The Fis protein has a stimulating role in initiation of replication in Escherichia coli in vivo.

The Fis protein is a nucleoid associated protein that has previously been reported to act negatively in initiation of replication in Escherichia coli. In this work we have examined the influence of this protein on the initiation of replication under different growth conditions using flow cytometry. The Fis protein was found to be increasingly important with increasing growth rate. During multi-fork replication severe under-initiation occurred in cells lacking the Fis protein; the cells initiated at an elevated mass, had fewer origins per cell and the origins were not initiated in synchrony. These results suggest a positive role for the Fis protein in the initiation of replication.

Mechanical constraints on Hin subunit rotation imposed by the Fis/enhancer system and DNA supercoiling during site-specific recombination.

Hin, a member of the serine family of site-specific recombinases, regulates gene expression by inverting a DNA segment. DNA inversion requires assembly of an invertasome complex in which a recombinational enhancer DNA segment bound by the Fis protein associates with the Hin synaptic complex at the base of a supercoiled DNA branch. Each of the four Hin subunits becomes covalently joined to the cleaved DNA ends, and DNA exchange occurs by translocation of a Hin subunit pair within the tetramer. We show here that, although the Hin tetramer forms a bidirectional molecular swivel, the Fis/enhancer system determines both the direction and number of subunit rotations. The chirality of supercoiling directs rotational direction, and the short DNA loop stabilized by Fis-Hin contacts limit rotational processivity, thereby ensuring that the DNA strands religate in the recombinant configuration. We identify multiple rotational conformers that are formed under different supercoiling and solution conditions.

Synergistic role of curli and cellulose in cell adherence and biofilm formation of attaching and effacing Escherichia coli and identification of Fis as a negative regulator of curli.

Curli are adhesive fimbriae of Escherichia coli and Salmonella enterica. Expression of curli (csgA) and cellulose (bcsA) is co-activated by the transcriptional activator CsgD. In this study, we investigated the contribution of curli and cellulose to the adhesive properties of enterohaemorragic (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) O127:H6. While single mutations in csgA, csgD or bcsA in EPEC and EHEC had no dramatic effect on cell adherence, double csgAbcsA mutants were significantly less adherent than the single mutants or wild-type strains to human colonic HT-29 epithelial cells or to cow colon tissue in vitro. Overexpression of csgD (carried on plasmid pCP994) in a csgD mutant, but not in the single csgA or bscA mutants, led to significant increase in adherence and biofilm formation in EPEC and EHEC, suggesting that synchronized over-production of curli and cellulose enhances bacterial adherence. In line with this finding, csgD transcription was activated significantly in the presence of cultured epithelial cells as compared with growth in tissue culture medium. Analysis of the influence of virulence and global regulators in the production of curli in EPEC identified Fis (factor for inversion stimulation) as a, heretofore unrecognized, negative transcriptional regulator of csgA expression. An EPEC E2348/69Deltafis produced abundant amounts of curli whereas a double fis/csgD mutant yielded no detectable curli production. Our data suggest that curli and cellulose act in concert to favour host colonization, biofilm formation and survival in different environments.

Studies on the expression of 6S RNA from E. coli: involvement of regulators important for stress and growth adaptation.

The small bacterial 6S RNA has been recognized as a transcriptional regulator, facilitating the transition from exponential to stationary growth phase by preferentially inhibiting E sigma 70 RNA polymerase holoenzyme transcription. Consistent with this function, the cellular concentration of 6S RNA increases with stationary phase. We have studied the underlying mechanisms responsible for the growth phase-dependent differences in 6S RNA concentration. To this aim, we have analyzed the effects of the typical bacterial growth phase and stress regulators FIS, H-NS, LRP and StpA on 6S RNA expression. Measurements of 6S RNA accumulation in strains deficient in each one of these proteins support their contribution as potential regulators. Specific binding of the four proteins to DNA fragments containing 6S RNA promoters was demonstrated by gel retardation and DNase I footprinting. Moreover, in vitro transcription analysis with both RNA polymerase holoenzymes, E sigma 70 and E sigma 38, demonstrated a direct inhibition of 6S RNA transcription by H-NS, StpA and LRP, while FIS seems to act as a dual regulator. In vitro transcription in the presence of ppGpp indicates that 6S RNA promoters are not stringently regulated. Our results underline that regulation of 6S RNA transcription depends on a complex network, involving a set of bacterial regulators with general importance in the adaptation to changing growth conditions.

Identification of regulatory network topological units coordinating the genome-wide transcriptional response to glucose in Escherichia coli.

BACKGROUND: Glucose is the preferred carbon and energy source for Escherichia coli. A complex regulatory network coordinates gene expression, transport and enzyme activities in response to the presence of this sugar. To determine the extent of the cellular response to glucose, we applied an approach combining global transcriptome and regulatory network analyses. RESULTS: Transcriptome data from isogenic wild type and crp- strains grown in Luria-Bertani medium (LB) or LB + 4 g/L glucose (LB+G) were analyzed to identify differentially transcribed genes. We detected 180 and 200 genes displaying increased and reduced relative transcript levels in the presence of glucose, respectively. The observed expression pattern in LB was consistent with a gluconeogenic metabolic state including active transport and interconversion of small molecules and macromolecules, induction of protease-encoding genes and a partial heat shock response. In LB+G, catabolic repression was detected for transport and metabolic interconversion activities. We also detected an increased capacity for de novo synthesis of nucleotides, amino acids and proteins. Cluster analysis of a subset of genes revealed that CRP mediates catabolite repression for most of the genes displaying reduced transcript levels in LB+G, whereas Fis participates in the upregulation of genes under this condition. An analysis of the regulatory network, in terms of topological functional units, revealed 8 interconnected modules which again exposed the importance of Fis and CRP as directly responsible for the coordinated response of the cell. This effect was also seen with other not extensively connected transcription factors such as FruR and PdhR, which showed a consistent response considering media composition. CONCLUSION: This work allowed the identification of eight interconnected regulatory network modules that includes CRP, Fis and other transcriptional factors that respond directly or indirectly to the presence of glucose. In most cases, each of these modules includes genes encoding physiologically related functions, thus indicating a connection between regulatory network topology and related cellular functions involved in nutrient sensing and metabolism.

Fis regulates transcriptional induction of RpoS in Salmonella enterica.

The sigma factor RpoS is known to regulate at least 60 genes in response to environmental sources of stress or during growth to stationary phase (SP). Accumulation of RpoS relies on integration of multiple genetic controls, including regulation at the levels of transcription, translation, protein stability, and protein activity. Growth to SP in rich medium results in a 30-fold induction of RpoS, although the mechanism of this regulation is not understood. We characterized the activity of promoters serving rpoS in Salmonella enterica serovar Typhimurium and report that regulation of transcription during growth into SP depends on Fis, a DNA-binding protein whose abundance is high during exponential growth and very low in SP. A fis mutant of S. enterica serovar Typhimurium showed a ninefold increase in expression from the major rpoS promoter (PrpoS) during exponential growth, whereas expression during SP was unaffected. Increased transcription from PrpoS in the absence of Fis eliminated the transcriptional induction as cells enter SP. The mutant phenotype can be complemented by wild-type fis carried on a single-copy plasmid. Fis regulation of rpoS requires the presence of a Fis site positioned at -50 with respect to PrpoS, and this site is bound by Fis in vitro. A model is presented in which Fis binding to this site allows repression of rpoS specifically during exponential growth, thus mediating transcriptional regulation of rpoS.

The effect of host-encoded nucleoid proteins on transposition: H-NS influences targeting of both IS903 and Tn10.

Nucleoid proteins are small, abundant, DNA-binding proteins that profoundly affect the local and global structure of the chromosome, and play a major role in gene regulation. Although several of these proteins have been shown to enhance assembly of transpososomes before initiating transposition, no systematic survey has been carried out examining the in vivo role(s) of these proteins in transposition. We have examined the requirement of the six most abundant nucleoid proteins in transposition for three different transposons, IS903, Tn10 and Tn552. Most notably, H-NS was required for efficient transposition of all three elements in a papillation assay, suggesting a general role for H-NS in bacterial transposition. Further studies indicated that H-NS was exerting its effect on target capture. Targeting preferences for IS903 into the Escherichia coli chromosome were dramatically altered in the absence of H-NS. In addition, the alterations observed in the IS903 target profile emphasized the important role that H-NS plays in chromosome organization. A defect in target capture was also inferred for Tn10, as an excised transposon fragment, a precursor to target capture, accumulated in in vivo induction assays. Furthermore, a transposase mutant that is known to increase target DNA bending and to relax target specificity eliminated this block to target capture. Together, these results imply a role for H-NS in target capture, either by providing regions of DNA more accessible to transposition or by stabilizing transpososome binding to captured targets immediately before strand transfer.

Topological analysis of Hin-catalysed DNA recombination in vivo and in vitro.

In vitro studies have demonstrated that Hin-catalysed site-specific DNA inversion occurs within a tripartite invertasome complex assembled at a branch on a supercoiled DNA molecule. Multiple DNA exchanges within a recombination complex (processive recombination) have been found to occur with particular substrates or reaction conditions. To investigate the mechanistic properties of the Hin recombination reaction in vivo, we have analysed the topology of recombination products generated by Hin catalysis in growing cells. Recombination between wild-type recombination sites in vivo is primarily limited to one exchange. However, processive recombination leading to knotted DNA products is efficient on substrates containing recombination sites with non-identical core nucleotides. Multiple exchanges are limited by a short DNA segment between the Fis-bound enhancer and closest recombination site and by the strength of Fis-Hin interactions, implying that the enhancer normally remains associated with the recombining complex throughout a single exchange reaction, but that release of the enhancer leads to multiple exchanges. This work confirms salient mechanistic aspects of the reaction in vivo and provides strong evidence for the propensity of plectonemically branched DNA in prokaryotic cells. We also demonstrated that a single DNA exchange resulting in inversion in vitro is accompanied by a loss of four negative supercoils.

Proteome analysis of Salmonella enterica serovar Typhimurium fis mutant.

Salmonella enterica serovar Typhimurium is an enteric pathogen and a principal cause of gastroenteritis in humans. The factor-for-inversion stimulation protein (Fis) is known to play a pivotal role in the expression of Salmonella pathogenicity island (SPI)-1 genes in addition to various cellular processes such as recombination, replication, and transcription. In order to understand Fis function in pathogenicity of Salmonella, we performed two-dimensional gel electrophoresis and identified proteins whose expression pattern is affected by Fis using mass spectrometry. The results revealed various proteins that can be grouped according to their respective cellular functions. These groups include the genes involved in the metabolism of sugar, flagella synthesis, translation, and SPI expression. Changes in SPI expression suggest the possibility that regulation of genes in SPI-2 as well as SPI-1 is affected by Fis.

Fis stabilizes the interaction between RNA polymerase and the ribosomal promoter rrnB P1, leading to transcriptional activation.

It has been shown that Fis activates transcription of the ribosomal promoter rrnB P1; however, the mechanism by which Fis activates rrnB P1 transcription is not fully understood. Paradoxically, although Fis activates transcription of rrnB P1 in vitro, transcription from the promoter containing Fis sites (as measured from rrnB P1-lacZ fusions) is not reduced in a fis null mutant strain. In this study, we further investigated the mechanism by which Fis activates transcription of the rrnB P1 promoter and the role of Fis in rRNA synthesis and cell growth in Escherichia coli. Like all other stringent promoters investigated so far, open complex of rrnB P1 has been shown to be intrinsically unstable, making open complex stability a potential regulatory step in transcription of this class of promoters. Our results show that Fis acts at this regulatory step by stabilizing the interaction between RNA polymerase and rrnB P1 in the absence of NTPs. Mutational analysis of the Fis protein demonstrates that there is a complete correlation between Fis-mediated transcriptional activation of rrnB P1 and Fis-mediated stabilization of preinitiation complexes of the promoter. Thus, our study indicates that Fis-mediated stabilization of RNA polymerase-rrnB P1 preinitiation complexes, presumably at the open complex step, contributes prominently to transcriptional activation. Furthermore, our in vivo results show that rRNA synthesis from the P1 promoters of several rRNA operons are reduced 2-fold in a fis null mutant compared with the wild type strain, indicating that Fis plays an important role in the establishment of robust rRNA synthesis when E. coli cells are emerging from a growth-arrested phase to a rapid growth phase. Thus, our results resolve an apparent paradox of the role of Fis in vitro and in vivo in the field.

The small nucleoid-binding proteins H-NS, HU, and Fis affect hilA expression in Salmonella enterica serovar Typhimurium.

hilA encodes an activator of Salmonella enterica serovar Typhimurium virulence genes and is transcriptionally modulated by environmental conditions. We show that H-NS represses hilA under low-osmolarity conditions. H-NS, HU, and Fis also appear to affect the derepression of hilA by HilD. Modulation of hilA by counteracting repressing and derepressing mechanisms may allow Salmonella serovar Typhimurium to regulate its virulence genes in response to different situations in vivo.

Selective regulation of ptsG expression by Fis. Formation of either activating or repressing nucleoprotein complex in response to glucose.

Transcription of ptsG encoding glucose-specific permease, enzyme IICB(Glc), in Escherichia coli is initiated from two promoters, P1 and P2. ptsG transcription is repressed by Mlc, a glucose-inducible regulator of carbohydrate metabolism. The regulation of ptsG P1 transcription is also under positive control by cyclic AMP receptor protein and cyclic AMP complex (CRP.cAMP) as observed in other Mlc regulon. We report here that Fis, one of the nucleoid-associated proteins, plays a key role in glucose induction of Mlc regulon. ptsG transcription was induced when wild-type cells were grown in the presence of glucose. However, in a fis mutant, the basal level of ptsG transcription was higher but decreased when cells were grown in the presence of glucose, which implies the possibility of regulatory interactions among Fis, Mlc, and CRP.cAMP. Footprinting experiments with various probes and transcription assays revealed that Fis assists both Mlc repression and CRP.cAMP activation of ptsG P1 through the formation of Fis.CRP.Mlc or Fis.CRP nucleoprotein complexes at ptsG P1 promoter depending on the availability of glucose in the growth medium. ptsG P2 transcription was inhibited by Fis and Mlc. Tighter Mlc repression and enhanced CRP.cAMP activation of ptsG P1 by Fis enable cells to regulate Mlc regulon efficiently by selectively controlling the concentration of enzyme IICB(Glc) that modulates Mlc activity.

Transcription factor as a topological homeostat.

Abundant prokaryotic chromatin architectural proteins often function also as global transcriptional regulators. In addition, some of this class of proteins modulate the activity of cellular topoisomerases and hence, the superhelical density of DNA. The relationships between the global effect of these proteins on DNA topology and their local effects exerted on particular promoter regions remain largely unexplored. One of the best-characterised examples of this class of proteins is the pleiotropic regulator of metabolism FIS, which reduces the activity of DNA gyrase and counteracts the increase of the overall superhelicity of DNA during early exponential growth phase. Binding of FIS to supercoiled DNA molecules in vitro leads to the formation of branched structures and consequent multiplication of apical loops, whereas on bending the upstream regions of stable RNA promoters FIS acts as a topological homeostat maintaining high local levels of supercoiling required for promoter activity. We argue that the coordinated effects of FIS on the global and local DNA architecture optimise gene expression by channelling the free energy of negative supercoiling to specific, biologically relevant sites.

Transient repressor effect of Fis on the growth phase-regulated osmE promoter of Escherichia coli K12.

Transcription of the gene osmE of Escherichia coli is osmotically inducible and regulated by the growth phase. Expression of osmE is directed by a single promoter, osmE (p), which is recognized by Esigma(70) and Esigma(s), two forms of RNA polymerase using, respectively, the sigma factors sigma(70) and sigma(s). Esigma(s) transcribes osmE (p) during entry into stationary phase. Esigma(70) is responsible for osmotic induction of osmE (p) during the exponential growth phase. In a search for proteins that can modulate osmE (p) expression in trans, we performed electrophoretic mobility shift experiments using a DNA fragment carrying osmE (p) and crude extracts from E. coli. One major retarded band was observed in these experiments. The Fis protein is responsible for this retarded band, and binds to several sites upstream and downstream of, and overlapping, the promoter region of osmE. In a fis mutant background, the kinetics of in vivo transcription of osmE (p) during growth demonstrated that Fis is not responsible for the repression of the promoter seen during early exponential phase. In contrast, expression of osmE (p) at elevated osmolarity during the mid-exponential growth phase is increased in the absence of Fis, demonstrating that Fis is able to act as a repressor in vivo at a particular stage of growth.

Equilibrium denaturation studies of the Escherichia coli factor for inversion stimulation: implications for in vivo function.

The Factor for Inversion Stimulation (FIS) is a dimeric DNA binding protein found in enteric bacteria that is involved in various cellular processes, including stimulation of certain specialized DNA recombination events and transcription regulation of a large number of genes. The intracellular FIS concentration, when cells are grown in rich media, varies dramatically during the early logarithmic growth phase. Its broad range of concentrations could potentially affect the nature of its quaternary structure, which in turn, could affect its ability to function in vivo. Thus, we examined the stability of FIS homodimers under a wide range of concentrations relevant to in vivo expression levels. Its urea-induced equilibrium denaturation was monitored by far- and near-UV circular dichroism (CD), tyrosine fluorescence, and tyrosine fluorescence anisotropy. The denaturation transitions obtained were concentration-dependent and showed similar midpoints (C(m)) and m values, suggesting a two-state denaturation process involving the native dimer and unfolded monomers (N(2) <--> 2U). The DeltaG(H(2)O) for the unfolding of FIS determined from global and individual curve fitting was 14.2 kcal/mole. At concentrations <9 microM, the FIS dimer began to dissociate, as noted by the change in CD signal and size-exclusion high-pressure liquid chromatography retention times and peak width. The estimated dimer dissociation constant based on the CD and size-exclusion chromatography data is in the micromolar range, resulting in a DeltaG(H(2)O) of at least 5 kcal/mole less than that calculated from the urea denaturation data. This discrepancy suggests a deviation from a two-state denaturation model, perhaps due to a marginally stable monomeric intermediate. These observations have implications for the stability and function of FIS in vivo.