human ETEC strains was colonization factor antigen I (CFA/I)1. CFA/I is a single fimbrial antigen found on ETEC producing only heat-stable (ST) enterotoxin and on ETEC producing both heat-labile and heat-stable enterotoxin (LT, ST) of at least 15 serogroups. Later CFA/11 was detected2 and was subsequently shown to consist of three distinct coli surface associated (CS) antigens, designated CS1, CS2 and CS33. Nearly all CFA/II-positive E.coli produce CS3, but those of serotype 06.H16 or 06. H- also express either CS1 or CS2 depending on their biotype: biotype A strains produce CS1 whereas those of biotypes B, C and F express CS23. Production of CS1 and CS3 has been reported in only one other ETEC strain. This strain was of serotype 0139.H28 and differs from other 0139.H28 strains that are CS3 only producers4.