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
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ABSTRACT |
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The complete dataset for the microarray analysis is presented as supplementary data with the online version of this paper (at http://mic.sgmjournals.org).
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INTRODUCTION |
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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.
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METHODS |
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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 ml1 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).
-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
-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
.
-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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RESULTS AND DISCUSSION |
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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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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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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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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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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
-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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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 CuZn-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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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 helixturnhelix 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 34 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.
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ACKNOWLEDGEMENTS |
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Received 2 April 2004;
revised 30 April 2004;
accepted 1 May 2004.
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