Since the discovery of the Escherichia coli leucine-responsive regulatory protein (Lrp) almost 50 years ago, hundreds of Lrp homologs have been discovered, occurring in 45% of sequenced bacteria and almost all sequenced archaea. Lrp-like proteins are often referred to as the feast/famine regulatory proteins (FFRPs), reflecting their common regulatory roles. Acting as either global or local transcriptional regulators, FFRPs detect the environmental nutritional status by sensing small effector molecules (usually amino acids) and regulate the expression of genes involved in metabolism, virulence, motility, nutrient transport, stress tolerance, and antibiotic resistance to implement appropriate behaviors for the specific ecological niche of each organism. Despite FFRPs' complexity, a significant role in gene regulation, and prevalence throughout prokaryotes, the last comprehensive review on this family of proteins was published about a decade ago. In this review, we integrate recent notable findings regarding E. coli Lrp and other FFRPs across bacteria and archaea with previous observations to synthesize a more complete view on the mechanistic details and biological roles of this ancient class of transcription factors.

Regulation of the uropathogenic Escherichia coli (UPEC) fimB and fimE genes was examined following type 1 pili binding to mannose-coated Sepharose beads. Within 25 min after mannose attachment, fimE expression dropped eightfold, whereas fimB transcription increased about two- to fourfold. Because both fim genes encode site-specific recombinases that affect the position of the fimS element containing the fimA promoter, the positioning of fimS was also examined. The fimS element changed to slightly more Phase-OFF in bacteria mixed with plain beads, whereas UPEC cells interacting with mannose-coated beads had significantly less Phase-OFF orientation of fimS under pH 7 conditions. On the other hand, Phase-OFF oriented fimS increased fourfold when UPEC cells were mixed with plain beads in a pH 5.5 environment. Positioning of fimS was also affected by fimH mutations, demonstrating that the FimH ligand binding to its receptor facilitates the changes. Moreover, enzyme immunoassays showed that UPEC cells had greater type 1 pili expression when mixed with mannose-coated beads versus plain beads. These results indicate that, after type 1 pilus binding to tethered mannose receptors, the physiology of the E. coli cells changes to maintain the expression of type 1 pili even when awash in an acidic environment.

This review provides a brief review of the current understanding of the structure-function relationship of the Escherichia coli nucleoid developed after the overview by Pettijohn focusing on the physical properties of nucleoids. Isolation of nucleoids requires suppression of DNA expansion by various procedures. The ability to control the expansion of nucleoids in vitro has led to purification of nucleoids for chemical and physical analyses and for high-resolution imaging. Isolated E. coli genomes display a number of individually intertwined supercoiled loops emanating from a central core. Metabolic processes of the DNA double helix lead to three types of topological constraints that all cells must resolve to survive: linking number, catenates, and knots. The major species of nucleoid core protein share functional properties with eukaryotic histones forming chromatin; even the structures are different from histones. Eukaryotic histones play dynamic roles in the remodeling of eukaryotic chromatin, thereby controlling the access of RNA polymerase and transcription factors to promoters. The E. coli genome is tightly packed into the nucleoid, but, at each cell division, the genome must be faithfully replicated, divided, and segregated. Nucleoid activities such as transcription, replication, recombination, and repair are all affected by the structural properties and the special conformations of nucleoid. While it is apparent that much has been learned about the nucleoid, it is also evident that the fundamental interactions organizing the structure of DNA in the nucleoid still need to be clearly defined.

We have demonstrated that SlyA activates fimB expression and hence type 1 fimbriation, a virulence factor in Escherichia coli. SlyA is shown to bind to two operator sites (O(SA1) and O(SA2)), situated between 194 and 167 base pairs upstream of the fimB transcriptional start site. fimB expression is derepressed in an hns mutant and diminished by a slyA mutation in the presence of H-NS only. H-NS binds to multiple sites in the promoter region, including two sites (H-NS2 and H-NS3) that overlap O(SA1) and O(SA2), respectively. Mutations that disrupt either O(SA1) or O(SA2) eliminate or reduce the activating effect of SlyA but have different effects on the level of expression. We interpret these results as reflecting the relative competition between SlyA and H-NS binding. Moreover we show that SlyA is capable of displacing H-NS from its binding sites in vitro. We suggest SlyA binding prevents H-NS binding to H-NS2 and H-NS3 and the subsequent oligomerization of H-NS necessary for full inhibition of fimB expression. In addition, we show that SlyA activates fimB expression independently of two other known regulators of fimB expression, NanR and NagC. It is demonstrated that the rarely used UUG initiation codon limits slyA expression and that low SlyA levels limit fimB expression. Furthermore, Western blot analysis shows that cells grown in rich-defined medium contain ~1000 SlyA dimers per cell whereas those grown in minimal medium contain >20% more SlyA. This study extends our understanding of the role that SlyA plays in the host-bacterial relationship.

The DNA-binding mode of archaeal feast/famine-regulatory proteins (FFRPs), i.e. paralogs of the Esherichia coli leucine-responsive regulatory protein (Lrp), was studied. Using the method of systematic evolution of ligands by exponential enrichment (SELEX), optimal DNA duplexes for interacting with TvFL3, FL10, FL11 and Ss-LrpB were identified as TACGA[AAT/ATT]TCGTA, GTTCGA[AAT/ATT]TCGAAC, CCGAAA[AAT/ATT]TTTCGG and TTGCAA[AAT/ATT]TTGCAA, respectively, all fitting into the form abcdeWWWedcba. Here W is A or T, and e.g. a and a are bases complementary to each other. Apparent equilibrium binding constants of the FFRPs and various DNA duplexes were determined, thereby confirming the DNA-binding specificities of the FFRPs. It is likely that these FFRPs recognize DNA in essentially the same way, since their DNA-binding specificities were all explained by the same pattern of relationship between amino-acid positions and base positions to form chemical interactions. As predicted from this relationship, when Gly36 of TvFL3 was replaced by Thr, the b base in the optimal DNA duplex changed from A to T, and, when Thr36 of FL10 was replaced by Ser, the b base changed from T to G/A. DNA-binding characteristics of other archaeal FFRPs, Ptr1, Ptr2, Ss-Lrp and LysM, are also consistent with the relationship.

Phase variation (PV) of surface molecules and other phenotypes is a major adaptive strategy of pathogenic and commensal bacteria. Phase variants are produced at high frequencies and in a reversible manner by hypermutation or hypervariable methylation in specific regions of the genome. The major mechanisms of PV involve site-specific recombination, homologous recombination, simple sequence DNA repeat tracts or epigenetic modification by the dam methylase. PV rates of some of these mechanisms are subject to the influence of genome maintenance pathways such as DNA replication, recombination and repair while others are independent of these pathways. For each of these mechanisms, the rate of generation of phase variants is controlled by intrinsic and dispensable factors. These factors can impart environmental regulation on switching rates while many factors are subject to heterogeneity both within isolates of a species and between species. A major gap in our understanding is whether these environmental and epidemiological variations in PV rate have a major impact on fitness. Experimental approaches to studying the biological relevance of differing PV rates are being developed, and a recent intriguing finding is of a co-ordination of switching rates in the phase variable P-pili of uropathogenic bacteria.

Pathogenic Escherichia coli 4787 (O115:KV165) causes septicemia in pigs and expresses the fimbriae F165(1) encoded by the foo operon that belongs to the P fimbrial family. fooI and fooB, encoding specific foo regulators, are divergently transcribed; their intergenic region is responsible for the regulation of foo expression. The role of global and local supercoiling (transcription-induced supercoiling within the intergenic region) on the regulation of foo expression was investigated. Expression of fooB was significantly altered when global negative supercoiling was reduced by a mutation that decreases DNA gyrase activity. Deletion of the topA gene, encoding for topoisomerase I that relaxes local negative supercoiling, further reduced fooB expression. This suggests that both global and local supercoiling can significantly affect fooB expression. Moreover, FooI, a positive regulator of fooB expression, has no effect on fooB expression in the topA null mutant. This study showed that divergent transcription from a strong promoter can significantly enhance fooB expression and compensate for the absence of FooI in a wild-type strain.

Phase-variable expression of type 1 fimbriae in Escherichia coli K-12 involves inversion by site-specific recombination of a 314 bp sequence containing the promoter for fim structural gene expression. The invertible sequence is flanked by 9 bp inverted repeats, and each repeat is in turn flanked by non-identical recombinase-binding elements (RBEs) to which the FimB or FimE site-specific recombinases bind. These proteins have distinct DNA inversion preferences: FimB inverts the switch in the ON-to-OFF and OFF-to-ON directions with similar efficiencies, whereas FimE inverts it predominantly in the ON-to-OFF direction. We have found that FimB and FimE invert the switch through a common mechanism. A genetic investigation involving base-by-base substitution combined with a biochemical study shows that the same DNA cleavage and religation sites are used within the 9 bp inverted repeats, and that each recombination involves a common 3 bp spacer region. A comprehensive programme of RBE exchanges and replacements reveals that FimB is much more tolerant of RBE sequence variation than FimE. The asymmetric location of conserved 5'-CA motifs at either side of each spacer region allows the inside and outside of the switch to be differentiated while the RBE sequence heterogeneity permits its ON and OFF forms to be distinguished by the recombinases.

Expression of the FimB recombinase, and hence the OFF-to-ON switching of type 1 fimbriation in Escherichia coli, is inhibited by sialic acid (Neu(5)Ac) and by GlcNAc. NanR (Neu(5)Ac-responsive) and NagC (GlcNAc-6P-responsive) activate fimB expression by binding to operators (O(NR) and O(NC1) respectively) located more than 600 bp upstream of the fimB promoter within the large (1.4 kb) nanC-fimB intergenic region. Here it is demonstrated that NagC binding to a second site (O(NC2)), located 212 bp closer to fimB, also controls fimB expression, and that integration host factor (IHF), which binds midway between O(NC1) and O(NC2), facilitates NagC binding to its two operator sites. In contrast, IHF does not enhance the ability of NanR to activate fimB expression in the wild-type background. Neither sequences up to 820 bp upstream of O(NR), nor those 270 bp downstream of O(NC2), are required for activation by NanR and NagC. However, placing the NanR, IHF and NagC binding sites closer to the fimB promoter enhances the ability of the regulators to activate fimB expression. These results support a refined model for how two potentially key indicators of host inflammation, Neu(5)Ac and GlcNAc, regulate type 1 fimbriation.

Fimbria-mediated interaction with the host elicits both innate and adaptive immune responses, and thus their expression may not always be beneficial in vivo. Furthermore, the metabolic drain of producing fimbriae is significant. It is not surprising, therefore, to find that fimbrial production in Escherichia coli and Salmonella enterica is under extensive environmental regulation. In many instances, fimbrial expression is regulated by phase variation, in which individual cells are capable of switching between fimbriate and afimbriate states to produce a mixed population. Mechanisms of phase variation vary considerably between different fimbriae and involve both genetic and epigenetic processes. Notwithstanding this, fimbrial expression is also sometimes controlled at the posttranscriptional level. In this chapter, we review key features of the regulation of fimbrial gene expression in E. coli and Salmonella. The occurrence and distribution of fimbrial operons vary significantly among E. coli pathovars and even among the many Salmonella serovars. Therefore, general principles are presented on the basis of detailed discussion of paradigms that have been extensively studied, including Pap, type 1 fimbriae, and curli. The roles of operon specific regulators like FimB or CsgD and of global regulatory proteins like Lrp, CpxR, and the histone-like proteins H-NS and IHF are reviewed as are the roles of sRNAs and of signalling nucleotide cyclic-di-GMP. Individual examples are discussed in detail to illustrate how the regulatory factors cooperate to allow tight control of expression of single operons. Molecular networks that allow coordinated expression between multiple fimbrial operons and with flagella in a single isolate are also presented. This chapter illustrates how adhesin expression is controlled, and the model systems also illustrate general regulatory principles germane to our overall understanding of bacterial gene regulation.

Site-specific recombinases of the integrase family usually require cofactors to impart directionality in the recombination reactions that they catalyze. The FimB integrase inverts the Escherichia coli fim switch (fimS) in the on-to-off and off-to-on directions with approximately equal efficiency. Inhibiting DNA gyrase with novobiocin caused inversion to become biased in the off-to-on direction. This directionality was not due to differential DNA topological distortion of fimS in the on and off phases by the activity of its resident P(fimA) promoter. Instead, the leucine-responsive regulatory (Lrp) protein was found to determine switching outcomes. Knocking out the lrp gene or abolishing Lrp binding sites 1 and 2 within fimS completely reversed the response of the switch to DNA relaxation. Inactivation of either Lrp site alone resulted in mild on-to-off bias, showing that they act together to influence the response of the switch to changes in DNA supercoiling. Thus, Lrp is not merely an architectural element organizing the fim invertasome, it collaborates with DNA supercoiling to determine the directionality of the DNA inversion event.