A global role for Fis in the transcriptional control of metabolism and type III secretion in Salmonella enterica serovar Typhimurium

Arlene Kelly1, Martin D. Goldberg2, Ronan K. Carroll1, Vittoria Danino2, Jay C. D. Hinton2 and Charles J. Dorman1

1 Department of Microbiology, Moyne Institute of Preventive Medicine, University of Dublin, Trinity College, Dublin 2, Ireland
2 Molecular Microbiology Group, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK

Correspondence
Charles J. Dorman
cjdorman{at}tcd.ie


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Fis is a key DNA-binding protein involved in nucleoid organization and modulation of many DNA transactions, including transcription in enteric bacteria. The regulon of genes whose expression is influenced by Fis in Salmonella enterica serovar Typhimurium (S. typhimurium) has been defined by DNA microarray analysis. These data suggest that Fis plays a central role in coordinating the expression of both metabolic and type III secretion factors. The genes that were most strongly up-regulated by Fis were those involved in virulence and located in the pathogenicity islands SPI-1, SPI-2, SPI-3 and SPI-5. Similarly, motility and flagellar genes required Fis for full expression. This was shown to be a direct effect as purified Fis protein bound to the promoter regions of representative flagella and SPI-2 genes. Genes contributing to aspects of metabolism known to assist the bacterium during survival in the mammalian gut were also Fis-regulated, usually negatively. This category included components of metabolic pathways for propanediol utilization, biotin synthesis, vitamin B12 transport, fatty acids and acetate metabolism, as well as genes for the glyoxylate bypass of the tricarboxylic acid cycle. Genes found to be positively regulated by Fis included those for ethanolamine utilization. The data reported reveal the central role played by Fis in coordinating the expression of both housekeeping and virulence factors required by S. typhimurium during life in the gut lumen or during systemic infection of host cells.


Abbreviations: FDR, false discovery rate; Fis, factor for inversion stimulation

The complete dataset for the microarray analysis is presented as supplementary data with the online version of this paper (at http://mic.sgmjournals.org).


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Salmonella enterica serovar Typhimurium (S. typhimurium) is the most common and best studied of the S. enterica serovars that infect humans (Finlay & Brumell, 2000). It is able to infect a range of animal species, including chicken, cattle and mice, and intensive study of this organism is providing important insights into key processes involved in bacterial pathogenesis. In the mouse, S. typhimurium is a facultative intracellular pathogen capable of invading epithelial cells and it has the ability to survive and proliferate within macrophages. The bacterium can be manipulated genetically with relative ease and the complete genome sequence is available (McClelland et al., 2001), allowing a combination of genetic analysis, cell biology and animal infection studies. This multidisciplinary approach has provided a picture of the major events involved when S. typhimurium infects the murine host. Following ingestion and passage through the stomach, the bacteria cross the lining of the intestine by invading the intestinal epithelium, predominantly via M cells. The Salmonellae are subsequently phagocytosed by macrophages before entering the blood stream and establishing a systemic infection (Finlay & Brumell, 2000; Galán, 2001; Groisman & Mouslim, 2000; Holden, 2002; Scherer & Miller, 2001).

S. typhimurium is dependent upon the products of a large number of genes (up to 200) to cause infection (Finlay & Brumell, 2000). Some of the virulence genes are located on a 90 kb pathogenicity plasmid, of which the spv genes are the best characterized (Holden, 2002; Libby et al., 2000, 2002; Paesold et al., 2002). However, most of the virulence genes are located within Salmonella pathogenicity islands (SPI) on the chromosome (Galán, 2001; Groisman & Ochman, 1993, 1997; Hacker & Kaper, 1999; Hensel, 2000; Hensel et al., 1999) of which SPI-1 and SPI-2 have been the most intensively studied and encode two of the three type III secretion systems of S. typhimurium. The Inv/Spa system is encoded by SPI-1 and exports proteins required for epithelial cell invasion (Hardt et al., 1998; Mills et al., 1995; Wood et al., 1996). The genes of the SPI-2 island encode an alternative type III secretion system that is required for survival within the macrophage (Cirillo et al., 1998; Hensel, 2000; Hensel et al., 1998; Ochman et al., 1996; Waterman & Holden, 2003) and for systemic infection of the mouse (Hensel et al., 1995; Shea et al., 1996).

The third type III secretion system in S. typhimurium is concerned with the production and deployment of flagella (Hirano et al., 2003; McClelland et al., 2001; Minamino & Macnab, 1999). In common with SPI-1 and SPI-2, the flagellar regulon is highly complex in terms of its regulation and in temporal expression (Chilcott & Hughes, 2000; Kalir et al., 2001; Macnab, 1996, 2003). Several studies have reported that the expression of pathogenicity island genes is coordinated with that of genes contributing to motility (Ellermeier & Slauch, 2003; Goodier & Ahmer, 2001; Lawhon et al., 2003; Lucas et al., 2000). This connection between virulence gene expression and motility is not confined to S. typhimurium (Goodier & Ahmer, 2001; Merrell et al., 2002) and probably reflects a need for the pathogen to coordinate its physical mobility with the expression of genes involved in niche invasion and adaptation. Moreover, motility is known to be required for Salmonella virulence (Schmitt et al., 2001).

The complexity of the pathogenic phenotype is apparent from the very large number of genes involved in its expression (Eriksson et al., 2003). A major challenge in this field is to understand the underlying regulatory mechanisms that control the expression of individual genes and groups of genes. Genetic studies have identified regulators that are specific to particular virulence genes. These include SpvR, a transcription factor that governs transcription of the spv virulence genes on the 90 kb plasmid (Grob & Guiney, 1996; Grob et al., 1997; Sheehan & Dorman, 1998), the HilA protein that regulates transcription of the SPI-1 island genes (Akbar et al., 2003; Bajaj et al., 1996; Boddicker et al., 2003) and the SsrA/SsrB two-component system that controls SPI-2 gene expression (Cirillo et al., 1998; Deiwick et al., 1999; Lee et al., 2000; Valdivia & Falkow, 1997). In addition, several regulators concerned with house-keeping functions, such as the EnvZ/OmpR and PhoP/PhoQ two-component regulatory systems, have also been shown to influence virulence gene expression (Feng et al., 2003a; Garmendia et al., 2003; Groisman, 2001; Lee et al., 2000).

Proteins with wide-ranging functions in bacterial gene regulation are known as global regulators and these include the nucleoid-associated proteins. Sometimes referred to as histone-like proteins, these molecules typically play roles in organizing the genetic material within the bacterial nucleoid as well as influencing transcription (for a recent review see Dorman & Deighan, 2003). The factor for inversion stimulation (Fis) is an 11·2 kDa DNA-binding protein comprising 98 amino acids that was first identified as a stimulator of inversion of the Hin invertible DNA element in S. typhimurium. This is the genetic switch that is responsible for phase-variable expression of the H1 and H2 flagellar antigens (Johnson, 2002). Fis binds to an enhancer element at the switch and organizes a nucleoprotein complex that facilitates site-specific recombination by the Hin recombinase (Heichman & Johnson, 1990).

Since its discovery it has become apparent that the roles of Fis extend beyond its involvement in DNA inversion (Finkel & Johnson, 1992; Wagner, 2000). In Escherichia coli, Fis has been shown to modulate transcription of many genes, including those encoding stable RNA. Fis is also required for oriC-directed DNA replication and influences the topological state of DNA in the cell by repressing DNA gyrase and activating topoisomerase I gene expression (Gonzalez-Gil et al., 1996; Ross et al., 1990; Schneider et al., 1999; Weinstein-Fischer et al., 2000). A degenerate consensus sequence has been identified for Fis where it introduces a bend of between 40° and 90° upon binding (Hengen et al., 1997). The E. coli Fis protein has a preference for binding sites located within regions of DNA curvature and is known to bind as a dimer (Wagner, 2000). The level of Fis in the cell is subject to complex and multifactorial control. Transcription of the fis gene is influenced by the stringent response, is autoregulated by Fis protein and is controlled by the intracellular concentration of cytosine triphosphate (Ball et al., 1992; Walker et al., 1999). The fis promoter is stimulated by negative supercoiling of the DNA (Schneider et al., 2000). When bacteria are subcultured in fresh medium there is a dramatic burst of Fis expression producing 50 000 to 100 000 dimers per cell. Thereafter, this high level falls as the cells divide until there are fewer than 500 dimers per cell at the onset of stationary phase (Appleman et al., 1998; Ball et al., 1992).

Many of the Fis-related observations made in E. coli are also true in S. typhimurium (Keane & Dorman, 2003; Osuna et al., 1995). Some differences in expression that have been reported reflect differences in the promoter sequence between the species (Osuna et al., 1995). A fis mutant of S. typhimurium has been described as having reduced motility, although the underlying reason was not established. The same fis mutant had an extended lag phase in a rich growth medium and the viability of the bacterium was compromised by constitutive expression of Fis during stationary phase (Osuna et al., 1995).

Recently, Fis has been implicated in the control of virulence gene expression in pathogenic strains of E. coli (Goldberg et al., 2001; Sheikh et al., 2001), in Shigella flexneri (Falconi et al., 2001) and in S. typhimurium, where it has been found to influence expression of genes within the SPI-1 pathogenicity island (Schechter et al., 2003; Wilson et al., 2001; Yoon et al., 2003). In this study, we have used DNA microarrays to investigate the extent of Fis involvement in the control of gene expression in S. typhimurium. We have now established that Fis regulates the expression of genes involved with metabolism, transport, flagellar biosynthesis and invasion. We also show that Fis is required for optimal expression of the SPI-2 pathogenicity island.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growth media.
The bacterial strains used in this study are listed in Table 1. S. typhimurium strain SL1344 (Hoiseth & Stocker, 1981) was used throughout the work and is the same isolate used in previous studies of S. typhimurium gene expression (Clements et al., 2002; Eriksson et al., 2003). A fis knockout mutant, SL1344fis : : cat (Keane & Dorman, 2003), was constructed by transducing the fis : : cat lesion from LT-2 strain TH2285 (a gift from K. T. Hughes) to SL1344 by bacteriophage P22 generalized transduction (Sternberg & Maurer, 1991). In the mutant the fis gene has undergone a 150 bp deletion of the 5' end of the ORF and a chloramphenicol acetyltransferase gene has been inserted in place of the deleted fis DNA. The absence of the Fis protein in SL1344fis : : cat was confirmed by Western blotting (data not shown). The promoter probe plasmid pQF50 (Table 1; Farinha & Kropinski, 1990) used to study ssrA and ssaG promoter activity has a copy number of ~10 per chromosome. Bacteria were grown routinely in Luria–Bertani (LB) broth or on LB agar plates at 37 °C (Sambrook & Russell, 2001). Motility assays were performed with swarm plates containing 1 % Bacto-Tryptone, 0·5 % NaCl and 0·3 % Bacto-Agar (Macnab, 1986). These plates were inoculated centrally with equal numbers of bacteria and incubated at 37 °C for 8 h.


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Table 1. Bacterial strains and plasmids

 
Western blot analysis.
For preparation of whole-cell proteins, 2·0 OD600 units of bacteria was harvested and resuspended in lysis buffer (10 % sucrose, 50 mM Tris/HCl, pH 7·5, 100 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol) with 200 µg lysozyme ml–1 and subsequently freeze-thawed to ensure complete lysis. Equal volumes were added to 2x SDS sample buffer. Proteins were resolved using 16 or 12 % SDS-polyacrylamide gels (for detection of Fis or FliC, respectively) and proteins were electroblotted onto Protran nitrocellulose membrane (Schleicher & Schuell). Equal loading and consistent transfer of protein to the nitrocellulose membrane were confirmed by staining with Ponceau S [0·2 % Ponceau S, 3 % (w/v) trichloroacetic acid, 3 % (w/v) sulphosalicylic acid] before blocking in 5 % (w/v) dried skimmed milk in PBS. Membranes were probed overnight with the anti-Fis antibody (1 : 1000) (Keane & Dorman, 2003) or for 1 h with the anti-FliC antibody (1 : 1000; Becton Dickinson) diluted in blocking solution. Membranes were washed in PBS and incubated with goat anti-rabbit horseradish peroxidase-conjugated antiserum (Cell Signalling). Membranes were treated with Supersignal chemiluminescent substrate (Pierce) and visualized on X-ray film (Kodak).

Microarray procedures.
A microarray analysis was carried out to elucidate the fis regulon of S. typhimurium during growth in LB broth and was performed as described previously (Clements et al., 2002) except that the microarrays were printed on Corning CMT-GAPS-coated slides. Each microarray contained 4414 coding sequences and was based on the S. typhimurium LT-2a genome sequence (McClelland et al., 2001). The microarray data analysis procedures used in this study were fully MIAME compliant.

RNA extraction.
Volumes (100 ml) of LB in 250 ml flasks were inoculated from overnight cultures of SL1344 or SL1344fis : : cat and grown at 37 °C with shaking. At 1 and 4 h post subculture, 4·0 OD600 units was harvested, transferred to 0·2 vols phenol/ethanol mix [5 % (v/v) phenol, 95 % (v/v) ethanol] and incubated on ice for at least 30 min to stabilize bacterial RNA (Tedin & Blasi, 1996). The bacteria were pelleted by centrifugation and RNA was isolated using the Promega SV total RNA purification kit as described at www.ifr.ac.uk/safety/microarrays/protocols.html. After elution the RNA was quantified, precipitated and resuspended at a concentration of 3 µg ml–1 in RNase-free water (Sigma).

Probe preparation, scanning and data analysis.
Microarray approaches have been discussed by Lucchini et al. (2001) and Thompson et al. (2001). RNA (10 µg) was fluorescently labelled during reverse transcription into cDNA. Fluorescently labelled genomic DNA (4 µg) from SL1344 was used as a reference channel in each experiment. For labelling protocols, see www.ifr.bbsrc.ac.uk/safety/microarray/protocols.html. Scanning and data analyses were performed as described by Eriksson et al. (2003). All RNA samples were hybridized to microarrays in quadruplicate and two biological replicates were performed. Only coding regions whose expression showed at least a twofold difference [false discovery rate (FDR)<=0·05 %] in the absence of Fis were regarded as being affected by the fis mutation. The complete dataset for the microarray analysis is presented as supplementary data with the online version of this paper (at http://mic.sgmjournals.org).

{beta}-Galactosidase assays.
Chromosomal merodiploid lacZY transcriptional fusions to the promoters of flhD, fliA, fliC, fliE and fljA (Goodier & Ahmer, 2001) were transferred into SL1344 and SL1344fis : : cat backgrounds by P22 transduction (Sternberg & Maurer, 1991) and assayed for {beta}-galactosidase activity according to the method of Miller (1992). Plasmid derivatives of the promoter probe vector containing either the ssrA or the ssaG promoter inserted upstream of a lacZ reporter gene were also assayed in the SL1344 and SL1344fis : : cat genetic backgrounds. The ssrA and the ssaG promoter fragments were amplified by PCR as 645 and 580 bp DNA fragments, respectively, and each was cloned into pQF50 that had been linearized at its multiple cloning site with BamHI and KpnI. The resulting ssrA-lacZ and ssaG-lacZ reporter plasmids were named pQFssrA and pQFssaG, respectively. Details of oligonucleotides are given in Table 2. {beta}-Galactosidase assays were performed in duplicate and the data expressed as the means of the two measurements. Standard deviations were calculated and are indicated in each figure. Experiments were performed on at least three independent occasions and typical results are shown.


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Table 2. Primers used in this study

 
DNA mobility shift assays.
A 723 bp fragment of the flhDC promoter, a 301 bp fragment of the fliA promoter, a 314 bp fragment of the fliC promoter, a 645 bp fragment of the ssrA promoter and a 580 bp fragment of the ssaG promoter were used in electrophoretic mobility shift assays. The fragments were amplified by PCR using the primer pairs, BSflhDfw and BSflhDrv, BSfliAfw and BSfliArv, BSfliCfw and BSfliCrv, ssrA_F and ssrA_R, and ssaG_F and ssaG_R (Table 2), electrophoresed through a 1·3 % agarose gel and the bands excised and extracted using the Concert Rapid Gel Extraction System (GibcoBRL). The DNA was labelled with [{gamma}-32P]ATP (NEN). Unincorporated label was removed and the DNA was purified using the High Pure PCR Product Purification Kit (Roche Molecular Biochemicals). In each reaction, 5 ng labelled probe was added to 20 mM Tris/HCl (pH 7·5), 80 mM NaCl, 1 mM EDTA, containing 50 µg poly(dI-dC) ml–1 and 300 µg BSA ml–1. Reactions therefore contained approximately 125-fold excess of non-specific synthetic competitor. Various quantities of purified Fis in Fis storage buffer (0·5 M NaCl, 20 mM Tris, pH 7·5, 0·1 mM EDTA, 50 % glycerol) were added, giving final concentrations of 0, 4, 20 or 60 ng Fis in each reaction. The reactions were then incubated at room temperature for 30 min, followed by electrophoresis on a 7 % (w/v) polyacrylamide gel in 0·5x TBE. Radioactive fragments were visualized by autoradiography. The S. typhimurium spvR promoter was amplified using the primer pair, spvR11 and spvR14 (Table 2) and was used as a negative control.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Determination of peak Fis expression in S. typhimurium
To establish the optimum time points for transcriptional profiling of SL1344 and its fis : : cat derivative, we used Western blotting analysis to monitor Fis protein in the wild-type strain throughout the growth cycle in batch culture in Luria–Bertani (LB) broth (Fig. 1). We found that peak expression of Fis protein occurred 1 h after diluting the overnight culture into fresh medium. No Fis protein was detectable by 3 h. In a parallel experiment, no Fis protein was detectable in the fis mutant (data not shown). We chose time points of 1 and 4 h to represent samples where the cells contained maximum and minimum levels of Fis, respectively.



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Fig. 1. Expression of the Fis protein in an LB culture of strain SL1344. Growth of SL1344 was monitored at 600 nm for 24 h (a). Total protein was extracted from an SL1344 culture at the time points indicated and Western blotting was used to examine Fis protein expression over a 24 h time course (b). The OD600 of the culture at each time point is given below the relevant lane.

 
Transcriptional profiling of SL1344 and SL1344fis : : cat
Stationary-phase overnight cultures of SL1344 and SL1344fis : : cat were used to inoculate fresh LB and total RNA was extracted from the bacterial cultures after 1 and 4 h of growth. The RNA was used to make cDNA that was labelled and hybridized to microarrays (see Methods). Gene expression profiles were normalized to SL1344 for either the 1 or 4 h culture and expressed as the ratio of fis mutant to wild-type such that genes activated by Fis have a value less than one. Robust microarray data were obtained by statistical filtering with an FDR of 0·05 %. Genes showing greater than a twofold change in expression between the wild-type and mutant strains were identified at the two time points.

An overview of the regulon was obtained by defining functional categories of genes based on the Kyoto Encyclopedia of Genes and Genomics (KEGG; www.genome.ad.jp/kegg/kegg2.html). Categories containing a high proportion of Fis-dependent genes were identified (Fig. 2). It is apparent that the majority of genes regulated by Fis are associated with virulence and motility/chemotaxis. Intriguingly, most Fis-dependent genes were observed at the 4 h time point, when we have shown Fis to be no longer detectable by Western blot analysis.



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Fig. 2. Categories of genes regulated by Fis. Fis-regulated genes were grouped into functional categories based on the Kyoto Encyclopedia of Genes and Genomics (KEGG). The histograms represent the percentage of genes in each category affected by the fis mutation at 1 and 4 h after subculture, with each functional category listed on the left. Filled bars indicate the percentage of genes more highly expressed in SL1344 than in the fis mutant (i.e. considered as Fis-activated); hatched bars represent the percentage of genes more highly expressed in the SL1344fis : : cat mutant than in the wild-type (considered as Fis-repressed).

 
At 1 h after subinoculation, 291 of the 2041 filtered SL1344 coding sequences with an FDR<=0·05 % showed >=twofold changes in expression. Of these differentially expressed genes, 167 showed higher levels of expression in the fis mutant while 124 genes showed a lower level of expression. At the 4 h time point a total of 844 genes showed statistically significant (FDR<=0·05 %) changes in expression level with 356 being more highly expressed in the mutant and 488 being repressed. Of the 167 genes showing increased expression in the mutant at 1 h, 78 were downregulated at 4 h. We also found that of 124 genes showing lower expression in the fis mutant at 1 h, 97 had elevated expression by 4 h (see supplementary data Table S1 at http://mic.sgmjournals.org). Thus, for 60 % of the ORFs showing an up or down response to the absence of Fis at 1 h (the time point at which the protein was most abundant in the wild-type) the response was transient. This pattern reflects the transient nature of Fis expression (Fig. 1). The fact that fewer genes were Fis-dependent at 1 h than at 4 h may reflect the involvement of additional regulators at different stages of the growth cycle. One must also consider the possibility that Fis effects at either time point may be indirect.

Fis and virulence gene expression
Among the most strongly Fis-activated genes were the virulence genes located within the SPI pathogenicity islands (see Fig. 2 and Fig. 3) and the chemotaxis/flagellar regulons (Fig. 4). Generally the effect of the fis mutation was to decrease gene expression, indicating a role for Fis as a transcription activator. The genes that were most downregulated in the fis mutant at 1 h were those in SPI-2 (Fig. 3b). In light of the role for SPI-2 in adaptation to the macrophage, it was interesting to observe that the macrophage-induced genes mig-3 and mig-14, and a number of PhoP-PhoQ-activated genes also showed a dependency on Fis (Table 3). SPI-1 genes were also Fis-dependent, in keeping with previous findings (Wilson et al., 2001); at the 4 h time point, no other class of genes showed as strong a dependency on Fis. We found that genes within SPI-5 were regulated positively by Fis, with pipC showing the strongest Fis dependence. Our data identify a coordinating role for Fis in the activation of virulence genes in SPI-1, SPI-2, some in SPI-3, SPI-4 and SPI-5, and are consistent with the previously demonstrated link between expression of SPI-5 genes and those of SPI-1 and SPI-2 (Knodler et al., 2002). Not all S. typhimurium virulence genes were regulated by Fis. For example, the spv genes on the 90 kb virulence plasmid were not affected by the fis mutation (supplementary data Tables S1 and S2 at http://mic.sgmjournals.org).



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Fig. 3. Effect of the fis mutation on expression of selected virulence genes located within S. typhimurium pathogenicity islands. All expression data were normalized to SL1344 for the 1 (filled bars) and 4 h (open bars) time points and the ratio of the mutant/wild-type was calculated for genes within SPI-1 and SPI-1 effectors (bold) (a), SPI-2 and SPI-2 effectors (bold) (b), and SPI-3, SPI-4 and SPI-5 (c). Expression ratios less than 1·0 indicate genes normally activated by Fis.

 


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Fig. 4. Flagellar gene regulation by Fis. Expression data were normalized to SL1344 for the 1 (filled bars) and 4 h (open bars) time points and the ratios for the mutant/wild-type were calculated. Expression ratios less than 1·0 indicate genes normally activated by Fis.

 

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Table 3. Other virulence genes regulated by Fis

 
The effect of the fis mutation on specific SPI-2 genes
Transcriptional fusions to virulence genes in the SPI-2 pathogenicity island were tested individually for Fis activation. The promoter of the ssrA regulatory gene and the promoter of the ssaG structural gene encoding part of the type III secretion apparatus were cloned upstream of the promoterless lacZ reporter gene in plasmid pQF50. {beta}-Galactosidase expression was measured in SL1344 and in SL1344fis grown in LB broth. The results showed that the SPI-2 promoters were significantly less active in the absence of Fis, in agreement with the DNA microarray data. In the fis mutant, ssrA-lacZ and ssaG-lacZ expression were 50 and 3 %, respectively, of the wild-type level.

Motility genes
Genes contributing to flagellar biosynthesis and motility were among the most strongly downregulated in the fis mutant. Few of these genes were affected by the absence of Fis at the 1 h time point (Fig. 4). However, after 4 h, as the bacteria were approaching stationary phase, we detected a significant reduction in flagellar gene expression in the fis mutant. This presumably reflects the influence of regulatory factors additional to Fis in the late-exponential-phase culture of the wild-type. Both regulatory and structural genes involved in most aspects of flagellar expression and function were affected by the fis mutation and included genes from the early, middle and late stages of flagellar biosynthesis (Macnab, 1996, 2003). Also downregulated in the fis mutant was the lipoprotein gene lpp (Table 4) which affects flagellar assembly (Dailey & Macnab, 2002).


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Table 4. Metabolism and transport genes regulated by Fis

 
The effect of the fis mutation on specific flagellar genes
Previous studies demonstrated a role for Fis in Salmonella motility (Osuna et al., 1995; Yoon et al., 2003). We used lacZ fusions to five different flagellar gene promoters to investigate the effects of the fis mutation in more detail. The genes chosen were flhD (the regulator of Class 2 flagellar operons), fliA (the sigma factor for Class 3 operon expression), flgA (assembly of the flagellar basal body P ring), fliC (phase 1 flagellin) and fliE (the MS ring/rod adapter in the basal body) (Macnab, 1996, 2003). In each case the chromosomally located merodiploid lac fusions were transduced into SL1344 and its fis : cat derivative to generate strains AK01–AK10 (see Methods).

All five flagellar genes showed a similar pattern of expression in the wild-type strain (Fig. 5). Following inoculation of fresh broth, expression declined rapidly to a minimum value at approximately 2·5 h. Thereafter, there was a strong increase in flagellar gene expression leading to a peak at approximately 5 h. Expression then declined as the bacteria entered stationary phase. The effect of the fis knockout mutation was negative in all cases and resulted in a reduction in expression of approximately twofold (Fig. 5).



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Fig. 5. Expression of flagellar gene fusions in the presence and absence of Fis. {beta}-Galactosidase assays were used to measure expression of lacZ in strains harbouring fusions to a selection of flagellar genes in the presence and absence of the Fis protein. Typical growth curves are presented for SL1344 (squares) and its fis mutant derivative (diamonds) (a) and gene expression data throughout the growth curve are presented for flhD (b), fliA (c), fliC (d), fliE (e) and flgA (f).

 
Fis binding to flagellar and SPI-2 promoters
To examine the interaction of Fis with the flagellar and SPI-2 virulence genes in greater detail, representative promoter regions were selected for use in electrophoretic mobility shift assays. The flagellar genes selected were from the early (flhD), middle (fliA) and late (fliC) stages of flagellar biosynthesis (Fig. 6a). The SPI-2 genes studied were the ssrA regulatory and ssaG structural genes (Fig. 6b). Like the flagellar genes, these had already been examined individually and shown to be regulated by Fis. A DNA sequence from the promoter of the spvR gene, known not to be Fis-regulated (our unpublished data; see also supplementary data Tables S1 and S2 at http://mic.sgmjournals.org), was used as a negative control. In the case of each of the flagellar and SPI-2 genes, a shift in electrophoretic mobility was seen at the lowest concentration of Fis used (Fig. 6). In contrast, the negative control underwent only a weak shift at the highest Fis concentration. These data show that Fis interacts directly with the flagellar and SPI-2 genes.



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Fig. 6. Binding of the Fis protein to flagellar and SPI-2 gene promoter regions in vitro. The interaction of the Fis protein with the transcription regulatory regions of three flagellar genes (a) and two SPI-2 genes (b) was assessed by electrophoretic mobility shift assay. The regulatory sequences were amplified by PCR, radiolabelled and incubated with 0, 4, 20 or 60 ng purified Fis protein and electrophoresed. Samples were resolved by electrophoresis in 7 % polyacrylamide gels. The spvR promoter from the 90 kb virulence plasmid was used as a negative control.

 
Fis and motility
The effect of Fis on the motility phenotype was established by tests on semi-solid agar plates. The fis mutant was clearly much less motile than the wild-type (Fig. 7). Moreover, full motility was restored when the fis lesion was complemented in trans using a plasmid-borne copy of the functional fis gene (Fig. 7).



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Fig. 7. Effect of a fis mutation on Salmonella motility. The wild-type strain SL1344, the fis knockout mutant SL1344fis : : cat and the complemented mutant SL1344fis : : cat (pFis349) were compared for motility. Equal numbers of bacteria were used to inoculate the centres of semi-solid swarming agar plates and incubated at 37 °C for 8 h.

 
To ensure that the production of phase 1 flagellin protein was genuinely Fis-dependent, we monitored the levels of FliC by Western blotting. Total protein was isolated from wild-type and fis mutant cultures grown for 4 h in LB. Protein extracted from a fliC mutant was used as a negative control. Probing with anti-FliC antibody showed that the level of FliC was strongly repressed in the fis mutant (Fig. 8). This finding was fully consistent with the data from the motility assays, the {beta}-galactosidase assays and the DNA microarrays.



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Fig. 8. Expression of the flagellar protein FliC in the presence or absence of Fis. Expression of the FliC protein was measured by Western blotting in wild-type strain SL1344 and its fis knockout derivative, SL1344fis : : cat following 4 h growth in LB at 37 °C. Strains TH6233 (negative control; FliC) and TH6232 (positive control; FliC+) were included for comparison. The migration positions of molecular mass markers are indicated.

 
Genes involved in metabolism and transport
The most strongly Fis-repressed genes identified by the microarrays were involved in metabolism and transport (Table 4). This confirmed that Fis acts as a transcriptional repressor as well as an activator. A large number of these genes are required for colonization of the gut by S. typhimurium (see below). Therefore, we suggest that Fis plays a role in coordinating the expression of house-keeping genes with that of virulence genes as part of a regulatory mechanism controlling the transition from a free-living mode in the gut lumen to an intracellular niche.

At the 1 h time point, the genes most highly up-regulated in the fis mutant were all involved in biotin synthesis (bioB, bioC and bioF) (Fig. 9). Biotin is a critical cofactor in carboxyl group transfer enzymes, such as biotin carboxylase, involved in an early step of lipid biosynthesis (Cronan & Rock, 1996). Other genes from lipid biosynthesis were also found to be up-regulated in the fis mutant. These included fabB encoding {beta}-ketoacyl-ACP synthase I (KAS I), which converts malonyl-ACP to acetoacetyl-ACP, fabD, the gene encoding malonyl-CoA : ACP transacylase, and psd which encodes phosphatidylserine decarboxylase (Cronan & Rock, 1996).



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Fig. 9. Effect of the fis mutation on expression of the rts regulatory genes and the bio and pdu metabolic genes. Fis is an activator for genes with a relative expression value below 1·0 and a repressor for genes with values above 1·0.

 
Several genes concerned with carbon utilization and energy generation were found to be repressed by Fis. These include genes encoding enzymes of the citric acid cycle and its glyoxylate bypass, glycolysis and anaerobic respiration (Table 4).

Genes involved in propanediol utilization by S. typhimurium were repressed by Fis. Of 18 pdu (propanediol utilization) genes for which data were available, 17 showed increased expression in the fis mutant at the 4 h time point (Fig. 9). Consistent with this was our finding that the fis mutant grew more rapidly than the wild-type in minimal medium supplemented with propanediol as carbon source (data not shown). Expression of pdu is dependent on the carbon storage regulator CsrA. This protein forms a regulatory link between propanediol utilization, ethanolamine utilization, vitamin B12 synthesis, flagellar gene expression and SPI-1 virulence gene expression (Lawhon et al., 2003). We found Fis to have a positive role in the expression of ethanolamine utilization genes (eut) at the 4 h time point with little or no effect at 1 h (Table 4). This was in contrast to the up-regulation of the pdu genes in the fis mutant. It was also contrary to the situation reported for CsrA which induces both pdu and eut expression (Lawhon et al., 2003). The significance of this difference is unknown. Vitamin B12 is required by the cell for the utilization of both propanediol and ethanolamine (Lawhon et al., 2003). Although Fis was not found to affect genes involved in B12 production, it did repress genes (btuB, btuC) contributing to its uptake at the 4 h time point (Table 4).

The aldB gene encodes aldehyde dehydrogenase, an enzyme that links propanediol and glyoxylate metabolism (Lin, 1996). Propanediol production is a consequence of L-fucose and L-rhamnose utilization, both of which occur during microbial growth in the mammalian gut (Lawhon et al., 2003). The aldB gene was repressed by Fis (Table 4) in agreement with previous data from E. coli (Xu & Johnson, 1995a, b). The fact that repression was seen only at the 4 h time point may reflect the fact that aldB is also dependent on RpoS for transcriptional activation (Xu & Johnson, 1995a, b). Multiple regulatory inputs of this nature may underlie the differences seen at the 1 and 4 h time points for many of the Fis-regulated genes detected in this study.

Polyamines are required for optimal growth of E. coli, but it is unclear which systems are directly affected by them (Glansdorff, 1996). Several S. typhimurium genes involved in polyamine metabolism showed elevated expression in the fis mutant (Table 4). These encoded lysine decarboxylase (cadA) which is required for the conversion of lysine to cadaverine, cadaverine transport (cadB), S-adenosylmethionine decarboxylase (speD) which feeds S-adenosylmethionine into the spermidine biosynthetic pathway, and putrescine/spermidine transport (potB and potC). The absence of the cadA gene from Shigella is important for full virulence in that pathogen (Maurelli et al., 1998). While it may be tempting to speculate that repression of cadA transcription by Fis may represent a step in the expression of virulence in S. typhimurium, it is not known if lysine decarboxylase activity plays any role in S. typhimurium virulence. However, it is known that cadA contributes to acid tolerance in S. typhimurium (Park et al., 1996) and this may point to a role for Fis in adaptation to pH stress.

Fis repressed transcription of ndk, the gene that encodes nucleoside diphosphate kinase (Table 4). The involvement of Fis in negative regulation of ndk was of interest given its similar role in the expression of the nupG and rbsC nucleoside transport genes, suggesting that Fis coordinates the expression of genes involved in pyrimidine metabolism. Furthermore, the ndk gene product catalyses the interconversion of GDP and GTP, a key step in regulating the size of the pppGpp and ppGpp pools that underlie the stringent response (Cashel et al., 1996). This response regulates many important genes in the cell, including the fis gene itself. This effect on ndk expression may reflect yet another route through which the Fis protein autoregulates fis gene expression.

Stress response genes and global regulators
Few classical stress response genes were Fis-dependent at the 1 h time point. However, by 4 h several genes known to be involved in adaptation to stress were Fis-activated (Table 5). These included the htrA heat-shock and cspC cold-shock genes, together with the proV and proX genes of the proU osmotic stress response locus. Also found to be Fis-dependent were the sodC gene, encoding the Cu–Zn-containing superoxide dismutase, the sodA and sodB genes, encoding the Mn- and Fe-containing superoxide dismutases, respectively, and the dsbA gene encoding the periplasmic protein disulphide isomerase (Table 5).


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Table 5. Stress response genes regulated by Fis

 
A number of genes encoding nucleoid-associated proteins with global regulatory roles were affected by the absence of Fis in the mutant (supplementary data Tables S1 and S2 at http://mic.sgmjournals.org). These included the cold-shock-responsive hns gene previously shown to be activated by Fis (Dersch et al., 1994; Falconi et al., 1996), the hha gene whose product can form heteromeric complexes with H-NS and (like H-NS) regulates several virulence genes in response to temperature (Madrid et al., 2002; Nieto et al., 2002), and the stpA gene that encodes a paralogue of H-NS and can also form heteromers with it (Deighan et al., 2003; Free et al., 2001; Johansson et al., 2001; Williams et al., 1996). It has been reported previously that Fis has no effect on stpA gene expression in E. coli at 30 min following subinoculation (Free & Dorman, 1997). Here, no effect of the fis mutation on stpA expression was detected at 1 h, although stpA expression was dependent on Fis when wild-type and mutant were compared at 4 h. The hupA and hupB genes encode the subunits of the heterodimeric DNA-binding protein HU (Hillyard et al., 1990; Oberto et al., 1994). In addition to its role in nucleoid organization, HU contributes to the osmotic stress response of the cell and normal regulation of the proU osmotic stress response operon (Manna & Gowrishankar, 1994). Expression of the hupA gene showed a strong requirement for Fis at 1 h and has been described previously as being activated by Fis in E. coli (Claret & Rouvière-Yaniv, 1996) (supplementary data Tables S1 and S2 at http://mic.sgmjournals.org). The repressive effect of Fis on hupB expression described in E. coli (Claret & Rouvière-Yaniv, 1996) was not detected under the conditions used in our study.

The RtsA and RtsB proteins have widespread effects on gene expression in S. typhimurium (Ellermeier & Slauch, 2003). RtsA shows homology to AraC-like proteins, while RtsB possesses a helix–turn–helix motif that is characteristic of DNA-binding proteins. These proteins are known to coordinate the expression of SPI-1 pathogenicity island genes and the genes of the flagellar regulon. Specifically, RtsA binds to the hilA regulatory gene promoter in SPI-1 while RtsB binds to the flhDC regulatory operon promoter in the flagellar regulon (Ellermeier & Slauch, 2003). Interestingly, the genes encoding these proteins, STM4315 (rtsA) and STM4314 (rtsB) were among the most strongly Fis-activated genes detected in our microarray study (supplementary data Tables S1 and S2 at http://mic.sgmjournals.org). This shows that Fis can act at multiple levels within a regulatory hierarchy. For example, RtsB expression depends on Fis (our data), RtsB interacts with the flhDC promoter (Ellermeier & Slauch, 2003) as does Fis, which also interacts with promoters at lower levels in the flagellar gene regulatory hierarchy (Fig. 5).

Concluding remarks
The data presented in this paper show that Fis exerts wide-ranging effects on gene expression in S. typhimurium, fully justifying its description as a global regulator. However, the major effects of Fis are confined largely to specific classes of genes (see Fig. 2). In particular, Fis regulates those genes encoding the type III secretion machinery and cognate effectors required by the bacterium for invasion of host epithelial cells, for survival in macrophage and for the deployment of flagella for motility. Therefore, this is not a general effect on all promoters arising from the ability of Fis to influence DNA topology. Our discovery that Fis regulates the expression of all three type III secretion systems in Salmonella is in keeping with other studies that have pointed to regulatory overlaps between virulence genes and flagella in Salmonella (Eichelberg & Galan, 2000; Ellermeier & Slauch, 2003; Lawhon et al., 2003) and other bacteria (Goodier & Ahmer, 2001; Grant et al., 2003). The effect of Fis on murein lipoprotein expression is also relevant here, since the lpp gene product is a structural component of the cell envelope. It is attractive to consider that Fis coordinates expression of lpp with that of type III secretion systems that require cell surface integrity for function.

The Salmonella pathogenicity islands are regarded as having been acquired by horizontal gene transfer, possibly from outside the enteric group of Gram-negative bacteria (Galán, 2001; Groisman & Ochman, 1993, 1997; Hacker & Kaper, 1999; Hensel, 2000; Hensel et al., 1999). The degeneracy associated with the binding site used by Fis may have aided its recruitment as a regulator of these horizontally acquired genes. Perhaps this represents a selective pressure acting on Fis to maintain its ability to bind to sites with a high degree of DNA sequence diversity.

None of the Fis-responsive genes found in this study is regulated by Fis alone. Each has at least one, and frequently more than one, additional regulator. By co-operating with or antagonizing the action of the other regulators, Fis appears to modulate and fine-tune gene expression in ways that benefit the cell during growth and adaptation to environmental change.

It is apparent that many genes of unknown function showed a positive or a negative response to Fis (supplementary data Tables S1 and S2 at http://mic.sgmjournals.org). Furthermore, our analyses involved S. typhimurium strain SL1344 coupled with microarrays which were based on the genome sequence of the strain LT2. The sequence of SL1344 is incomplete (www.sanger.ac.uk/Projects/Salmonella/), but it is already clear that this strain contains a number of genes not found in LT2. This means that knowledge of the full Fis regulon remains incomplete at the global level. At a local level, much must be done to unravel the detail of the contributions made by Fis at specific promoters to allow the regulon to be appreciated more fully. This will contribute in a significant way to a deepening of our appreciation of the gene regulatory circuits used by bacteria, leading to a much more complete understanding of the workings of the cell.

We found that Fis acts to modulate expression of genes involved in aspects of metabolism and transport that are relevant to S. typhimurium during life in the gut. These are genes involved in propanediol utilization, ethanolamine utilization, acetate and fatty acid utilization (Table 4; Fig. 9). This points to an even wider role for this protein in coordinating the gene expression programme of the bacterium. It is intuitively appealing that S. typhimurium can benefit from coordinating the expression of its major virulence factors with its metabolism and motility, and that it should use a global regulator such as Fis to accomplish this. In particular Fis seems to be well placed to coordinate the expression of genes involved in the transition from a free-living life in the gut lumen to the intracellular niche.

A striking feature of the Fis regulon is the fact that considerably more Fis-dependent genes were expressed or repressed in late, rather than early, exponential phase. This phenomenon is reminiscent of observations made in enteropathogenic E. coli where several Fis-dependent virulence genes encoded by the Locus for Enterocyte Effacement (LEE) were found to be maximally expressed in late stationary phase (Goldberg et al., 2001). Similarly, the effect of a fis mutation on gyr gene transcription is most acute in stationary phase in both S. typhimurium (Keane & Dorman, 2003) and E. coli (Schneider et al., 1999). This seems paradoxical since Fis protein levels peak 1 h after diluting stationary-phase cultures into fresh medium (Fig. 1). The levels of Fis declined rapidly such that by 3–4 h the protein was no longer detectable by Western blotting. A number of factors may explain this phenomenon. First, we speculate that the tolerance shown by Fis for degeneracy in the sequence of its binding site allows it to bind to a range of sites with different affinities and that high-affinity sites will continue to be occupied even as Fis protein levels decline. Promoters with such high affinity sites may be regarded as privileged in that they continue to be occupied by Fis when lower affinity sites become vacant. Second, the involvement of additional growth-phase-dependent factors may assist Fis in widening the range of its effects at later stages of the growth cycle. This may involve cooperativity between the additional factors and the remaining Fis molecules to target Fis to the promoters. Third, the absence of Fis is known to alter the structural dynamics of the genome, even at late time points when Fis levels in wild-type cells are very low (Schneider et al., 1999). Therefore, variations in local DNA topology may contribute to differences in the gene expression patterns of the wild-type and fis mutant, even at the 4 h time point. Finally, it should also be borne in mind that many of the effects of the fis mutation may be indirect, regardless of the stage of growth. These complex issues will be important topics for future research.


   ACKNOWLEDGEMENTS
 
We thank Kelly T. Hughes for bacterial strains, Isabelle Hautefort, Kelly T. Hughes, Joyce Karlinsey, Sacha Lucchini, Mikael Rhen, Arthur Thompson, the late Robert Macnab and members of the Dorman lab for useful and stimulating discussions. We acknowledge financial support from Science Foundation Ireland, the Health Research Board and the BBSRC Core Strategic Grant.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Akbar, S., Schechter, L. M., Lostroh, C. P. & Lee, C. A. (2003). AraC/XylS family members, HilD and HilC, directly activate virulence gene expression independently of HilA in Salmonella typhimurium. Mol Microbiol 47, 715–728.[CrossRef][Medline]

Appleman, J. A., Ross, W., Salomon, J. & Gourse, R. L. (1998). Activation of Escherichia coli rRNA transcription by Fis during a growth cycle. J Bacteriol 180, 1525–1532.[Abstract/Free Full Text]

Bajaj, V., Lucas, R. L., Hwang, C. & Lee, C. A. (1996). Co-ordinate regulation of Salmonella typhimurium invasion genes by environmental and regulatory factors is mediated by control of hilA expression. Mol Microbiol 22, 703–714.[Medline]

Ball, C. A., Osuna, R., Ferguson, K. C. & Johnson, R. C. (1992). Dramatic changes in Fis levels upon nutrient upshift in Escherichia coli. J Bacteriol 174, 8043–8056.[Abstract]

Boddicker, J. D., Knosp, B. M. & Jones, B. D. (2003). Transcription of the Salmonella invasion gene activator, hilA, requires HilD activation in the absence of negative regulators. J Bacteriol 185, 525–533.[Abstract/Free Full Text]

Cashel, M., Gentry, D. R., Hernandez, V. J. & Vinella, D. (1996). The stringent response. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn, pp. 1458–1496. Edited by F. C. Neidhardt & others. Washington, DC: American Society for Microbiology.

Chilcott, G. S. & Hughes, K. T. (2000). Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar Typhimurium and Escherichia coli. Microbiol Mol Biol Rev 64, 694–708.[Abstract/Free Full Text]

Cirillo, D., Valdivia, R., Monack, D. & Falkow, S. (1998). Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival. Mol Microbiol 30, 175–188.[CrossRef][Medline]

Claret, L. & Rouvière-Yaniv, J. (1996). Regulation of HU alpha and HU beta by CRP and FIS in Escherichia coli. J Mol Biol 263, 126–139.[CrossRef][Medline]

Clements, M. O., Eriksson, S., Thompson, A., Lucchini, S., Hinton, J. C. D., Normark, S. & Rhen, M. (2002). Polynucleotide phosphorylase is a global regulator of virulence and persistency in Salmonella enterica. Proc Natl Acad Sci U S A 99, 8784–8789.[Abstract/Free Full Text]

Cronan, J. E., Jr & Rock, C. O. (1996). Biosynthesis of membrane lipids. In Escherichia Coli and Salmonella: Cellular and Molecular Biology, 2nd edn, pp. 612–636. Edited by F. C. Neidhardt & others. Washington, DC: American Society for Microbiology.

Dailey, F. E. & Macnab, R. M. (2002). Effects of lipoprotein biogenesis mutations on flagellar assembly in Salmonella. J Bacteriol 184, 771–776.[Abstract/Free Full Text]

Deighan, P., Beloin, C. & Dorman, C. J. (2003). Three-way interactions among the Sfh, StpA and H-NS nucleoid-structuring proteins of Shigella flexneri 2a strain 2457T. Mol Microbiol 48, 1401–1416.[CrossRef][Medline]

Deiwick, J., Nikolaus, T., Erdogan, S. & Hensel, M. (1999). Environmental regulation of Salmonella pathogenicity island 2 gene expression. Mol Microbiol 31, 1759–1773.[CrossRef][Medline]

Dersch, P., Kneip, S. & Bremer, E. (1994). The nucleoid-associated DNA-binding protein H-NS is required for the efficient adaptation of Escherichia coli K-12 to a cold environment. Mol Gen Genet 245, 255–259.[Medline]

Dorman, C. J. & Deighan, P. (2003). Regulation of gene expression by histone-like proteins in bacteria. Curr Opin Genet Dev 13, 179–184.[CrossRef][Medline]

Eichelberg, K. & Galan, J. E. (2000). The flagellar sigma factor FliA ({sigma}28) regulates the expression of Salmonella genes associated with the centisome 63 type III secretion system. Infect Immun 68, 2735–2743.[Abstract/Free Full Text]

Ellermeier, C. D. & Slauch, J. M. (2003). RtsA and RtsB coordinately regulate expression of the invasion and flagellar genes in Salmonella enterica serovar Typhimurium. J Bacteriol 185, 5096–5108.[Abstract/Free Full Text]

Eriksson, S., Lucchini, S., Thompson, A., Rhen, M. & Hinton, J. C. D. (2003). Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica. Mol Microbiol 47, 103–118.[CrossRef][Medline]

Falconi, M., Brandi, A., La Teana, A., Gualerzi, C. O. & Pon, C. L. (1996). Antagonistic involvement of FIS and H-NS proteins in the transcriptional control of hns expression. Mol Microbiol 19, 965–975.[Medline]

Falconi, M., Proseda, G., Giangrossi, M., Beghetto, E. & Colonna, B. (2001). Involvement of Fis in the H-NS-mediated regulation of virF gene in Shigella and enteroinvasive Escherichia coli. Mol Microbiol 42, 439–452.[CrossRef][Medline]

Farinha, M. A. & Kropinski, A. M. (1990). Construction of broad-host range plasmid vectors for easy visible selection and analysis of promoters. J Bacteriol 172, 3496–3499.[Medline]

Feng, X., Oropeza, R. & Kenney, L. J. (2003a). Dual regulation by phospho-OmpR of ssrA/B gene expression in Salmonella pathogenicity island 2. Mol Microbiol 48, 1131–1143.[CrossRef][Medline]

Finkel, S. E. & Johnson, R. C. (1992). The Fis protein: it's not just for DNA inversion anymore. Mol Microbiol 6, 3257–3265.[Medline]

Finlay, B. B. & Brumell, J. H. (2000). Salmonella interactions with host cells: in vitro and in vivo. Philos Trans R Soc Lond B Biol Sci 355, 623–631.[CrossRef][Medline]

Free, A. & Dorman, C. J. (1997). The Escherichia coli stpA gene is transiently expressed during growth in rich medium and is induced in minimal medium and by stress conditions. J Bacteriol 197, 909–918.

Free, A., Porter, M. E., Deighan, P. & Dorman, C. J. (2001). Requirement for the molecular adapter function of StpA at the Escherichia coli bgl promoter depends upon the level of truncated H-NS protein. Mol Microbiol 42, 903–917.[CrossRef][Medline]

Galán, J. E. (2001). Salmonella interactions with host cells: type III secretion at work. Annu Rev Cell Dev Biol 17, 53–86.[CrossRef][Medline]

Garmendia, J., Beuzon, C. R., Ruiz-Albert, J. & Holden, D. W. (2003). The roles of SsrA–SsrB and OmpR–EnvZ in the regulation of genes encoding the Salmonella typhimurium SPI-2 type III secretion system. Microbiology 149, 2385–2396.[Abstract/Free Full Text]

Glansdorff, N. (1996). Biosynthesis of arginine and polyamines. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn, pp. 408–433. Edited by F. C. Neidhardt & others. Washington, DC: American Society for Microbiology.

Goldberg, M. D., Johnson, M., Hinton, J. C. D. & Williams, P. H. (2001). Role of the nucleoid-associated protein Fis in the regulation of virulence properties of enteropathogenic Escherichia coli. Mol Microbiol 41, 549–559.[CrossRef][Medline]

Gonzalez-Gil, G., Bringmann, P. & Kahmann, R. (1996). FIS is a regulator of metabolism in Escherichia coli. Mol Microbiol 22, 21–29.[Medline]

Goodier, R. I. & Ahmer, B. M. (2001). SirA orthologs affect both motility and virulence. J Bacteriol 183, 2249–2258.[Abstract/Free Full Text]

Grant, A. J., Farris, M., Alefounder, P., Williams, P. H., Woodward, M. J. & O'Connor, C. D. (2003). Co-ordination of pathogenicity island expression by the BipA GTPase in enteropathogenic Escherichia coli (EPEC). Mol Microbiol 48, 507–521.[CrossRef][Medline]

Grob, P. & Guiney, D. G. (1996). In vitro binding of the Salmonella dublin virulence plasmid regulatory protein SpvR to the promoter regions of spvA and spvR. J Bacteriol 178, 1813–1820.[Abstract]

Grob, P., Kahn, D. & Guiney, D. G. (1997). Mutational characterization of promoter regions recognized by the Salmonella dublin virulence plasmid regulatory protein SpvR. J Bacteriol 179, 5398–5406.[Abstract]

Groisman, E. A. (2001). The pleiotropic two-component regulatory system PhoP-PhoQ. J Bacteriol 183, 1835–1842.[Free Full Text]

Groisman, E. A. & Mouslim, C. (2000). Molecular mechanisms of Salmonella pathogenesis. Curr Opin Infect Dis 13, 519–522.[Medline]

Groisman, E. A. & Ochman, H. (1993). Cognate gene clusters govern invasion of host epithelial cells by Salmonella typhimurium and Shigella flexneri. EMBO J 12, 3779–3787.[Abstract]

Groisman, E. A. & Ochman, H. (1997). How Salmonella became a pathogen. Trends Microbiol 5, 343–349.[CrossRef][Medline]

Hacker, J. & Kaper, J. (1999). The concept of pathogenicity islands. In Pathogenicity Islands and Other Mobile Virulence Elements, pp. 1–11. Edited by J. Kaper & J. Hacker. Washington DC: American Society for Microbiology.

Hardt, W.-D., Urlaub, H. & Gálan, J. E. (1998). A substrate of the centisome 63 type III protein secretion system of Salmonella typhimurium is encoded by a cryptic bacteriophage. Proc Natl Acad Sci U S A 95, 2574–2579.[Abstract/Free Full Text]

Heichman, K. A. & Johnson, R. C. (1990). The Hin invertasome: protein-mediated joining of distant recombination sites at the enhancer. Science 249, 511–517.[Medline]

Hengen, P. N., Bartram, S. L., Stewart, L. E. & Schneider, T. D. (1997). Information analysis of Fis binding sites. Nucleic Acids Res 25, 4994–5002.[Abstract/Free Full Text]

Hensel, M. (2000). Salmonella pathogenicity island 2. Mol Microbiol 36, 1015–1023.[CrossRef][Medline]

Hensel, M., Shea, J. E., Gleeson, C., Jones, M. D., Dalton, E. & Holden, D. W. (1995). Simultaneous identification of bacterial virulence genes by negative selection. Science 269, 400–403.[Medline]

Hensel, M., Shea, J. E., Waterman, S. R. & 7 other authors (1998). Genes encoding putative effector proteins of the type III secretion of Salmonella pathogenicity island 2. Mol Microbiol 30, 163–174.[CrossRef][Medline]

Hensel, M., Nikolaus, T. & Egelseer, C. (1999). Molecular and functional analysis indicates a mosaic structure of Salmonella pathogenicity island 2. Mol Microbiol 31, 489–498.[CrossRef][Medline]

Hillyard, D. R., Edlund, M., Hughes, K. T., Marsh, M. & Higgins, N. P. (1990). Subunit-specific phenotypes of Salmonella typhimurium HU mutants. J Bacteriol 172, 5402–5407.[Medline]

Hirano, T., Minamino, T., Namba, K. & Macnab, R. M. (2003). Substrate specificity classes and the recognition signal for Salmonella type III flagellar export. J Bacteriol 185, 2485–2492.[Abstract/Free Full Text]

Hoiseth, S. K. & Stocker, B. A. D. (1981). Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291, 238–239.[Medline]

Holden, D. W. (2002). Trafficking of the Salmonella vacuole in macrophages. Traffic 3, 161–169.[CrossRef][Medline]

Johansson, J., Eriksson, S., Sondén, B., Wai, S. N. & Uhlin, B. E. (2001). Heteromeric interactions among nucleoid-associated bacterial proteins: localization of StpA-stabilizing regions in H-NS of Escherichia coli. J Bacteriol 183, 2343–2347.[Abstract/Free Full Text]

Johnson, R. C. (2002). Bacterial site-specific DNA inversion systems. In Mobile DNA II, pp. 230–271. Edited by L. Craig, R. Craigie, M. Gellert & A. M. Lambowitz. Washington, DC: American Society for Microbiology.

Kalir, S., McClure, J., Pabbaraju, K., Southward, C., Ronen, M., Leibler, S., Surette, M. G. & Alon, U. (2001). Ordering genes in a flagella pathway by analysis of expression kinetics from living bacteria. Science 292, 2080–2083.[Abstract/Free Full Text]

Keane, O. M. & Dorman, C. J. (2003). The gyr genes of Salmonella enterica serovar Typhimurium are repressed by the factor for inversion stimulation, Fis. Mol Gen Genomics 270, 56–65.[CrossRef][Medline]

Knodler, L. A., Celli, J., Hardt, W. D., Vallance, B. A., Yip, C. & Finlay, B. B. (2002). Salmonella effectors within a single pathogenicity island are differentially expressed and translocated by separate type III secretion systems. Mol Microbiol 43, 1089–1103.[CrossRef][Medline]

Lawhon, S. D., Frye, J. G., Suyemoto, M., Porwollik, S., McClelland, M. & Altier, C. (2003). Global regulation by CsrA in Salmonella typhimurium. Mol Microbiol 48, 1633–1645.[CrossRef][Medline]

Lee, A. K., Detweiler, C. S. & Falkow, S. (2000). OmpR regulates the two-component system SsrA-SsrB in Salmonella pathogenicity island 2. J Bacteriol 182, 771–781.[Abstract/Free Full Text]

Libby, S. J., Lesnick, M., Hasegawa, P., Weidenhammer, E. & Guiney, D. G. (2000). The Salmonella virulence plasmid spv genes are required for cytopathology in human monocyte-derived macrophages. Cell Microbiol 2, 49–58.[CrossRef][Medline]

Libby, S. J., Lesnick, M., Hasegawa, P., Kurth, M., Belcher, C., Fierer, J. & Guiney, D. G. (2002). Characterization of the spv locus in Salmonella enterica serovar Arizona. Infect Immun 70, 3290–3294.[Abstract/Free Full Text]

Lin, E. C. C. (1996). Dissimilatory pathways for sugars, polyols, and carboxylates. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn, pp. 307–342. Edited by F. C. Neidhardt & others. Washington, DC: American Society for Microbiology.

Lucas, R. L., Lostroh, C. P., DiRusso, C. C., Spector, M. P., Wanner, B. L. & Lee, C. A. (2000). Multiple factors independently regulate hilA and invasion gene expression in Salmonella enterica serovar Typhimurium. J Bacteriol 182, 1872–1882.[Abstract/Free Full Text]

Lucchini, S., Thompson, A. & Hinton, J. C. D. (2001). Microarrays for microbiologists. Microbiology 147, 1403–1414.[Free Full Text]

Macnab, R. M. (1986). Proton-driven bacterial flagellar motor. Methods Enzymol 125, 563–581.[Medline]

Macnab, R. M. (1996). Flagella and motility. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn, pp. 123–145. Edited by F. C. Neidhardt & others. Washington, DC: American Society for Microbiology.

Macnab, R. M. (2003). How bacteria assemble flagella. Annu Rev Microbiol 57, 77–100.[Medline]

Madrid, C., Nieto, J. M. & Juarez, A. (2002). Role of the Hha/YmoA family of proteins in the thermoregulation of the expression of virulence factors. Int J Med Microbiol 291, 425–432.[Medline]

Manna, D. & Gowrishankar, J. (1994). Evidence for involvement of proteins HU and RpoS in transcription of the osmoresponsive proU operon in Escherichia coli. J Bacteriol 176, 5378–5384.[Abstract]

Maurelli, A. T., Fernandez, R. E., Bloch, C. A., Rod, C. K. & Fasano, A. (1998). ‘Black holes' and bacterial pathogenicity: a large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli. Proc Natl Acad Sci U S A 95, 3943–3948.[Abstract/Free Full Text]

McClelland, M., Sanderson, K. E., Spieth, J. & 23 other authors (2001). Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413, 852–856.[CrossRef][Medline]

Merrell, D. S., Butler, S. M., Qadri, F., Dolganov, N. A., Alam, A., Cohen, M. B., Calderwood, S. B., Schoolnik, G. K. & Camilli, A. (2002). Host-induced epidemic spread of the cholera bacterium. Nature 417, 642–645.[CrossRef][Medline]

Miller, J. H. (1992). A Short Course in Bacterial Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Mills, D. M., Bajaj, V. & Lee, C. A. (1995). A 40 kilobase chromosomal fragment encoding Salmonella typhimurium invasion genes is absent from the corresponding region of the Escherichia coli K-12 chromosome. Mol Microbiol 15, 749–759.[Medline]

Minamino, T. & Macnab, R. M. (1999). Components of the Salmonella flagellar export apparatus and classification of export substrates. J Bacteriol 181, 1388–1394.[Abstract/Free Full Text]

Nieto, J. M., Madrid, C., Miquelay, E., Parra, J. L., Rodriguez, S. & Juarez, A. (2002). Evidence for direct protein–protein interaction between members of the enterobacterial Hha/YmoA and H-NS families of proteins. J Bacteriol 184, 629–635.[Abstract/Free Full Text]

Oberto, J., Drlica, K. & Rouvière-Yaniv, J. (1994). Histones, HMG, HU, IHF: Même combat. Biochimie 76, 901–908.[CrossRef][Medline]

Ochman, H., Soncini, F. C., Solomoin, F. & Groisman, E. A. (1996). Identification of a pathogenicity island required for Salmonella survival in host cells. Proc Natl Acad Sci U S A 93, 7800–7804.[Abstract/Free Full Text]

Osuna, R., Lienau, D., Hughes, K. T. & Johnson, R. C. (1995). Sequence, regulation, and functions of fis in Salmonella typhimurium. J Bacteriol 177, 2021–2032.[Abstract]

Paesold, G., Guiney, D. G., Eckmann, L. & Kagnoff, M. F. (2002). Genes in the Salmonella pathogenicity island 2 and the Salmonella virulence plasmid are essential for Salmonella-induced apoptosis in intestinal epithelial cells. Cell Microbiol 4, 771–781.[CrossRef][Medline]

Park, Y. K., Bearson, B., Bang, S. H., Bang, I. S. & Foster, J. W. (1996). Internal pH crisis, lysine decarboxylase and the acid tolerance response of Salmonella typhimurium. Mol Microbiol 20, 605–611.[Medline]

Ross, W., Thompson, J. F., Newlands, J. T. & Gourse, R. L. (1990). E. coli Fis protein activates ribosomal RNA transcription in vitro and in vivo. EMBO J 9, 3733–3742.[Abstract]

Sambrook, J. & Russell, D. W. (2001). Molecular Cloning, a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Schechter, L. M., Jain, S., Akbar, S. & Lee, C. A. (2003). The small nucleoid-binding proteins H-NS, HU, and Fis affect hilA expression in Salmonella enterica serovar Typhimurium. Infect Immun 71, 5432–5435.[Abstract/Free Full Text]

Scherer, C. A. & Miller, S. I. (2001). Molecular pathogenesis of Salmonellae. In Principles of Bacterial Pathogenesis, pp. 265–333. Edited by E. A. Groisman. San Diego: Academic Press.

Schmitt, C. K., Ikeda, J. S., Darnell, S. C., Watson, P. R., Bispham, J., Wallis, T. S., Weinstein, D. L., Metcalf, E. S. & O'Brien, A. D. (2001). Absence of all components of the flagellar export and synthesis machinery differentially alters virulence of Salmonella enterica serovar Typhimurium in models of typhoid fever, survival in macrophages, tissue culture invasiveness, and calf enterocolitis. Infect Immun 69, 5619–5625.[Abstract/Free Full Text]

Schneider, R., Travers, A., Kutateladze, T. & Muskhelishvili, G. (1999). A DNA architectural protein couples cellular physiology and DNA topology in Escherichia coli. Mol Microbiol 34, 953–964.[CrossRef][Medline]

Schneider, R., Travers, A. & Muskhelishvili, G. (2000). The expression of the Escherichia coli fis gene is strongly dependent on the superhelical density of DNA. Mol Microbiol 38, 167–175.[CrossRef][Medline]

Shea, J. E., Hensel, M., Gleeson, C. & Holden, D. W. (1996). Identification of a virulence locus encoding a type III secretion system in Salmonella typhimurium. Proc Natl Acad Sci U S A 93, 2593–2597.[Abstract/Free Full Text]

Sheehan, B. J. & Dorman, C. J. (1998). In vivo analysis of the interactions of the LysR-like regulator SpvR with the operator sequences of the spvA and spvR virulence genes of Salmonella typhimurium. Mol Microbiol 30, 91–105.[CrossRef][Medline]

Sheikh, J., Hicks, S., D'Agnol, M., Philips, A. D. & Nataro, J. P. (2001). Roles for Fis and YafK in biofilm formation by enteroaggregative Escherichia coli. Mol Microbiol 41, 983–997.[CrossRef][Medline]

Sternberg, N. L. & Maurer, R. (1991). Bacteriophage-mediated generalized transduction in Escherichia coli and Salmonella typhimurium. Methods Enzymol 204, 18–43.[Medline]

Tedin, K. & Blasi, U. (1996). The RNA chain elongation rate of the lambda late mRNA is unaffected by high levels of ppGpp in the absence of amino acid starvation. J Biol Chem 271, 17675–17686.[Abstract/Free Full Text]

Thompson, A., Lucchini, S. & Hinton, J. C. D. (2001). It's easy to build your own microarrayer! Trends Microbiol 9, 154–156.[CrossRef][Medline]

Valdivia, R. H. & Falkow, S. (1997). Fluorescence-based isolation of bacterial genes expressed within host cells. Science 277, 2007–2011.[Abstract/Free Full Text]

Wagner, R. (2000). Transcription Regulation in Prokaryotes. Oxford: Oxford University Press.

Walker, K. A., Atkins, C. L. & Osuna, R. (1999). Functional domains of the Escherichia coli fis promoter: roles of the –35, –10, and transcription initiation regions in the response to stringent control and growth phase-dependent regulation. J Bacteriol 181, 1269–1280.[Abstract/Free Full Text]

Waterman, S. R. & Holden, D. W. (2003). Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system. Cell Microbiol 5, 501–511.[CrossRef][Medline]

Weinstein-Fischer, D., Elgrably-Weiss, M. & Altuvia, S. (2000). Escherichia coli response to hydrogen peroxide: a role for DNA supercoiling, topoisomerase I and Fis. Mol Microbiol 35, 1413–1420.[CrossRef][Medline]

Williams, R. M., Rimsky, S. & Buc, H. (1996). Probing the structure, function, and interactions of the Escherichia coli H-NS and StpA proteins by using dominant negative derivatives. J Bacteriol 178, 4335–4343.[Abstract]

Wilson, R. L., Libby, S. J., Freet, A. M., Boddicker, J. D., Fahlen, T. F. & Jones, B. D. (2001). Fis, a DNA nucleoid-associated protein, is involved in Salmonella typhimurium SPI-1 invasion gene expression. Mol Microbiol 39, 79–88.[CrossRef][Medline]

Wood, M. W., Rosqvist, R., Mullan, P. B., Edwards, M. H. & Galyov, E. E. (1996). SopE, a secreted protein of Salmonella dublin, is translocated into the target eukaryotic cell via a sip-dependent mechanism and promotes bacterial entry. Mol Microbiol 22, 327–338.[Medline]

Xu, J. & Johnson, R. C. (1995a). aldB, an RpoS-dependent gene in Escherichia coli encoding an aldehyde dehydrogenase that is repressed by Fis and activated by Crp. J Bacteriol 177, 3166–3175.[Abstract]

Xu, J. & Johnson, R. C. (1995b). Identification of genes negatively regulated by Fis: Fis and RpoS comodulate growth-phase-dependent gene expression in Escherichia coli. J Bacteriol 177, 938–947.[Abstract]

Yoon, H., Lim, S., Heu, S., Choi, S. & Tyu, S. (2003). Proteome of Salmonella enterica serovar Typhimurium fis mutant. FEMS Microbiol Lett 226, 391–396.[CrossRef][Medline]

Received 2 April 2004; revised 30 April 2004; accepted 1 May 2004.



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