The leucine-responsive regulatory protein binds to the fim switch to control phase variation of type 1 fimbrial expression in Escherichia coli K-12

Phase variation of type 1 fimbriation in Escherichia coli is associated with the site-specific recombination of a 314-bp DNA invertible element. The fim switch directs transcription of fimA, the major fimbrial subunit gene, in one orientation (on) but not the other (off). Switching requires either fimB (on-to-off or off-to-on inversion) or fimE (on-to-off inversion only) and is reduced sharply in strains containing lrp::Tn10 mutations. Both fimE-promoted switching and fimB-promoted switching are stimulated by the amino acids alanine, isoleucine, leucine, and valine, and this regulation requires lrp. Here it is shown that the leucine-responsive regulatory protein (Lrp) binds in and adjacent to the fim switch. Mutations in fim that lower Lrp binding in vitro have corresponding effects on both fimB-promoted switching and fimE-promoted switching in vivo. Lrp initiates binding at one of two sites within the fim switch. Additional cooperative binding results in an extensive region of protection from both DNase I and 1,10-phenanthroline-copper complex-activated DNA cleavage. The region of protection can extend to within 12 bp of the right inverted repeat (switch off) and occupies over one-third of the switch. It is proposed that wrapping of fim DNA around an Lrp complex is required to form a recombination-proficient structure

Modelling fim expression in Escherichia coli K12

Fimbriae are structures in Escherichia coli, the expression of which is controlled by the fim operon. Understanding this expression is important because the fimbriae are important virulence factors. This expression can be studied using targeted mutations to the DNA, which can be used to disable binding or transcription of a protein. How- ever, this can be problematic as only the net effect is observed. Turning off expression of a protein may enhance fim expression, but deactivating this protein may also repress another protein that functions as an activator of fim expression. The net result may be that fim expression goes down, so it would seem at first glance that the disabled protein was an activator of fim expression and not a repressor. In order to understand this complex network of interactions, an agent based model of fim expression has been created. The subject of this paper is to introduce this model and to use it to disambiguate between a number of hypotheses about this system. Parameters such as binding probability will be optimised using a genetic algorithm. The final model and parameters show a good match to experimental data

Orientational Control is an Efficient Control Mechanism for Phase Switching in the E coli fim System

The 'fim' system in 'E. coli' controls the expression of type-1 fimbriae. These are hair-like structures that can be used to attach to host cells. Fimbriation is controlled by a mechanism called ''orientational control''. We present two families of models for orientational control to understand the details of how it works. We find that the main benefits of orientational control are that (i) it allows rapid adjustment of fimbriation levels in response to a change of environmental conditions while (ii) keeping the overall frequencies with which a cell switches between the fimbriate state and the afimbriate state low. The main reason for the efficiency of orientational control in regulation of fimbriation levels is that it keeps the system far from its steady state

A theoretical interpretation of the transient sialic acid toxicity of a nanR mutant of Escherichia coli

This article reports on experimental evidence that an Escherichia coli nanR mutant shows inhibited growth in N-acetylneuraminic acid. This effect is prevented when inocula are grown in an excess of glucose, but not in an excess of glycerol. The nanATEK operon is controlled by catabolite repression, suggesting that diminished expression of the nanATEK operon in the presence of glucose explains the inocula effects. Neither double nanR-nagC nor nanR dam mutants show growth inhibition in the presence of N-acetylneuraminic acid. A theoretical model of N-acetylneuraminic acid metabolism (i.e., in particular of the nanATEK and nagBACD operons) is presented; the model suggests an interpretation of this effect as being due to transient high accumulations of GlcNAc-6P in the cell. This accumulation would lead to suppression of central metabolic functions of the cell, thus causing inhibited growth. Based on the theoretical model and experimental data, it is hypothesised that the nanATEK operon is induced in a two-step mechanism. The first step is likely to be repressor displacement by N-acetylneuraminic acid. The second stage is hypothesised to involve Dam methylation to achieve full induction

Sialic acid and N-acetylglucosamine Regulate type 1 Fimbriae Synthesis.

Type 1 fimbriae of E. coli, a chaperon-usher bacterial adhesin, are synthesized by the majority of strains of the bacterium. Although frequently produced by commensal strains, the adhesin is nevertheless a virulence factor in Extraintestinal Pathogenic E. coli (ExPEC). The role of the adhesin in pathogenesis is best understood in Uropathogenic E. coli (UPEC). Host attachment and invasion by type 1 fimbriate bacteria activates inflammatory pathways, with TLR4 signaling playing a predominant role. In a mouse model of cystitis, type 1 fimbriation not only enhances UPEC adherence to the surface of superficial umbrella cells of the bladder urothelium, but is both necessary and sufficient for their invasion. Moreover the adhesin plays a role in the formation of transient intracellular bacterial communities (IBCs) within the cytoplasm of urothelial cells as part of UPEC cycles of invasion. The expression of type 1 fimbriation is controlled by phase variation at the transcriptional level, a mode of gene regulation in which bacteria switch reversibly between fimbriate and afimbriate phases. Phase variation has been widely considered to be a mechanism enabling immune evasion. Notwithstanding the apparently random nature of phase variation, switching of type 1 fimbrial expression is nevertheless controlled by a range of environmental signals that include the amino sugars sialic acid and N-acetylglucosamine (GlcNAc). Sialic acid plays a pivotal role in innate immunity, including signaling by the toll-like receptors. Here how sialic acid and GlcNAc control type 1 fimbriation is described and the potential significance of this regulatory response is discussed

The regulation of pap and type 1 fimbriation in Escherichia coli

The ability of bacterial pathogens to bind to the host mucosa is a critical step in the pathogenesis of many bacterial infections and, for Escherichia coli, a large number of different fimbrial adhesins have been implicated as virulence factors. In this chapter, our current understanding of the regulatory mechanisms that control the expression of two of the best characterized fimbrial adhesins, pyelonephritis-associated pilus (encoded by pap) and the type 1 fimbria (encoded by fim), will be described. The expression of both fimbrial adhesins is controlled by phase variation (the reversible and apparently random switching between expressing ('on') and non-expressing ('off') states), and is regulated in response to environmental conditions. The phase variation of pap (and of some other fimbriae in Escherichia coli) is determined by the formation of alternative nucleoprotein complexes that either activate (phase 'on') or suppress (phase 'off') transcription of the fimbria genes. Formation of each complex protects a single Dam methylation site (5' GATC) from modification (GATCdist in phase 'on' cells and GATCprox in phase 'off' cells). Furthermore, complex formation is inhibited by methylation of the two 5' GATC sites. Both the phase variation of pap and the transcription of the pap genes in phase 'on' cells, are regulated and expression is subject to both positive and negative feedback control. In contrast to pap, the phase variation of fim is determined by the site-specific inversion of a short element of DNA (the fim switch). In phase 'on' cells, a promoter within the invertible element directs the transcription of the fim structural genes, whereas in phase 'off' cells transcription of the fimbrial genes is silenced. Despite the very different molecular mechanisms controlling the expression of pap and fim, the two systems share many features in common and have probably evolved to fulfill the same function. In addition to details about the molecular mechanisms that control pap and fim, the possible physiological significance of the observed regulation will be discussed

Modulation of the Sensitivity of FimB Recombination to Branched-Chain Amino Acids and Alanine in Escherichia coli K-12

Phase variation of type 1 fimbriae of Escherichia coli requires the site-specific recombination of a short invertible element. Inversion is catalyzed by FimB (switching in either direction) or FimE (inversion mainly from on to off) and is influenced by auxiliary factors integration host factor (IHF) and leucine-responsive regulatory protein (Lrp). These proteins bind to sites (IHF site II and Lrp sites 1 and 2) within the invertible element to stimulate recombination, presumably by bending the DNA to enhance synapses. Interaction of Lrp with a third site (site 3) cooperatively with sites 1 and 2 (termed complex 1) impedes recombination. Inversion is stimulated by the branched-chain amino acids (particularly leucine) and alanine, and according to a current model, the amino acids promote the selective loss of Lrp from site 3 (complex 2). Here we show that the central portion of the fim invertible element, situated between Lrp site 3 and IHF site II, is dispensable for FimB recombination but that this region is also required for full amino acid stimulation of inversion. Further work reveals that the region is likely to contain multiple regulatory elements. Lrp site 3 is shown to bind the regulatory protein with low affinity, and a mutation that enhances binding to this element is found both to diminish the stimulatory effects of IVLA on FimB recombination and to inhibit recombination in the absence of the amino acids. The results obtained emphasize the importance of Lrp site 3 as a control element but also highlight the complexity of the regulatory system that affects this site

Direct observation of type 1 fimbrial switching

The defining feature of bacterial phase variation is a stochastic 'all-or-nothing' switching in gene expression. However, direct observations of these rare switching events have so far been lacking, obscuring possible correlations between switching events themselves, and between switching and other cellular events, such as division and DNA replication. We monitored the phase variation of type 1 fimbriae in individual Escherichia coli in real time and simultaneously tracked the chromosome replication process. We observed distinctive patterns of fim (fimbriae) expression in multiple genealogically related lineages. These patterns could be explained by a model that combines a single switching event with chromosomal fim replication, as well as the epigenetic inheritance of expressed fim protein and RNA, and their dilution by growth. Analysis of the moment of switching at sub-cell-cycle resolution revealed a correlation between fim switching and cell age, which challenges the traditional idea of phase variation as a random Poissonian phenomenon