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Originally published In Press as doi:10.1074/jbc.M406910200 on August 30, 2004

J. Biol. Chem., Vol. 279, Issue 46, 47543-47554, November 12, 2004
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Novel Phenotypes of Escherichia coli tat Mutants Revealed by Global Gene Expression and Phenotypic Analysis*{boxs}

Bérengère Ize{ddagger}§, Ida Porcelli¶||, Sacha Lucchini**, Jay C. Hinton**, Ben C. Berks¶, and Tracy Palmer{ddagger}§{ddagger}{ddagger}

From the {ddagger}Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom, the §School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom, the Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom, and the **Molecular Microbiology Group, Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, United Kingdom

Received for publication, June 21, 2004 , and in revised form, August 11, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The Tat protein export system serves to export folded proteins harboring an N-terminal twin arginine signal peptide across the cytoplasmic membrane. In this study, we have used gene expression profiling of Escherichia coli supported by phenotypic analysis to investigate how cells respond to a defect in the Tat pathway. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are defective in the integrity of their cell envelope because of the mislocalization of two amidases involved in cell wall metabolism (Ize, B., Stanley, N. R., Buchanan, G., and Palmer, T. (2003) Mol. Microbiol. 48, 1183–1193). To distinguish between genes that are differentially expressed specifically because of the cell envelope defect and those that result from other effects of the tatC deletion, we also analyzed two different transposon mutants of the {Delta}tatC strain that have their outer membrane integrity restored. Approximately 50% of the genes that were differentially expressed in the tatC mutant are linked to the envelope defect, with the products of many of these genes involved in self-defense or protection mechanisms, including the production of exopolysaccharide. Among the changes that were not explicitly linked to envelope integrity, we characterized a role for the Tat system in iron acquisition and copper homeostasis. Finally, we have demonstrated that overproduction of the Tat substrate SufI saturates the Tat translocon and produces effects on global gene expression that are similar to those resulting from the {Delta}tatC mutation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The cell envelope of Gram-negative bacteria such as Escherichia coli is composed of an inner (or cytoplasmic) membrane and an outer membrane, which delimits the periplasm and contains the murein layer. A central process in the construction of a bacterial cell is the localization of proteins to the different cellular compartments. In most bacteria, protein translocation across the inner membrane toward the periplasm proceeds by one of two pathways. The majority of proteins are translocated by the general Sec (secretory) protein export pathway. This system transports proteins in an unfolded state and is driven by a combination of ATP hydrolysis and the transmembrane proton electrochemical gradient (1, 2). However, a growing subset of proteins, often containing bound redox cofactors and playing important roles in energy conservation, are transported by the more recently discovered Tat (twin arginine translocation) pathway (3–5). Proteins are specifically targeted to the Tat system by cleavable N-terminal signal peptides harboring a distinctive (S/T)RRXFLK motif, in which the consecutive arginine residues are almost invariant (6). The Tat machinery translocates fully folded proteins, or even enzyme complexes, using the energy provided by the transmembrane protonmotive force (7, 8). Recently, it was shown than the Tat system is also involved in the post-translational biogenesis of certain integral membrane proteins (9).

In E. coli, the Tat translocase consists of the integral inner membrane proteins TatA, TatB, TatC, and TatE (10–15). The core Tat transporter is probably a large oligomeric complex of TatA, TatB, and TatC, with the TatA protein acting as the conducting channel (12, 16–18). Although TatA or TatE, TatB, and TatC are the only essential components of the translocon, DeLisa et al. (19) showed recently that PspA (phage shock protein A) may increase Tat translocation efficiency in vivo. Genes encoding the Tat proteins are expressed constitutively, indicating a role for Tat under all growth conditions (20).

The cell envelope of Gram-negative bacteria functions as a selective barrier: the lipopolysaccharide leaflet prevents the diffusion of toxic compounds through the outer membrane (21), whereas porins and other specific receptors allow passive or active transport of nutrients. Cell protection is also mediated by efflux systems that pump toxic compounds out into the extracellular medium (22). The murein layer determines the shape of the cell envelope and provides resistance to osmotic and mechanical stresses. It has been observed that those E. coli tat mutants that are completely defective in Tat-dependent protein translocation have impaired outer membrane integrity (23). These tat mutant strains leak periplasmic proteins, are hypersensitive to drugs and detergents, and display a distinctive chain-forming phenotype when visualized by microscopy. Inspection of the long chains of cells suggests that cell division is retarded at a relatively late stage in the {Delta}tatC mutant because the murein septa are fully formed (23). We (25) and others (24) have recently shown that two of the three E. coli N-acetylmuramyl-L-alanine amidases, AmiA and AmiC, are substrates of the Tat pathway. Amidases are involved in splitting the murein septum at a late stage in cell division, physically separating the two new daughter cells (26). Failure of these proteins to be correctly localized accounts for the cell envelope defect of tat mutant strains, and thus, a combined chromosomal deletion of the genes encoding amiA and amiC or of DNA covering just the signal peptide coding regions closely mimics the tat envelope phenotype (24, 25). Interestingly, overproduction of the Sec-targeted AmiB protein can compensate for the outer membrane and cell division defects of the {Delta}tatC mutant (25).

Genome sequencing studies indicate that the Tat system is found in many bacterial pathogens of animals and plants, including E. coli pathotypes K1 and 0157, Salmonella typhimurium, Neisseria meningitidis, Pseudomonas aeruginosa, and Agrobacterium tumefaciens, and it is becoming increasingly apparent that the Tat system makes an important contribution to the virulence of at least some of these organisms (27–30). In P. aeruginosa, the Tat system operates in parallel with the Sec system to secrete virulence factors via the type II secretion pathway (31). For example, the P. aeruginosa Tat system is required for the translocation of two phospholipases and of proteins involved in iron siderophore biosynthesis and uptake and in biofilm formation (29, 31). Additionally, if, as predicted (25), the N-acetylmuramyl-L-alanine amidases of P. aeruginosa are Tat-dependent, then defects in the integrity of the cell envelope would also be expected to reduce the virulence of the P. aeruginosa tat mutant. Indeed, Ochsner et al. (29) reported abnormal flagellum and pilus function in the tat mutant, a phenotype that could be the result of cell envelope defects.

In this study, we have used gene expression profiling supported by phenotypic analysis to investigate the global effects of a tat null mutation on E. coli. We found that ~50% of the differential gene regulation observed is a consequence of the cell envelope defect of the tat mutant strain. Some of the remaining genes that are not affected by the defective cell envelope encode proteins involved in iron acquisition and copper homeostasis, and we demonstrate that the Tat system is required for these types of metal ion metabolism. Finally, we show that overexpression of a native Tat substrate protein saturates the transport capacity of the Tat pathway, leading to effects on global gene expression similar to those found in a tat null mutant.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Bacterial Strains, Plasmids, and Growth Conditions—The strains and plasmids used and constructed in this work are listed in Table I. The plasmid pQEFhuD expresses fhuD with a sequence coding for a hexahistidine tag fused in-frame at the 3'-end. fhuD was amplified by PCR using primers incorporating NcoI and BglII restriction sites. The product was digested and cloned into the polylinker region of pQE60 (QIAGEN Inc.). The insert was sequenced to ensure that no mutations had been introduced. Plasmid pNR30 expresses sufI under the control of the lac promoter. It was constructed by excision of the sufI gene from pNR14 (32) with EcoRI/XbaI and cloning into pBluescript SK (Stratagene) using the same sites.


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  		TABLE I
Strains and plasmids used in this study

 
All bacterial cultures were grown at 37 °C unless stated otherwise. For microarray experiments, cells were cultured in minimal MOPS1 medium, which is both defined and reproducible, supplemented with glycerol (0.5%) as a carbon source (www.genome.wisc.edu/resources/protocols/mopsminimal.htm) (33). Aerobic growth was achieved in flasks that contained 0.1 volume of growth medium with constant aeration at 250 rpm. For anaerobic cultures, MOPS was supplemented in addition with 0.4% fumarate to serve as a terminal electron acceptor. Anaerobic cultures were grown in 15-ml capped tubes filled to the top and incubated at 37 °C. Cultures of the each strain were inoculated from overnight cultures grown under identical conditions to give an absorbance at 600 nm of 0.05 for aerobic cultures and of 0.015 for anaerobic cultures in MOPS medium. Four independent cultures for each strain were grown in parallel. The absorbance at 600 nm of one culture for each strain was measured and used as a reference. The cells of the three remaining cultures were harvested at mid-log phase, which was A600 ~ 1.25 for aerobic growth (final A600 for aerobically grown cells was typically 2.1) and A600 ~ 0.45 for anaerobic growth (final A600 was typically 0.9), and their RNAs were isolated (see below). For biofilm formation assays, cells were grown in colonizing factor antigen medium (1% (w/v) casamino acids, 0.15% (w/v) yeast extract, 0.005% (w/v) MgSO4, and 0.0005% (w/v) MnCl2) (34). For protein expression studies, strains were grown aerobically in LB medium (35). Concentrations of antibiotics were as described previously (10). For trimethylamine-N-oxide (TMAO) reductase assays, cells were cultured in the modified medium of Cohen and Rickenberg (36) supplemented with 0.2% (w/v) glucose and 0.4% (w/v) TMAO.

RNA Isolation—For the microarray and reverse transcription (RT)-PCR experiments, total bacterial RNA was isolated from mid-log cultures with the SV total RNA isolation system (Promega) according to themethoddescribed(www.ifr.bbsrc.ac.uk/safety/microarrays/protocols.html). The quality of RNA was checked using the RNA nanochip (Labchip on an Agilent 2100 Bioanalyzer). The concentration of RNA was determined by measuring the absorbance at 260 nm. One A260 unit corresponds to 40 µg/ml RNA. The RNA was then precipitated by adding 0.1 volume of sodium acetate and 2.5 volumes of ethanol and resuspended in sterile RNase-free distilled H2O to obtain a concentration of 2 µg/µl. RNA samples were stored at –80 °C.

cDNA Preparation, Hybridization, and Data Analysis—We used a common reference (E. coli MG1655 genomic DNA) as the co-hybridized control for one channel on all microarrays (37), as this method has the advantage of allowing the direct comparison of multiple samples and has been shown to be comparable with the traditional comparison of two cDNA samples on one microarray.2 Total RNA was used to generate cDNA according to the protocol described (www.ifr.bbsrc.ac.uk/safety/microarrays/protocols.html). Fluorescently labeled cDNA probes were prepared by random priming. A total of 16 µg of total RNA was transcribed and labeled with Cy5-conjugated dUTP (Amersham Biosciences) using 200 units of Stratascript (Stratagene) and random primers (Invitrogen). RNA strands were removed by addition of 0.1 M NaOH at 70 °C for 15 min, followed by neutralization with 0.1 M HCl. cDNA was purified further with a QIAquick PCR purification kit (QIAGEN Inc.) and then dried by vacuum centrifugation in a Speedvac Plus 210C. E. coli MG1655 genomic DNA (2 µg), isolated using a QIAGEN genomic DNA kit, was labeled with Cy3-conjugated dCTP (Amersham Biosciences) using 1 µl of Klenow enzyme and random primers from the BioPrime DNA labeling kit (Invitrogen). The reactions were then incubated overnight at 37 °C, cleaned using the QIAquick PCR purification kit, and dried in a Speedvac Plus 210C. Finally, dried labeled chromosomal DNA was resuspended in 10 µl of H2O. Labeled reference and test cDNAs were then combined and hybridized to the microarrays. RNAs from two biological replicates were hybridized to microarrays. The E. coli microarrays used in this study have been described previously (39).

After hybridization and washing, slides were scanned using an Axon 4000A scanner. The location and intensity of each spot were determined for both channels (Cy3 and Cy5) using the software GenPix Pro 3.0 (Axon Instruments, Inc.). The local background intensity was then substracted from the spot intensity for each spot for both channels. Spots with a signal for the reference channel below the background plus 3 standard deviations of the background as well as spots showing obvious blemishes were not considered for subsequent analysis (denoted nd in Fig. 3). Finally, the Cy5/Cy3 ratio was calculated. To compensate for unequal dye incorporation, the Cy5/Cy3 ratios were centered using the median Cy5/Cy3 ratio. The data centering was performed separately for each block, one block being defined as a group of DNA spots deposited by the same pin on the slide using Excel. Data visualization and data mining were performed using GeneSpring 6.1 (Silicon Genetics)



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  		FIG. 3.
Expression of the cps operon is induced under anaerobic conditions in the {Delta}tatC strain. The organization of the cps operon is shown, with the induction ratios obtained for the {Delta}tatC mutant compared with the wild-type strain indicated under each gene. nd, not determined.

 
RT-PCR—Total RNA was converted to cDNA using Superscript II (Invitrogen) as described by the manufacturer. The cDNA was then used directly as a template for PCR. The total number of PCR cycles ranged from 15 to 35. RT-PCR products were analyzed by agarose gel electrophoresis. Total RNA was also used directly (without reverse transcription) as a template for PCR to ensure that there was no contaminating DNA.3

Kolter Assay for Biofilm Formation—The capacity of E. coli strains to form biofilms was assayed as described previously (40) with a few modifications. Briefly, bacteria were grown overnight in colonizing factor antigen medium at 37 °C with agitation and standardized to A600 = 0.175, which is equivalent to 107 cells/ml. This culture was then diluted by 1:10 to make a total volume of 100 µl for aerobic cultures and of 350 µl for anaerobic cultures. Cultures were grown at 30 °C in a 96-well microtiter plate. After incubation periods of 24, 48, and 72 h, the plates were washed three times with sterile deionized water and dried in a 60 °C oven for 30 min. To observe biofilm formation, 130 µl of a 1% crystal violet solution was added to each well, and the plates were incubated at room temperature for 30 min. The crystal violet solution was withdrawn, and the plates were washed extensively with deionized H2O until all unbound crystal violet was removed. Plates were dried at 37 °C for ~1 h, after which wells were filled with 150 µl of 20% acetone in ethanol and incubated for 10 min at room temperature. To visualize the amount of cells attached to the surface, which reflects the formation of biofilm, absorbance was measured at 590 nm. Blank values were obtained from wells containing only medium.

Copper Sensitivity Determination—For the copper sensitivity assay, overnight cultures of the various strains were diluted to A600 = 0.05 with LB medium containing various concentrations of copper chloride. Cells were grown at 250 rpm, and a final A600 measurement was taken after 15 h of growth. 100% survival is defined as the absorbance of each strain after 180 min of growth in LB medium without CuCl2. The LD50 is defined as the concentration of CuCl2 that killed 50% of the cells after 15 h of growth.

Growth Promotion Test on Agar Plates—The growth experiments with different iron sources were assayed as described previously (41) with a few modifications. The strains were grown overnight in LB medium. The cultures were diluted to ~107 cells/ml, and 100 µl of the diluted cells was mixed with 3 ml of nutrient broth soft agar (0.3% agar; Oxoid) and poured onto nutrient broth plates (1.5% agar), both containing the iron chelator 2,2'-dipyridyl (0.2 mM; Fluka). Sterile filter disks (6-mm diameter) were placed onto the plates, onto which 10 µl of either 10 mM ferric citrate (Sigma) or 10 mM ferrichrome (Sigma) was then spotted. Plates were incubated at 30 °C for 48 h before photographing.

Protein Methods—Subcellular fractions were prepared by lysozyme/EDTA/cold osmotic shock (42). Proteins were separated by SDS-PAGE on 15% acrylamide gels (43). Immunoblotting was performed using the ECL method (Amersham Biosciences) according to the manufacturer's instructions. Anti-hexahistidine tag antiserum was obtained from Invitrogen. Protein concentration was estimated according to the method of Lowry et al. (44) using bovine serum albumin as a standard. TMAO-benzyl-viologen oxidoreductase activity was measured as described previously (45).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Experimental Design and Rationale—The goal of our experiments was to ascertain, at a global level, how E. coli responds to a defect in the Tat protein export pathway. To this end, we compared the transcriptional profiles of E. coli strain MC4100 with those of a derivative (B1LK0) that lacks a functional Tat pathway because of an in-frame deletion of the tatC gene. Bioinformatic analyses have suggested that there are at least 27 substrates of the E. coli Tat system, 13 of which have been experimentally confirmed (5). Approximately two-thirds of the E. coli Tat substrates are predicted to be cofactor-containing proteins, many of which contribute to the anaerobic respiratory flexibility of the organism. Therefore, it was important to perform our comparative analyses under both aerobic and anaerobic growth conditions. The most striking phenotypes of E. coli tat mutants are the formation of long chains of cells and the compromised outer membrane integrity, both of which arise because of the failure to transport AmiA and AmiC, two sequence-related N-acetylmuramyl-L-alanine amidases. Because these phenotypes might be expected to mask other, more subtle effects of the tat mutation, we additionally undertook global analysis of two transposon insertion mutants of the {Delta}tatC strain that are repaired, to differing degrees, in the integrity of their outer membrane and in their ability to divide. Strain BMI1 (B1LK0 yjeF::Tn10) overproduces the Sec-targeted cell wall amidase AmiB, a functional homolog of the two Tat-targeted amidases, and this results in the restoration of cell envelope integrity. In strain BMI6 (B1LK0 fimH::Tn10), the transposon is inserted into the fimH gene, which encodes a receptor-recognizing element of type 1 fimbriae (25). It is not clear how an insertion in this gene, which encodes a component of the pilus fiber (46), can reduce the severity of the cell envelope defect of a {Delta}tatC mutant, and we note that this insertion provides only a partial repair phenotype (25).

To compare gene expression profiles of E. coli strains with and without severe envelope defects, it was important to determine the growth rates for each strain. Cells were grown at 37 °C in minimal MOPS medium containing the non-fermentable carbon source glycerol either in the presence of oxygen or supplemented with fumarate in the absence of oxygen as described under "Experimental Procedures." Fig. 1 (A and B) shows the growth profiles of strains grown under aerobic and anaerobic conditions, respectively. The specific growth rates during exponential phase were 0.53 h–1 for the parental strain MC4100, 0.51 h–1 for the {Delta}tatC mutant (B1LK0), 0.53 h–1 for strain BMI1 ({Delta}tatC yjeF::Tn10), and 0.55 h–1 for strain BMI6 ({Delta}tatC fimH::Tn10) under aerobic growth conditions and 0.13 h–1 for MC4100, {Delta}tatC, and BMI1 under anaerobic growth conditions. Strain MC4100/pNR30 is the parental strain expressing multicopy sufI and will be described below. Because the growth curves were very similar for the five strains grown under aerobic conditions and for the three strains grown under anaerobic conditions, we harvested the cells for genomic expression analysis at mid-exponential phase, with A600 = 1.25 for aerobic cultures and 0.45 for anaerobic cultures.



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  		FIG. 1.
Growth of E. coli strains in MOPS medium. Strains MC4100 (parental; {blacksquare}), B1LK0 ({Delta}tatC; {circ}), BMI1 ({Delta}tatC yjeF::Tn10; {triangleup}), BMI6 ({Delta}tatC fimH::Tn10; {diamondsuit}), and MC4100/pNR30 ({square}) were grown in the presence (A) or absence (B) of oxygen as described under "Experimental Procedures." Each point on the growth curve is an average of three experiments. Cells were harvested for gene expression profiling at A600 = 1.25 for aerobic growth and 0.45 for anaerobic growth. Error bars represent S.E.

 
Comparative Global Analysis of Gene Expression Profiles— Total RNA isolated from each of the cultures described above was converted to cDNA and fluorescently labeled prior to hybridization of the DNA microarrays, which were carried out in duplicate; each experiment was repeated with RNA prepared from independent cultures (biological replicates). The data were quantified as described under "Experimental Procedures." Our data analysis was performed on 4262 of the 4279 protein-coding sequences identified in E. coli K12 strain MG1655 (47). Genes were considered differentially expressed if they were up- or down-regulated by ≥2-fold in each of the two biological replicates compared with the parental strain MC4100. The expression patterns and predicted functions of differentially expressed genes are listed in Supplemental Table SI. The annotations for the genes listed here were updated by BLAST and domain structure searches (ProDom) of the EcoCyc (ecocyc.org/), NCBI (www.ncbi.nlm.nih.gov/), and Swiss Protein (ca.expasy.org/) Databases (as of March 15, 2004). The genes were then categorized and assigned to functional groups. A comparison of the data obtained under aerobic growth conditions for the B1LK0 ({Delta}tatC), BMI1 ({Delta}tatC yjeF::Tn10), and BMI6 ({Delta}tatC fimH::Tn10) strains with those obtained for the parental strain identified 153, 157, and 91 genes up-regulated and 136, 281, and 113 genes down-regulated (Supplemental Table SI), respectively. This indicates that between 2.2 and 7.7% of the E. coli MC4100 genes are differentially regulated between the different strains during aerobic growth. Under anaerobic growth conditions, a comparison of the data obtained for B1LK0 and BMI1 with those obtained for the parental strain identified 41 and 32 genes up-regulated and 34 and 50 genes down-regulated (Supplemental Table SI), respectively. This represents a differential expression of ~1% of the genes in the E. coli MC4100 genome under anaerobic conditions.

Validation of the Microarray Data—To confirm the microarray data, we used RT-PCR to measure the expression level changes in a selection of the up-regulated genes. For these experiments, we used cDNA derived from the total RNA used in the microarray experiments as well as total RNA isolated from three independent exponentially growing cultures. Analysis of the tatAB genes, which are constitutively expressed (20), was used as a positive control. Fig. 2 shows the results of RT-PCR analysis of the tatAB, aroF, ylcB, fhuA, and fhuD genes.



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  		FIG. 2.
Validation of the microarray data by RT-PCR. Shown are the results from agarose gel analysis of fragments obtained after RT-PCR of total cDNA synthesized from RNA isolated from the following strains grown aerobically in MOPS medium: MC4100 (lane 1), B1LK0 ({Delta}tatC; lane 2), BMI1 ({Delta}tatC yjeF::Tn10; lane 3), BMI6 ({Delta}tatC fimH::Tn10; lane 4), and MC4100/pNR30 (lane 5). Equal volumes of PCR were loaded for each sample. Specific primers were used for amplification of the tatAB, aroF, cusC, fhuA, and fhuD genes. The relative expression levels (-fold) obtained for each gene in the microarray experiment (Supplemental Table SI) are shown boxed under each panel.

 
It is clear that the expression of the tatAB and fhuA genes as examined by RT-PCR did not vary between the samples, in accordance with the lack of apparent regulation of these genes seen in the microarray analysis. The aroF gene, which was highly up-regulated in the B1LK0 ({Delta}tatC) and BMI1 ({Delta}tatC yjeF::Tn10) strains, was also markedly up-regulated when analyzed by RT-PCR. In addition, as shown in Fig. 2, the cusC and fhuD genes also showed up-regulation relative to the expression level in the parental strain, in agreement with the results of the microarray analysis. It should be noted that these results were achieved using the minimal number of RT-PCR cycles that produced a product from the DNA derived from the {Delta}tatC mutants ({Delta}tatC/{Delta}tatC yjeF::Tn10/{Delta}tatC fimH::Tn10) and that the result shown for each strain is representative of those observed using five independent samples. Higher numbers of cycles resulted in the appearance of RT-PCR products from both wild-type and mutant samples, consistent with the observation that these genes are expressed in both strains but at quite different levels. Taken together, the RT-PCR results provide a clear and independent validation of our microarray data.

It has been demonstrated previously that, in the BMI1 strain, the transposon has inserted into the yjeF gene, resulting in the up-regulation of the downstream genes (yjeE, amiB, mutL, and miaA) that form a superoperon (25, 48). Accordingly, we found each of these four genes highly upregulated in BMI1 compared with the parental strain under both aerobic and anaerobic conditions (Supplemental Table SI). This observation provides additional evidence to support the reliability of our microarray data.

Up-regulation of a Set of Genes Involved in Cell Envelope-associated Functions—Comparison of the gene expression profiles obtained showed that there were 153 genes up-regulated in the {Delta}tatC mutant (B1LK0) relative to the parental strain under aerobic growth conditions and 41 genes up-regulated during anaerobic growth (Supplemental Table SI). 15 of these genes were common to both growth conditions. Of these 153 up-regulated genes, 93 (48%) of them showed a parental type pattern of expression in the two transposon insertion strains, BMI1 and BMI6. Because induction of the expression of these genes, which are listed in Table II, is lost when the cell envelope defect is repaired by the transposon insertions, their transcriptional changes in the tat mutant can be assigned to the response of the cell to the cell envelope defect.


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  		TABLE II
Genes up-regulated (≥2-fold) relative to the parental strain in the E. coli B1LK0 ({Delta}tatC) strain but not in the BMI1 (B1LK0 yjeF::Tn10) and BMI6 (B1LK0 fimH::Tn10) strains under aerobic and anaerobic conditions

Asterisks indicate genes of the Rcs regulon (58, 59). Genes shown to be induced by ampicillin are indicated in boldface (49).

 
Among these 93 genes, 31 have no ascribed function, and 11 have only a putative function assigned to them (Table II). The majority of genes of known function are associated with the cell envelope (cell processes and cell structure categories) (Table II). A recent report has described the global transcriptional response of E. coli after exposure to the antibacterial compound ampicillin (49). Ampicillin is a {beta}-lactam that inhibits cell wall synthesis by irreversibly inactivating the penicillin-binding proteins. Significantly, 60% of the genes up-regulated in response to the cell envelope defect of the tatC mutation overlap with those induced by ampicillin treatment (indicated in boldface in Table II). These include genes induced by osmotic shock (osm and ots) (50, 51) and severe envelope stress (e.g. spy, which is induced by spheroplasting, and psp genes, which are induced by a number of conditions that lead to inner or outer membrane stress) (52, 53). This is entirely consistent with the fact that the primary envelope lesion in tat mutant strains is a defect in cell wall metabolism.

Three signal transduction pathways involved in sensing periplasmic or envelope stress ({sigma}E, Cpx, and Bae) have been described in E. coli (54–56). Each of these pathways probably senses envelope stress through the accumulation of different subsets of misfolded envelope proteins (57). However, none of the genes encoded by any of these signal transduction regulons is differentially expressed in the {Delta}tatC mutant (with the exception of spy, which is subject to a complex regulatory pattern), indicating that the three known stress responses are not induced.

Interestingly, however, many of the genes of the Rcs (regulator of capsule synthesis) regulon are induced as a result of the cell envelope phenotype of the {Delta}tatC strain (indicated by asterisks in Table II) (58, 59). Genes under the control of the Rcs two-component pathway probably contribute to the modification of the bacterial surface in response to changes in the environment (for example, in response to desiccation or changes in osmolarity) (57). Thus, it appears that the cell responds to the cell envelope defect of the tat mutant by specifically up-regulating genes of the Rcs signal transduction pathway.

A number of genes normally involved in anaerobic energy metabolism were found to be up-regulated under aerobic conditions in the cell envelope-compromised {Delta}tatC mutant strain. This suggests that the cell envelope defect affects aerobic energy metabolism. This might offer an additional explanation for induction of the expression of the psp genes since PspA has been implicated in the maintenance of the transmembrane proton electrochemical gradient, and its expression is known to be induced by agents that interfere with energy production (60, 61).

A total of 27 genes were specifically down-regulated aerobically and 21 were down-regulated anaerobically in response to the cell envelope defect of the {Delta}tatC mutant (Supplemental Table SI). Notably, only four of these genes are linked to cell structure and cell processes. These observations suggest that the cell responds to envelope stress arising from the {Delta}tatC mutation primarily by specific up-regulation (rather than down-regulation) of genes involved in cell envelope processes.

E. coli tat Mutants Are Defective in Biofilm Formation—As discussed above, the expression of a number of genes of the Rcs regulon was induced as a result of the cell envelope defect of the tatC strain. Indeed, almost all of the genes of the wca operon were up-regulated in response to the cell envelope defect of {Delta}tatC mutant strain B1LK0 (Table II). The wca or cps (capsular polysaccharide synthesis) operon comprises 23 kb of DNA that encodes 21 open reading frames involved in the polymerization, transport, and modification of colonic acid, which is integral to cell capsule formation (Fig. 3 and Table II) (62). Consistent with this, tat mutants routinely appeared highly mucoid in comparison with the wild-type strain when incubated on solid medium for extended periods of time (data not shown). The cell capsule is known to protect E. coli from environmental assaults that might damage or perturb the outer membrane (63), and the induction of cps genes is consistent with the cell envelope lesion in the {Delta}tatC strain.

In addition to its protective role, colanic acid is also an important component in the development of E. coli biofilms. Biofilms are sessile bacterial communities that live attached to solid surfaces and are likely to be the predominant form of bacterial growth in natural settings. Colanic acid makes a vital contribution to the spatial arrangement of E. coli cells within biofilms and to biofilm thickness (64, 65). Notably, a number of genes that were up-regulated in the {Delta}tatC strain as a result of the cell envelope defect, including cpxP, spy, the psp genes, and ycfJ (Table II), are also up-regulated in E. coli cells growing in biofilms (66). Interestingly, it was also reported that the tatE gene, which encodes a minor component of the Tat machinery, is also up-regulated in biofilm-grown cells (66). Given our observations, we sought to test whether the {Delta}tatC mutation affects, positively or negatively, biofilm formation. As shown in Fig. 4 (A and B), the parental strain was capable of time-dependent biofilm formation when cultured in colonizing factor antigen medium under both aerobic and anaerobic growth conditions, respectively. However, the magnitude of biofilm formation observed under anaerobic conditions was lower than that observed under aerobic conditions, presumably reflecting the reduced growth rate. In contrast to the parental strain, the {Delta}tatC mutant (B1LK0) was significantly impaired in biofilm formation. However, strain BMI1 ({Delta}tatC yjeF::Tn10), which is repaired in the cell envelope defect, was able to form biofilms almost as well as the parental strain (Fig. 4). These observations indicate that the {Delta}tatC strain fails to form biofilms because of defects in the cell envelope.



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  		FIG. 4.
Detection of biofilm formation by E. coli strains on an abiotic surface under aerobic (A) and anaerobic (B) growth conditions in colonizing factor antigen medium. Biofilm formation was assessed using the Kolter assay described under "Experimental Procedures" after 24 h (white bars), 48 h (gray bars), and 72 h (black bars). In all cases, the data are the average of four replicates; error bars represent S.E. Strains are MC4100 (wild-type (WT)), B1LK0 ({Delta}tatC), BMI1 ({Delta}tatC yjeF::Tn10), and TAN-3 ({Delta}wcaM).

 
The WcaM protein, which is encoded in the wca gene cluster, is synthesized with a twin arginine signal peptide that is capable of engaging with the Tat pathway.4 To examine whether WcaM mislocalization might also contribute to the failure of the {Delta}tatC strain to form biofilms, we tested the biofilm-forming ability of the E. coli {Delta}wcaM strain TAN-3 (23). Deletion of wcaM did not negatively affect biofilm formation, indicating that mislocalization of the WcaM protein alone is not responsible for the lack of biofilm formation by the {Delta}tatC strain (Fig. 4).

Gene Expression Changes Linked to the {Delta}tatC Mutation That Are Independent of the Cell Envelope Defect—To examine the effects of the {Delta}tatC mutation that are not linked to the cell envelope defect, we identified the genes that were differentially expressed in the B1LK0 ({Delta}tatC), BMI1 ({Delta}tatC yjeF::Tn10), and BMI6 ({Delta}tatC fimH::Tn10) strains relative to the parental strain under aerobic growth conditions and in both B1LK0 and BMI1 relative to the parental strain under anaerobic growth conditions (Table III). Analysis of the results showed that 33 genes (0.79% of the genome) were differentially regulated under aerobic growth conditions, whereas 17 genes (0.4% of the genome) were differentially regulated under anaerobic conditions. As expected, tatC is down-regulated in all of the tatC mutant strains and under both growth conditions.


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  		TABLE III
Up-regulated and down-regulated genes (≥ or ≤2-fold) in E. coli strains B1LK0 ({Delta}tatC), BMI1 ({Delta}tatC yjeF::Tn10), BMI6 ({Delta}tatC fimH::Tn10), and MC4100/pNR30 (parental strain carrying multicopy suf1) relative to the parental strain under aerobic (B1LK0, BMI1, BMI6, and MC4100/pNR30) and anaerobic (B1LK0 and BMI1) growth conditions

Asterisks indicate genes that show a >2-fold average in expression level, but one of the two replicates was <2-fold.

 
Role of the Tat System in Iron Transport—Of the 33 genes differentially regulated under aerobic conditions that are not linked to the cell envelope defect, fhuD is one of the most highly up-regulated (average of 3.3-fold). Interestingly, it is the only gene predicted to encode a protein with a plausible twin arginine signal sequence (Fig. 5A). fhuD is the third gene of the fhuACDB operon and was up-regulated in all three of the {Delta}tatC strains (Table III). Surprisingly, fhuA and fhuC did not qualify as differentially expressed. The fhuA gene was up-regulated 1.23-, 1.24-, and 1.49-fold in {Delta}tatC, BMI1 ({Delta}tatC yjeF::Tn10), and BMI6 ({Delta}tatC fimH::Tn10), respectively, whereas fhuC was up-regulated 1.32-, 1.33-, and 1.39-fold in the same strains (Supplemental Table SI). We confirmed that fhuD (but not fhuA) was up-regulated by RT-PCR (Fig. 2). This unusual observation suggests that the fhuA region of the fhu transcript is unstable or that an unknown internal promoter allows independent transcription of fhuD.



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  		FIG. 5.
FhuD is an authentic Tat substrate. A, the FhuD signal sequence (NCBI accession number AAC73263 [GenBank] . Residues common to the twin arginine motif are shown in boldface. The hydrophobic region is underlined. B, growth stimulation of E. coli strains by either ferrichrome or ferric citrate as the sole iron source. Cells of E. coli strains MC4100 (wild-type (WT)) and B1LK0 ({Delta}tatC) were embedded into a top layer of soft agar on a nutrient broth plate containing the iron chelator 2,2'-dipyridyl. The photographs show the growth zones around the filter disks soaked with solution of ferrichrome (F) or ferric citrate (C) after 48 h at 30 °C. C, analysis of the spheroplast (lanes 1 and 2) and periplasmic (lanes 3 and 4) fractions prepared from strains MC4100 (parental strain; lanes 1 and 3) and B1LK0 (MC4100 {Delta}tatC; lanes 2 and 4), each carrying plasmid pQEFhuD. In each case, the fractions (spheroplast, 40 µg of protein; and periplasm, 12 µg of protein) were separated by SDS-PAGE (15%) and analyzed by immunoblotting using anti-hexahistidine tag antibody as described under "Experimental Procedures." P, precursor form of FhuD; M, mature form of FhuD. The molecular masses (in kilodaltons) are indicated on the left.

 
FhuD is the periplasmic shuttle protein involved in the Fe(III)-ferrichrome import system (Fhu) of E. coli (67). To solubilize iron prior to transport, E. coli synthesizes and secretes high affinity extracellular ferric iron-chelating siderophores (68). Ferri-siderophore complexes are then imported via a chain of specific outer membrane receptors, periplasmic binding proteins, and inner membrane ABC transporters. E. coli has six known siderophore receptors providing specificity for several ferri-siderophore complexes. To test the involvement of the Tat system in the translocation of the shuttle protein FhuD and indirectly in the import of ferrichrome, we assessed the growth of the wild-type and {Delta}tatC strains in limited iron medium, with either ferrichrome or ferric citrate as the sole iron source, as described under "Experimental Procedures." Ferric citrate internalization is not dependent on the Fhu system and requires only the fecABCDE genes, the expression of which is not altered in all three {Delta}tatC mutant strains. As expected, both strains clearly grew around filter disks that had been soaked in solutions of ferric citrate (Fig. 5B). In contrast, the {Delta}tatC mutant completely failed to grow using ferrichrome as the sole iron source, whereas the wild-type strain grew normally, strongly suggesting that FhuD was mislocalized in the {Delta}tatC mutant (Fig. 5B). To rule out the involvement of the outer membrane defect in the mislocalization of FhuD in the {Delta}tatC mutant, we additionally tested strain MCDSSAC (25). This strain carries chromosomal deletions in the signal peptide-coding regions for amiA and amiC and therefore mimics the cell envelope defect phenotype of the {Delta}tatC strain while retaining a fully functional Tat pathway. We confirmed that strain MCDSSAC was able to grow using either ferric citrate or ferrichrome (data not shown), indicating that iron transport is affected in the {Delta}tatC mutant independently of the outer membrane defect.

To directly demonstrate that FhuD is a substrate for the Tat translocation pathway, we constructed an epitope-tagged version of the protein and assessed its subcellular localization in the parental and {Delta}tatC strains. Immunoblotting identified the FhuD polypeptide in both strains (Fig. 5C). Upon cellular fractionation, FhuD was found predominantly in the periplasmic fraction of the wild-type strain, as expected (Fig. 5C, lanes 1 and 3). In contrast, FhuD was located exclusively in the spheroplast fraction of the {Delta}tatC mutant (Fig. 5C, lanes 2 and 4), indicating the Tat dependence of FhuD translocation. It should be noted that Rohrback et al. (69) have shown that FhuD is folded in the cytoplasm prior to translocation, as expected of a Tat substrate. We conclude from these experiments that FhuD is an authentic Tat substrate and that mislocalization of this protein most probably accounts for the iron transport defect of the {Delta}tatC mutant. It is possible that the cell senses the inability to transport ferrichrome and up-regulates fhuD expression and/or stabilizes its mRNA accordingly. The fact that fhuD is up-regulated only under aerobic conditions probably reflects the availability of highly soluble Fe(II) as an iron source under anaerobic conditions.

Role of the Tat System in Copper Homeostasis—We observed that most of the genes in the cusCFBA operon (formerly known as ylcBCD-ybdE) (70) were up-regulated in all three {Delta}tatC strains (B1LK0, BMI1, and BMI6) when grown under aerobic conditions (Fig. 6A). To determine whether these expression level changes were reproducible, we performed RT-PCR analysis on the first gene of the cus operon, cusC. As shown in Fig. 2, cusC was demonstrably up-regulated in all of the {Delta}tatC mutant strains.



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  		FIG. 6.
Effect of the {Delta}tatC mutation on the copper tolerance of E. coli. A, expression levels of the different genes of the cus operon in E. coli strains B1LK0 ({Delta}tatC), BMI1 ({Delta}tatC yjeF::Tn10), and BMI6 ({Delta}tatC fimH::Tn10) versus MC4100 under aerobic growth conditions. The asterisk indicates a gene with a >2-fold average in expression level, but one of the two replicates was <2-fold. B, copper sensitivity of the E. coli tatC strain. Overnight cultures of strains MC4100 (parental strain; {blacksquare}), B1LK0 (MC4100 {Delta}tatC; ), BMI1 (B1LK0 yjeF::Tn10; {blacksquare}), MCDSSAC (MC4100 amiA{Delta}2–33, amiC{Delta}2–32; {square}), and NRS-4 (MC4100 {Delta}cueO; {diamond}) were diluted 1:100 into fresh LB medium supplemented with the indicated concentrations of CuCl2 as described under "Experimental Procedures." 100% survival is defined as the absorbance of each strain after 15 h of growth in LB medium without CuCl2. Each point on the growth curve is an average of four experiments; error bars represent S.E.

 
The cusCFBA operon encodes a copper tolerance system. Copper is an important trace element for E. coli, but can be toxic even at low concentrations (71, 72). To cope with this duality, two pathways control intracellular copper availability in E. coli (73, 74): Cue (for Cu efflux), which is the primary aerobic system, and Cus (for Cu sensing), which becomes necessary when copper concentration is increased. The Cue system consists of a periplasmic oxidase (CueO, formerly YacK), a copper efflux P-type ATPase (CopA), and a copper-responsive metalloregulatory protein (CueR) (73). CueO requires oxygen as a substrate and thus has a strictly oxygen-dependent function even though expression of the cueO gene has also been observed under anaerobic conditions (75). Notably, CueO bears a twin arginine signal peptide and is mislocalized in tat mutant strains (11, 32). Moreover, copA was down-regulated 2.46-, 1.95-, and 1.81-fold in {Delta}tatC, BMI1 ({Delta}tatC yjeF::Tn10), and BMI6 ({Delta}tatC fimH::Tn10), respectively, under aerobic conditions (Supplemental Table SI). We observed up-regulation of the cus operon in the three {Delta}tatC strains only under aerobic conditions. It is known that the Cus system plays an important role in copper tolerance under anaerobic growth conditions and, under extreme copper stress, under aerobic growth conditions. Moreover, the induction of the cusC promoter occurs at a higher copper concentration under aerobic conditions (200 µM) than under anaerobic conditions (70 µM) (76). We therefore hypothesized that, in the {Delta}tatC strain under aerobic conditions, the primary copper export system (Cue) is defective because of CueO mislocalization, which allows the build-up of copper within the cell to a level that triggers up-regulation of the Cus system, subsequently allowing the maintenance of copper homeostasis within the cell.

To ascertain whether up-regulation of the cus operon is related to the mislocalization of CueO in the {Delta}tatC strains, we tested the tolerance of the {Delta}tatC mutant (B1LK0) to copper. The results shown in Fig. 6B demonstrate that, whereas the wild-type strain showed an LD50 for copper of 3.6 mM, disruption of tatC led to a significant increase in copper sensitivity relative to the parental strain (LD50 = 2 mM). Strain NRS-4, which harbors an in-frame deletion in the cueO gene, was, similarly, more sensitive to copper than the parental strain (Fig. 6B). However, NRS-4 was not as sensitive as {Delta}tatC to increasing copper concentrations, indicating that mislocalization of CueO in the {Delta}tatC mutant is not completely responsible for the copper sensitivity of the {Delta}tatC strain. To determine whether the cell envelope defect of the tat mutant also contributes to the copper sensitivity, we tested the susceptibility of strains MCDSSAC (Tat+, cell envelope-impaired) and BMI1 (Tat–, cell envelope-repaired) to extraneous copper (Fig. 6B). Like the cueO mutant strain, both MCDSSAC and BMI1 exhibited a copper resistance phenotype that was intermediate between the wild-type and {Delta}tatC mutant strains. Taken together, these results indicate that the copper sensitivity phenotype of the {Delta}tatC strain most probably results from a combined effect of both CueO mislocalization and a pleiotropic defect in the cell envelope.

Overexpression of SufI Mimics a {Delta}tatC Mutant—The gene expression profiles of the parental and tatC strains represent a unique resource to address the question of how a Tat wild-type E. coli cell responds to the saturating overexpression of a Tat substrate. For our analysis, we transformed the tat+ strain MC4100 with a multicopy plasmid (pNR30) that carries the sufI gene. SufI is a monomeric 50-kDa protein that is exported to the periplasm by the Tat system (11, 32). It was first identified as a multicopy suppressor of the cell division phenotype of an fstI mutant (77), although its cellular function is still not known. SufI does not bind a cofactor and has thus been widely used as substrate for in vivo and in vitro Tat transport studies (7, 8, 11, 32). Expression of sufI from construct pNR30 is under the control of the lac promoter and is therefore constitutive in strain MC4100, which harbors the {Delta}lacU169 mutation.

To confirm that SufI is produced at levels that saturate the Tat pathway in our experimental system, we looked at the effect of sufI overexpression from pNR30 on the export of the Tat substrate TMAO reductase (TorA). Strains MC4100, B1LK0 ({Delta}tatC), and MC4100/pNR30 were cultured anaerobically in the presence of TMAO and fractionated, and the TorA activity of the periplasmic and spheroplast fractions was determined. As expected, upon cellular fractionation, TorA was found predominantly in the periplasmic fraction of the Tat+ strain, but was absent in the same fraction of the {Delta}tatC strain (Fig. 7). The Tat+ strain overexpressing sufI (MC4100/pNR30) showed a lower periplasmic TorA activity than the parental strain, suggesting partial saturation of the Tat pathway (Fig. 7). This is consistent with previous work reporting that overproduction of SufI or CueO can saturate export by the Tat system (7, 19).



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  		FIG. 7.
Overexpression of sufI competes with translocation of TorA by the Tat pathway. E. coli strains MC4100 (wild-type (WT)), B1LK0 ({Delta}tatC), and MC4100 overexpressing sufI (WT/pNR30) were cultured anaerobically in CR medium (36) in the presence of TMAO, followed by fractionation into spheroplasts (gray bars) and periplasmic fractions (white bars). TorA activity was measured as described under "Experimental Procedures." Results represent the average of three samples; error bars represent S.E.

 
MC4100 and MC4100 carrying pNR30 were cultured aerobically in glycerol/MOPS medium at 37 °C. The specific growth rates during exponential phase were 0.53 h–1 for MC4100 and 0.52 h–1 for MC4100/pNR30 (Fig. 1A), and the cells of each strain were harvested for gene expression analysis at midexponential phase (A600 = 1.25). Comparison of the two strains identified 47 genes (1% of the genome) that were up-regulated and 172 genes (4% of the genome) that were down-regulated in the strain overexpressing sufI (Supplemental Table SI). These results were additionally validated by RT-PCR (Fig. 2). Of the 33 genes differentially regulated in all three of the {Delta}tatC, BMI1 ({Delta}tatC yjeF::Tn10) and BMI6 ({Delta}tatC fimH::Tn10) mutants relative to the parental strain under aerobic growth conditions, 27 of these were also differentially up-regulated in the wild-type strain overexpressing sufI (Table III). These results are consistent with the conclusion that overproduction of SufI leads to saturation of the Tat translocation system. The remaining genes that are differentially regulated in the strain overexpressing sufI may be associated with the presence of a multicopy plasmid or related to the cellular function of SufI itself.

Concluding Remarks—In this work, we have demonstrated that the Tat system plays an important role in the general physiology of E. coli. Global analysis of gene regulation has shown that expression of 6.9 and 1.8% of E. coli genes varies by a factor of >2-fold in response to inactivation of the Tat system under aerobic and anaerobic growth conditions, respectively. We were surprised to note that only a small percentage of these genes were differentially expressed under both growth conditions, but perhaps this is consistent with the profound effects on gene expression caused by anaerobic growth, which causes differential expression of 50% of all genes (78).

One of the most striking effects of mutating the tat system in E. coli is the failure of cells to undergo complete cell division, which is associated with a defect in the outer membrane barrier. This pleiotropic cell envelope phenotype is caused by the mislocalization of AmiA and AmiC, two cell wall amidases that are substrates of the Tat pathway. By also analyzing global gene expression in two additional {Delta}tatC mutant strains that are repaired in the pleiotropic cell envelope phenotype by transposon insertion, we showed that almost half of the differentially expressed genes in the {Delta}tatC strain are linked directly to the failure to export AmiA/AmiC. A large proportion of these genes are also up-regulated upon treatment of E. coli with the antimicrobial compound ampicillin, which targets cell wall synthesis (49), which is entirely consistent with the role of the amidases in cell wall processes. In keeping with the nature of the cell envelope defect, many of these differentially expressed genes encode products with roles in cell envelope-related processes, including the production of capsular polysaccharide. There have been reports of the effects of tat mutation on the motility of E. coli and flagellar assembly, which are, most likely, also secondary effects related to the cell envelope phenotype (28). We have noted here that the same pleiotropic defect also prevents the {Delta}tatC mutant from forming biofilms, and it is highly likely that the cell envelope defect is the major contributing factor to the attenuated virulence observed for tat mutant strains of E. coli K1 and 0157 (27, 28).

Interestingly, our analysis of genes that were differentially regulated in all three {Delta}tatC strains (i.e. that were not directly linked to the cell envelope response) has identified roles for the Tat pathway in copper detoxification and iron acquisition. Indeed, phenotypic analysis of the {Delta}tatC strains has indicated that the genes encoding the anaerobic copper-detoxifying system (Cus) are up-regulated under aerobic growth conditions, at least in part because of the mislocalization of CueO, a Tat-dependent multicopper oxidase. Moreover, we have also demonstrated that FhuD, a periplasmic shuttle protein involved in the import of the iron siderophore ferrichrome, is a novel Tat substrate and that the {Delta}tatC mutant is unable to grow aerobically with ferrichrome as the sole source of iron. Given the importance of iron in the pathogenicity of many bacterial species, we speculate that the mislocalization of FhuD may also be a contributing factor to the attenuation of E. coli tat mutant strains. Overall, these discoveries show that the Tat protein export system has a surprisingly pleiotropic effect on global gene expression in E. coli and is involved in some unexpected cell processes.


   FOOTNOTES
 
Note Added in Proof—Supplemental Table SI may also be downloaded from the following Web address: www.jic.bbsrc.au.uk/staff/tracy-palmer/.

* This work was in part supported by Grant 83/P16414 and a core strategic grant from the Biotechnology and Biological Sciences Research Council (to J. C. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Supplemental Table SI. Back

|| Supported by a Ph.D. studentship from the Department of Biochemistry, University of Oxford. Back

{ddagger}{ddagger} Royal Society Research Fellow. To whom correspondence should be addressed: Dept. of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK. Tel.: 44-1603-450726; Fax: 44-1603-450778; E-mail: tracy.palmer{at}bbsrc.ac.uk.

1 The abbreviations used are: MOPS, 4-morpholinepropanesulfonic acid; TMAO, trimethylamine-N-oxide; RT, reverse transcription. Back

2 Speed, T. P., and Yang, Y. H. (2002) Stanford Microarray Repository; available at genome-www5.stanford.edu/MicroArray/SMD/helpindex.html. Back

3 Primer sequences for RT-PCRs are available upon request. Back

4 D. Tullman, P. Iranpour, E. Strauch, B. Ribnicky, M. P. DeLisa, Y. Kawarasaki, T. Palmer, and G. Georgiou, unpublished data. Back


   ACKNOWLEDGMENTS
 
We acknowledge Drs. Frank Sargent, David Clarke, Ben Field, and Isabelle Caldelari and Professors Ian Booth and George Georgiou for helpful discussions and Professor Georgiou for critical reading of the manuscript. We thank Dr. Nicola Stanley for the construction of pNR30 and Shea Hamilton for help with the biofilm assay.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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