JOURNAL OF BACTERIOLOGY, Mar. 2005, p. 17631772 0021-9193/05/$08.000 doi:10.1128/JB.187.5.17631772.2005 Copyright 2005, American Society for Microbiology.

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Vol. 187, No. 5

Genome-Wide Analyses of Escherichia coli Gene Expression Responsive to the BaeSR Two-Component Regulatory System
Kunihiko Nishino,1,2,3,4 Takeshi Honda,4 and Akihito Yamaguchi1,2,3*
Department of Cell Membrane Biology, Institute of Scientic and Industrial Research, Osaka University, Ibaraki,1 and Core Research Evolutional Science and Technology (CREST), Japan Science and Technology Corporation,2 and Faculty of Pharmaceutical Science3 and Department of Bacterial Infections, Research Institute for Microbial Diseases,4 Osaka University, Osaka, Japan
Received 21 July 2004/Accepted 16 November 2004

sis. 2 comp. presentes en euk.

The BaeSR two-component regulatory system controls expression of exporter genes conferring drug resistance in Escherichia coli (S. Nagakubo, K. Nishino, T. Hirata, and A. Yamaguchi, J. Bacteriol. 184:41614167, 2002; N. Baranova and H. Nikaido, J. Bacteriol. 184:41684176, 2002). To understand the whole picture of BaeSR regulation, a DNA microarray analysis of the effect of BaeR overproduction was performed. BaeR overproduction activated 59 genes related to two-component signal transduction, chemotactic responses, agellar biosynthesis, maltose transport, and multidrug transport, and BaeR overproduction also repressed the expression of the ibpA and ibpB genes. All of the changes in the expression levels were also observed by quantitative real-time reverse transcription-PCR analysis. The expression levels of 15 of the 59 BaeR-activated genes were decreased by deletion of baeSR. Of 11 genes induced by indole (a putative inducer of the BaeSR system), 10 required the BaeSR system for induction. Combination of the expression data sets revealed a BaeR-binding site sequence motif, 5-TTTTTCTCCATDATTGGC-3 (where D is G, A, or T). Several genes up-regulated by BaeR overproduction, including genes for maltose transport, chemotactic responses, and agellar biosynthesis, required an intact PhoBR or CreBC two-component regulatory system for up-regulation. These data indicate that there is cross-regulation among the BaeSR, PhoBR, and CreBC two-component regulatory systems. Such a global analysis should reveal the regulatory network of the BaeSR system. and acrD, which encode multidurug exporter systems (15, 16, 36). Overproduction of BaeR, in the background of a deciency of the E. coli major multidrug exporter AcrB, confers resistance against -lactams, novobiocin, sodium dodecyl sulfate, and bile salts. However, the physiological role of the BaeSR system has remained unknown. We hypothesized that the BaeSR system controls the expression of a wide range of genes. E. coli microarrays have been successfully used to quantify the entire complement of individual mRNA transcripts (40, 46). Therefore, to reveal the whole picture of the BaeSR-controlled genes, a microarray analysis of genes affected by BaeR overproduction was performed in this study. The expression levels of all of the BaeR-affected genes were also investigated by quantitative real-time reverse transcription-PCR (qRT-PCR) analysis. Also, we investigated the effect of baeSR deletion on the levels of expression of genes by qRT-PCR analysis. In order to understand the response of the BaeSR system to signals, we examined the effect of addition of indole on gene expression levels. Combination of these expression data sets revealed a BaeR-binding site sequence motif. Furthermore, we examined the effects of deletion of phoBR or creBC on the levels of expression of the BaeR-induced genes to elucidate the genetic network of the BaeSR system.
MATERIALS AND METHODS Bacterial strains and growth conditions. The strains used in this work were E. coli K-12 derivatives (Table 1). They were grown at 37C in Luria-Bertani broth

Bacteria have developed signaling systems for eliciting a variety of adaptive responses to their environments. These adaptive responses are often mediated by two-component regulatory systems. A typical two-component regulatory system is composed of a histidine kinase sensor residing in the inner membrane and a cognate response regulator in the cytoplasm. Similar systems control the expression of genes for nutrient acquisition, virulence, antibiotic resistance, and numerous other pathways in diverse bacteria (2, 17, 47, 48). There are also analogous signaling systems in cells of eukaryotes, including fungi, amoebae, and plants (19, 31, 59, 60, 62). In Escherichia coli, 29 histidine kinase sensors, 32 response regulators, and one HPt (histidine-containing phosphotransmitter) domain protein have been found during analyses of the E. coli K-12 genome (34). Each sensor responds to specic environ29 HK, mental changes to cope with the numerous conditions that E. 32 RR coli faces. The functions of many of these systems remain E. coli undetermined. In a previous study, it was found that the BaeSR two-component system modulates the drug resistance of E. coli by regulating the expression of drug transporter genes (5, 36). The response regulator BaeR modulates the expression of mdtABC

* Corresponding author. Mailing address: Institute of Scientic and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki-shi, Osaka 567-0047, Japan. Phone: 81-6-6879-8545. Fax: 81-6-6879-8549. E-mail: akihito@sanken.osaka-u.ac.jp.

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NISHINO ET AL. TABLE 1. Strains and plasmids

J. BACTERIOL.

Strain or plasmid

Genotype

Source or reference

E. coli strains W3110 KAM3 NKE41 NKE42 NKE10 NKE11 NKE52 NKE56 NKE57 NKE122 NKE124 Plasmids pUC118 pUCbaeR

Wild type acrB-decient mutant of TG1 KAM3/pUC118 KAM3/pUCbaeR W3110/pUC118 W3110/pUCbaeR W3110 baeSR W3110 creBC/pUCbaeR W3110 phoBR/pUCbaeR W3110 BaeR binding motif for acrD, pUCbaeR W3110 BaeR binding motif for mdtA, pUCbaeR BamHI-PstI fragment containing baeR with 100-bp upstream anking sequence cloned into pUC118

Laboratory stock 32 This study This study This study This study This study This study This study This study This study Takara Bio, Inc. This study

in the absence of isopropyl--D-thiogalactopyranoside (56). The cells were rapidly collected for total RNA extraction when the culture reached an optical density at 600 nm of 0.6. Plasmid construction. The baeR gene was amplied from W3110 genoimic DNA by using primers 5-CGCGGATCCTTGAAGCACATAATGGTCGCA-3 and 5-CGCCTGCAGCTAAACGATGCGGCAGGCGTC-3, which introduced BamHI and PstI sites at the ends of the amplied fragment. This fragment contained baeR with a 100-bp upstream sequence. The PCR fragment was cloned between the BamHI and PstI sites of vector pUC118 (Takara Bio Inc., Shiga, Japan), which resulted in the baeR gene being arranged in the same orientation as the lactose promoter of the pUC118 vector. The nucleotide sequence of the recombinant plasmid was determined with an ABI PRISM 3100-Avant genetic analyzer (Applied Biosystems). Susceptibility testing. The antibacterial activities of agents were determined on L agar (1% tryptone, 0.5% yeast extract, 0.5% NaCl) plates containing novobiocin or sodium deoxycholate (Sigma) at various concentrations. Agar plates were prepared by the twofold agar dilution technique recommended by the Japan Society of Chemotherapy (20, 21). Organisms were tested by using a nal inoculum size of 104 CFU/spot, delivered with a multipoint inoculator (Sakuma Seisakusyo, Tokyo, Japan), and were incubated at 37C for 18 h in air. The MICs of compounds were dened as the lowest concentrations that severely inhibited bacterial cell growth. Construction of gene deletion mutants. Gene disruption was performed by the method of Datsenko and Wanner (11). The chloramphenicol resistance gene (cat), anked by Flp recognition target sites, was amplied by PCR with primers with 40-nucleotide extensions that were homologous to the beginning and end of the coding sequence of the gene or of the BaeR-binding motif to be disrupted. Plasmid pKD3 was used as a template. The resulting PCR product was used to transform the recipient W3110 strain expressing Red recombinase, and recombinant clones were isolated as chloramphenicol-resistant colonies. Chromosomal DNA was isolated from the mutants obtained, and the structures of the deleted loci were conrmed by performing a series of PCRs with primers complementary to cat and to adjacent regions. cat was eliminated by using plasmid pCP20 as described previously (11). RNA extraction. Total RNA was isolated from bacterial cultures with an RNeasy Protect Bacteria mini kit (QIAGEN) and RNase-free DNase (QIAGEN) as described previously (40, 43, 45). The absence of genomic DNA in DNase-treated RNA samples was conrmed by both inspecting nondenaturing agarose electrophoresis gels and performing PCR with primers known to target the genomic DNA. The RNA concentration was determined spectrophotometrically (56). DNA microarray analysis. Total RNA from strains NKE10 and NKE11 was extracted from mid-exponential-phase cultures as described above. Preparation of uorescently labeled cDNA, microarray hybridization, and data analysis were performed as described previously (46). In brief, cDNA labeled with Cy3-dUTP (NKE10) and Cy5-dUTP (NKE11) was synthesized from each lot of total RNA by random priming. Labeled cDNA probes were puried and hybridized to glass slide microarrays (IntelliGene E. coli CHIP, version 2.0; Takara Bio, Inc.). The slides were scanned for uorescence intensity by using a 428 array scanner (Affymetrix). The signal density of each spot in an array was quantied by using

the ImaGene software, version 4.2 (BioDiscovery). Basically, a normalized relative Cy5/Cy3 ratio of 4 or above was considered a signicant increase in expression, and a ratio of 0.25 or below was considered a signicant decrease in expression. Determination of specic transcript levels by quantitative, real-time PCR following reverse transcription. Bulk cDNA samples were synthesized from total RNA derived from E. coli cells by using TaqMan reverse transcription reagents (PE Applied Biosystems) and random hexamers as primers. Specic primer pairs were designed with the ABI PRISM Primer Express software (PE Applied Biosystems). rrsA of the 16S rRNA gene was chosen as the normalizing gene. Real-time PCR was performed with each specic primer pair by using SYBR Green PCR master mixture (PE Applied Biosystems). The reactions were performed with an ABI PRISM 7000 sequence detection system (PE Applied Biosystems), and during the reactions the uorescence signal due to SYBR Green intercalation was monitored to quantify the double-stranded DNA product formed in each PCR cycle.

RESULTS Effect of overexpression of baeR on gene expression. To determine the effect of overexpression of baeR on gene expression, we constructed high-copy-number plasmid pUCbaeR containing the baeR gene (Table 1). The baeR gene was in the same orientation as the lactose promoter of the pUC118 vector. Thus, baeR was expected to be expressed from the lactose promoter. This plasmid conferred novobiocin and deoxycholate resistance to acrB-decient strain KAM3. The MICs of novobiocin for NKE41 (KAM3/pUC118) and NKE42 (KAM3/ pUCbaeR) were 2 and 16 g/ml, respectively. The MICs of deoxycholate for these strains were 1,250 and 40,000 g/ml, respectively. pUC118 and pUCbaeR were used in the following microarray experiments. DNA microarrays, which contain most of the genomic open reading frames of E. coli (46), allow comprehensive studies of BaeR-controlled E. coli gene expression. We used the E. coli W3110 strain as a host for microarray analysis, because the DNA microarrays were made from DNA fragments of W3110 (Takara Bio, Inc.). pUCbaeR did not confer drug resistance to wild-type E. coli strain W3110, because the intrinsic multidrug exporter AcrB masks the effect of baeR overexpression. pUCbaeR conferred multidrug resistance to W3110 acrB and to KAM3 (data not shown). NKE10 has a single copy of baeR in its chromosome and harbors the vector plasmid pUC118, while NKE11 bears high-copy-number plasmid pUCbaeR (Ta-

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ble 1). The comprehensive transcript proles of these two strains prepared from exponential-phase cells were compared. The increased baeR gene dosage in NKE11 resulted in a 24fold increase in the expression of cognate baeR transcripts, and the expression of 58 other genes (open reading frames) was elevated more than fourfold, while the expression of two genes (ibpA and ibpB) was repressed more than fourfold (Table 2). We reinvestigated the BaeR-dependent induction of these genes by qRT-PCR analysis. All changes in gene expression greater than fourfold were also observed by qRT-PCR. The degree of induction determined by microarray analysis was usually lower than that determined by qRT-PCR, probably because the dynamic range of the former analysis is narrower than that of the latter (Table 2) (40). Known genes in the BaeR regulon. In previous studies, it was found that overproduction of BaeR increases the expression of mdtABCD (yegMNOB) and acrD (5, 15, 36). Also, Raffa and Raivio have reported that envelope stress induces the expression of spy via a BaeSR signal transduction pathway (49). Both in the DNA microarray analysis and in the qRT-PCR analysis, enhancement of mdtA, mdtB, mdtC, mdtD, acrD, and spy expression was found (Table 2) (Fig. 1). We found that BaeR increased the expression of the outer membrane channel tolC gene (7.9-fold as determined by microarray analysis and 12fold as determined by qRT-PCR), which is required for the function of the MdtABC and AcrD multidrug export systems (Table 2) (41, 42). Enhanced expression of gene clusters due to BaeR overproduction. The expression of 11 gene clusters was increased by BaeR overproduction. These 11 gene clusters are located at different positions on the E. coli chromosome (Fig. 1). Mainly, these clusters contain genes related to two-component signal transduction, chemotactic responses, agellar biosynthesis, maltose transport, and multidrug transport. Elevated expression of two-component regulatory system genes. The PhoBR two-component system controls the genes of the phosphate (Pho) regulon for assimilation of alternative P sources (65, 66). The microarray and qRT-PCR results showed that the expression levels of phoB and phoR were increased by baeR amplication (Fig. 1). BaeR overproduction increased the expression of baeS, which encodes a cognate sensor of the BaeR response regulator (37) and is located downstream of mdtABCD (42). It is thought that both mdtABCD and baeSR are regulated by BaeR, because they form an operon and are transcribed from the same promoter (Fig. 1) (6, 36). BaeR also increased the expression of the creB response regulator gene of the creBC two-component system. The CreBC system appears to be connected to carbon and energy metabolism (67). According to the microarray analysis results, the level of enhancement of expression of creB was 2.6-fold. Increased expression of the CreB regulon members (4), creD, malE, yidS, and yieI was observed by both microarray and qRT-PCR analyses (Table 2). Elevated expression of genes related to agellar synthesis and chemotaxis due to baeR overexpression. In E. coli, agellar biosynthesis, chemotaxis, and aerotaxis are coordinately regulated by hDC (10). Flagellar genes are expressed in three stages, early, middle, and late. The two early genes, hD and hC, form an operon through which environmental control of agellar synthesis is coordinated. Microarray analysis showed

no signicant effect of overexpression of baeR on hD expression (1.0-fold increase) or hC expression (1.2-fold increase). However, the expression of genes regulated by hDC, including gL, iACDST, and motAB, was signicantly increased (at least fourfold as determined by microarray analysis) (Fig. 1). These genes comprise both middle and late genes required for the synthesis and assembly of agella, as well as transcriptional regulators of agellar gene expression (gM and iA). Also regulated by hDC are genes associated with chemotaxis and aerotaxis, including cheAMRWZ, tap, tsr, and aer. Expression of most of these genes was also increased more than fourfold in the baeR-overexpressing strain (Fig. 1); the exception was aer (3.1-fold increase as determined by microarray analysis). Increased expression of the maltose operon due to baeR overexpression. Maltose and maltodextrins are present at high concentrations in the intestinal tracts of animals as by-products of starch metabolism. Maltose and maltodextrin are transported through a pore consisting of maltoporin encoded by lamB, which serves as a channel for sugar migration across the outer membrane. Both the transport and the utilization of these compounds are regulated by MalT, a regulator required for transcription at mal promoters (9). Genomic analysis demonstrated that there was increased expression of the maltose system in the baeR-overexpressing strain (Fig. 1). The ratio of expression of malT in the baeR-overexpressing strain to expression of malT in the host strain was increased only modestly (to 1.7). The expression of genes under the control of MalT was, however, signicantly increased (approximately 5- to 20-fold) (Table 2 and Fig. 1). BaeR increased the expression of malE, which encodes a maltose-binding protein, and malFGK, which encode the translocation complex. The genes used for maltose and maltodextrin metabolism also showed increased expression, as follows: malP, which encodes maltodextrin phosphorylase, 2.2-fold; malS, which encodes a nonessential maltodextrin-metabolizing enzyme, periplasmic -amylase, 2.9-fold; and malM, a periplasmic protein with an unknown function, 12fold. The expression of lamB, encoding maltoporin, the specic pore for maltodextirns and the receptor for phage in E. coli, was increased 12-fold. Enhanced expression of other operons. Elevated transcription of the gar operon (garPLRK) and garD was observed in the strain bearing the baeR amplication. Transcription of these genes was elevated 13-, 6.0-, 2.1-, 2.1-, and 6.5-fold, respectively (Fig. 1). These genes are related to D-galactarate metabolism (18, 35). BaeR increased the expression of three other clusters, one of which contains the amn and yeeN genes, one of which contains the gsp and yghU genes, and one of which contains the yieI and yieJ genes (Fig. 1). It has been reported that the amn gene encodes an AMP nucleosidase (26, 27) and that the gsp gene encodes a bifunctional glutathionylspermidine synthetase/amidase (8). The functions of the other genes are unknown (Table 2). Effect of the baeSR deletion on gene expression. To elucidate BaeSR regulation further, we investigated the effects of baeSR deletion on the gene expression levels. Total RNA from exponential-phase cells of W3110 and NKE52 (W3110 baeSR) was collected, and the gene expression levels were compared by the qRT-PCR method (Table 3). We chose target genes whose expression levels were enhanced by baeR amplication, as described above. As shown in Table 3, the expression of 15 genes

TABLE 2. E. coli genes whose relative expression levels were increased or decreased by baeR amplication
Effect of BaeR on gene expression (fold change) Microarray Real-time PCR

Gene

b no.

Known or predicted function

Increased expression creD spy mdtA (yegM) mdtB (yegN) baeR malE amn yiaD ansB yeeN malK garP (yhaU) gsp malM lamB yfjB yieI mdtC (yegO) tsr baeS tolC yieJ phoB elaA yghU cheM (tar) iC garD (yhaG) tap garL (yhaF) cheR yidS ypfH phnD malF sulA aroF gL malG iD glpF iT phnJ ycaC cheA icdA phoR xA iS motB b1141 motA agaZ cheW cheZ ybiC tsx acrD mdtD (yegB) Decreased expression ibpB ibpA
a b c

b4400 b1743 b2074 b2075 b2079 b4034 b1982 b3552 b2957 b1983 b4035 b3127 b2988 b4037 b4036 b2615 b3716 b2076 b4355 b2078 b3035 b3717 b0399 b2267 b2989 b1886 b1923 b3128 b1885 b3126 b1884 b3690 b2473 b4105 b4033 b0958 b2601 b1083 b4032 b1924 b3927 b1926 b4098 b0897 b1888 b1136 b0400 b1566 b1925 b1889 b1141 b1890 b3132 b1887 b1881 b0801 b0411 b2470 b2077 b3686 b3687

Tolerance to colicin E2 Periplasmic protein related to spheroblast formation Membrane protein inolved in drug resistance Multidrug transport protein (RND family) Response regulator in two-component regulatory system with BaeS Maltose transport protein, chemotaxis (ABC superfamily) AMP nucleosidase Putative outer membrane protein Periplasmic L-asparaginase II Conserved protein Maltose transport protein (ABC superfamily) (N terminal): phenotypic repressor of mal operon (C terminal) Putative (D)-glucarate/galactarate transport protein (MFS family) Glutathionylspermidine amidase (N terminal); glutathionylspermidine synthetase (C terminal) Periplasmic protein of mal regulon Maltoporin, high-afnity receptor for maltose and maltose oligosaccharides NAD kinase Putative membrane protein Multidrug transport protein (RND family) Methyl-accepting chemotaxis protein 1, serine sensor receptor Sensory histidine kinase in two-component regulatory system wtih BaeR Outer membrane channel; tolerance to colicin E1 Conserved hypothetical protein Response regulator in two-component regulatory system with PhoR (or CreC) Putative transferase Putative S-transferase Methyl-accepting chemotaxis protein II, aspartate sensor receptor Flagellar biosynthesis (D)-Galactarate dehydrogenase Methyl-accepting chemotaxis protein IV, peptide sensor receptor Alpha-dehydro-beta-deoxy-D-glucarate aldolase Glutamate methyltransferase, chemotactic response regulator Putative oxidoreductase with FAD/NAD(P)-binding domaind Conserved protein Phosphonate transport protein (ABC superfamily, peri_bind) Maltose transport protein (ABC superfamily, membrane) Suppressor of lon; inhibitor of cell division and FtsZ ring formation upon DNA damage or inhibition 3-Deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHP synthetase), tyrosine repressible Flagellar biosynthesis; hook-lament junction protein Maltose transport protein (ABC superfamily, membrane) Flagellar biosynthesis; lament capping protein; enables lament assembly MIP channel, glycerol diffusion Flagellar biosynthesis; putative export chaperone for FliD Conserved protein in phn operon Putative cysteine hydrolase Chemotactic sensory histidine kinase (soluble) in two-component regulatory system with CheB and CheY e14 prophage; isocitrate dehydrogenase, specic for NADP Sensory histidine kinase in two-component regulatory system with PhoB Qin prophage Flagellar biosynthesis; repressor of class 3a and 3b operons (RA activity) Enables agellar motor rotation, linking torque machinery to cell wall e14 prophage; putative exisionase Proton conductor component of motor, torque generator Tagatoso-6-phosphate aldolase I, subunit together with AgaY Purine-binding chemotaxis protein; regulation Chemotactic response, CheY protein phophatase Putative dehydrogenase Nucleoside channel; receptor of phage T6 and colicin K Aminoglycoside/multidrug efux pump (RND family) Putative multidrug transport protein (MFS family) Small heat shock protein Small heat shock protein

180 140 140 24 24 22 22 20 19 15 14 13 13 12 12 10 9.3 9.1 8.9 8.0 7.9 7.8 7.8 7.3 7.2 7.1 6.6 6.5 6.0 6.0 5.8 5.7 5.6 5.6 5.5 5.4 5.4 5.4 5.3 5.3 5.2 5.0 5.0 5.0 5.0 4.9 4.9 4.7 4.7 4.5 4.5 4.4 4.4 4.3 4.1 4.1 4.1 4.1 4.0 0.25 0.21

35,000 640 490 280 15,000 37 21 79 76 17 73 86 29 35 26 18 50 190 14 120 12 48 18 15 14 6.3 6.4 18 7.4 32 9.1 130 9.1 140 28 5.2 9.5 5.9 19 8.1 10 13 60 7.3 8.1 31 15 7.7 6.2 6.4 19 6.6 23 6.9 39 8.2 5.8 35 110 0.17 0.16

Based on information from the EcoGene database (http://bmb.med.maiami.edu/ecogene/ecoweb/) (55). Blattner number. Based on information from the E. coli Genome Project database (http://www.genome.wisc.edu/) (6). Based on information from the Gene ProtEC database (http://genprotec.mbl.edu/) (52, 53). d FAD, avin adenine dinucleotide.

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FIG. 1. Gene clusters whose expression was increased by BaeR overproduction. The expression changes for genes detected by real-time qRT-PCR are indicated under the gene names. The numbers in parentheses indicate the expression level changes detected by microarray analysis. The kb (kilobase pair) data indicate the positions on E. coli chromosomal DNA, as annotated on the Colibri website (http://genolist.Pasteur.fr /Colibri/). baeR was overexpressed from the high-copy-number plasmid pUCbaeR. N.D., not determined.

was decreased by baeSR deletion by more than a factor of two. qRT-PCR revealed that expression of mdtA, mdtB, mdtC, mdtD, yidS, phnJ, cheR, cheZ, ycaC, phnD, b1141, spy, acrD, lamB, and tap decreased by factors of 9.6, 2.0, 2.0, 2.0, 8.3, 3.3, 3.1, 2.9, 3.0, 2.8, 2.8, 2.2, 2.0, 2.0, and 2.0, respectively.

Effect of addition of indole on gene expression. Previously, Garbe et al. found that the Spy protein level is elevated when E. coli is grown in the presence of indole (13). Also, Raffa and Raivio found that the BaeSR system controls the expression of spy in response to the addition of indole (49). To help us

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NISHINO ET AL. TABLE 3. Expression levels of E. coli genes in the baeSR deletion mutant

J. BACTERIOL. TABLE 4. Effect of addition of indole on the levels of gene expression

Fold increasea Gene Strain W3110 Strain NKE52 (baeSR)

Gene

Effect of baeSR on gene expression (fold decrease)a

creD ........................................................................................... spy .............................................................................................. mdtA (yegM) ............................................................................. mdtB (yegN).............................................................................. baeR ........................................................................................... malE .......................................................................................... amn ............................................................................................ yiaD ........................................................................................... ansB ........................................................................................... yeeN ........................................................................................... malK .......................................................................................... garP (yhaU)............................................................................... gsp .............................................................................................. malM ......................................................................................... lamB .......................................................................................... yfjB ............................................................................................. yieI ............................................................................................. mdtC (yegO) ............................................................................. tsr ............................................................................................... baeS ........................................................................................... tolC ............................................................................................ yieJ ............................................................................................. phoB .......................................................................................... elaA ............................................................................................ yghU ........................................................................................... cheM (tar) ................................................................................. iC ............................................................................................. garD (yhaG).............................................................................. tap .............................................................................................. garL (yhaF)............................................................................... cheR ........................................................................................... yidS ............................................................................................ ypfH ........................................................................................... phnD .......................................................................................... malF .......................................................................................... sulA ............................................................................................ aroF ........................................................................................... gL............................................................................................. malG.......................................................................................... iD ............................................................................................. glpF ............................................................................................ iT ............................................................................................. phnJ ........................................................................................... ycaC ........................................................................................... cheA ........................................................................................... icdA ........................................................................................... phoR .......................................................................................... xA............................................................................................. iS .............................................................................................. motB .......................................................................................... b1141 ......................................................................................... motA .......................................................................................... agaZ ........................................................................................... cheW .......................................................................................... cheZ ........................................................................................... ybiC ............................................................................................ tsx ............................................................................................... acrD ........................................................................................... mdtD (yegB)..............................................................................

1.9 2.2 9.6 2.0 NE 1.9 1.2 1.1 0.9 1.6 0.8 0.7 1.7 0.8 2.0 1.1 1.8 2.0 1.4 NE 1.3 1.4 1.7 1.3 1.3 1.5 1.7 1.0 2.0 1.0 3.1 8.3 1.5 2.8 0.7 1.2 1.4 1.8 0.8 1.3 0.7 1.8 3.3 3.0 1.6 1.3 1.4 1.1 1.2 1.4 2.8 1.4 0.9 1.4 2.9 1.1 0.7 2.0 2.0

creD spy mdtA (yegM) mdtB (yegN) baeR malE amn yiaD ansB yeeN malK garP (yhaU) gsp malM lamB yfjB yieI mdtC (yegO) tsr baeS tolC yieJ phoB elaA yghU cheM (tar) iC garD (yhaG) tap garL (yhaF) cheR yidS ypfH phnD malF sulA aroF gL malG iD glpF iT phnJ ycaC cheA icdA phoR xA iS motB b1141 motA agaZ cheW cheZ ybiC tsx acrD mdtD (yegB)

5.2 14 2.4 2.4 1.6 0.6 1.4 1.8 0.6 0.5 0.2 0.4 1.2 0.5 0.3 1.0 1.2 2.3 0.1 1.4 1.4 1.1 1.0 1.2 2.2 0.2 0.1 0.3 0.2 0.6 0.3 1.9 1.3 1.2 0.5 1.0 1.1 0.2 0.7 0.1 20 0.1 1.2 2.3 0.1 2.1 1.4 0.1 0.1 0.1 1.4 0.1 1.1 0.2 0.4 1.5 0.3 2.3 2.4

1.0 1.5 0.7 0.8 NE 0.6 1.1 1.9 0.4 0.5 0.2 0.2 0.8 0.3 0.4 0.7 0.8 0.9 0.1 NE 0.9 0.7 0.7 0.8 1.3 0.1 0.1 0.2 0.1 0.3 0.2 0.7 0.9 0.1 0.4 0.6 0.8 0.1 0.5 0.0 13 0.0 0.4 1.1 0.1 1.1 0.7 0.0 0.1 0.1 0.9 0.1 0.6 0.1 0.1 1.0 0.2 0.5 1.1

a The values in boldface type indicate decreases of more than a factor of two compared to the expression levels of the genes in W3110. NE, no expression.

a The values in boldface type indicate increases of more than twofold compared to the expression levels of the genes in W3110 without addition of indole. NE, no expression.

understand the role of BaeSR regulation in response to indole, W3110 cells were grown in L-broth with or without 2 mM indole, after which total RNA was collected for qRT-PCR. A total of 59 genes were examined, and the expression of 11 of these genes was increased more than twofold when E. coli was

grown in the presence of indole (Table 4). qRT-PCR showed that expression of glpF, spy, creD, mdtA, mdtB, mdtC, mdtD, ycaC, acrD, yghU, and icdA increased by factors of 20, 14, 5.2, 2.4, 2.4, 2.3, 2.4, 2.3, 2.3, 2.2, and 2.1, respectively. We also

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TABLE 5. Effect of deletion of the BaeR-binding site sequence

Fold increasea Gene Strain NKE11 (wild type, pUCbaeR) Strain NKE122 (BaeR binding motif for acrD, pUCbaeR) Strain NKE124 (BaeR binding motif for mdtA, pUCbaeR)

FIG. 2. Intersections of genomic data sets. The diagram summarizes the data for 59 genes whose expression levels were increased by BaeR overproduction and for which there were valid data for expression proles of the baeSR mutant and wild type with the addition of indole. The expression levels of 15 of the 59 genes were decreased by deletion of baeSR, and 11 genes were identied as indole-regulated genes. The seven genes in the overlap of all three data sets were identied as components of the BaeSR regulon, as described in the text.

mdtA (yegM) mdtB (yegN) mdtC (yegO) mdtD (yegB) baeS acrD tolC spy baeR

490 280 190 110 120 35 12 640 15,000

510 300 190 120 130 1.7 13 600 17,000

1.1 0.6 0.4 1.1 0.6 38 12 590 11,000

a The values in boldface type indicate increases of more than twofold compared to the expression levels of the genes in NKE10 (W3110/pUC118).

investigated whether this gene induction is dependent on the BaeSR system. Most of the expression levels (10 genes; the exception was glpF) were not increased in the baeSR strain following growth in the presence of indole. Thus, indole appears to induce the expression of these genes via the BaeSR two-component signal transduction system. BaeR-binding site sequence motifs. We summarize the expression data sets in Fig. 2. Our microarray analysis identied 59 genes whose expression was increased by baeR overexpression. The expression of 15 genes was decreased by baeSR, and the expression of 11 genes was increased by indole. Using these three expression data sets, we identied seven genes as members of the BaeSR regulon (Fig. 2). This regulon contains the spy, mdtABCD, acrD, and ycaC genes. These genes are most likely regulated by BaeR directly. To identify the BaeR-binding motif, we analyzed upstream regions of spy, the mdtABCD operon, acrD, and ycaC. Analysis with the motif-nding program Align ACE (54) revealed a highly conserved 18-bp sequence in the upstream regions of spy, mdtA, and acrD (Fig. 3). The 18-bp consensus sequence is 5-TTTTTCTCCATDATTG

FIG. 3. Consensus sequence in the upstream regions of BaeR-regulated genes. Consensus sequences were found in the upstream regions of mdtA, spy, acrD, and ycaC. The numbering is relative to the start codon of the genes.

GC-3 (where D is G, A, or T). The program also found a similar sequence in the upstream region of ycaC, but the level of identity was low (50%) (Fig. 3). To test whether this sequence is required for gene induction by BaeR, we investigated the effect of deletion of the BaeR-binding motif on gene induction. We deleted the BaeR-binding motifs for the acrD gene and for the mdt operon. Overexpression of BaeR did not increase the expression levels of these genes without the BaeRbinding motifs (Table 5). Baranova and Nikaido reported that BaeR binds to the upstream region of mdtA (5). Recently, our group found that BaeR binds to the upstream regions of both mdtA and acrD (unpublished data). Also, the consensus sequence was found in the promoter region of spy. Thus, we suggest that expression of spy, mdtA, and acrD is directly regulated by the BaeSR two-component system. Searches of the regions upstream (400 bp) of all 59 genes whose expression was increased by BaeR overexpression did not reveal sequences matching the consensus sequence other than spy, mdtA, acrD, and ycaC. Effect of deletion of phoBR or creBC on BaeR-induced gene expression. As described above, expression of phoBR twocomponent system genes was increased by baeR amplication (Table 2). Overproduction of BaeR also increased expression of the CreB regulon (4), including expression of creD, malE, yidS, and yieI (Table 2) and the creB response regulator gene (2.6-fold increase as determined by microarray analysis). These data indicate that there is a possibility of cross-regulation among the PhoBR, CreBC, and BaeSR two-component systems. To test this hypothesis, we investigated the effects of deletion of phoBR and creBC on gene induction by baeR amplication. Total RNA was collected from exponential-phase cells of NKE57 (W3110 phoBR/pUCbaeR), NKE56 (W3110 creBC/pUCbaeR), and NKE10 (W3110/pUC118) for qRTPCR analysis. The expression levels of genes in NKE57 and NKE56 were compared to those in NKE10 (Table 6). The amplication of baeR in NKE56 (8,400-fold) was lower than that in NKE57 (21,000-fold), probably because the growth of NKE56 was slower than the growth of NKE57 (data not shown). Overproduction of BaeR induced the expression of 40 genes in the phoBR strain and the expression of 38 genes in the creBC strain by more than twofold. To put it differently,

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TABLE 6. Effect of deletion of phoBR and creBC on expression levels of genes that were increased by baeR amplication
Fold increasea Gene Strain NKE57 (phoBR) Strain NKE56 (creBC)

creD spy mdtA (yegM) mdtB (yegN) baeR malE amn yiaD ansB yeeN malK garP (yhaU) gsp malM lamB yfjB yieI mdtC (yegO) tsr baeS tolC yieJ phoB elaA yghU cheM (tar) iC garD (yhaG) tap garL (yhaF) cheR yidS ypfH phnD malF sulA aroF gL malG iD glpF iT phnJ ycaC cheA icdA phoR xA iS motB b1141 motA agaZ cheW cheZ ybiC tsx acrD mdtD (yegB)

1,400 650 380 54 21,000 0.0 16 23 44 8.6 0.0 18 14 0.0 0.0 11 20 100 11 50 6.0 14 NE 6.4 8.2 0.6 0.4 6.0 1.1 4.3 1.2 7.3 3.4 300 0.1 2.8 11 0.9 0.1 0.8 3.7 0.9 31 11 1.1 6.0 NE 3.5 0.7 2.9 4.3 2.7 5.1 0.6 0.6 4.8 3.3 75 94

31 240 310 53 8,400 0.2 8.8 23 27 4.2 0.1 18 10 0.5 0.2 7.6 1.0 47 4.5 28 3.0 0.8 9.1 3.4 6.6 0.8 0.5 6.1 0.8 3.3 1.1 1.0 3.3 70 0.3 2.1 8.0 0.6 0.3 0.9 0.4 1.0 16 3.9 0.9 6.4 5.2 2.9 0.8 2.8 3.2 2.7 5.7 0.9 1.1 4.4 4.0 22 42

PhoBR system and the CreBC system (63, 64). These 17 genes are malEFGKM-lamB (related to maltose transport), cheAM RWZ-tap (related to chemotactic response), and iCDST/gL (related to agellar biosynthesis). The expression of yieIJ, yidS, and glpF was not increased by baeR overexpression only in the creBC strain. These data indicate that there is a novel interaction among the BaeSR, PhoBR, and CreBC two-component systems. DISCUSSION In this work we examined the utility of microarray analysis for determining the global effects of baeR gene dosage amplication. In this study, we found a lot of genes whose BaeR dependence was not known previously. We discovered that overproduction of BaeR affects the expression of gene clusters related to two-component signal transduction, maltose transport, chemotatic responses, agellar biosynthesis, and multidrug transport. Since a large amount of BaeR may cause indirect regulation and this may not occur under the normal growth conditions, we also investigated the effect of deletion of baeSR on gene expression levels by using qRT-PCR methods. Also, in order to understand the whole picture of BaeSR regulation in response to signals, we investigated the effect of addition of indole on gene expression by qRT-PCR analysis. However, the data obtained, like all the expression prole data, cannot be used to distinguish direct targets from indirect targets, a critical distinction when regulatory networks are mapped. In this study, we combined the three expression data sets to dene the members of the BaeSR regulon. Analysis of upstream regions of the BaeSR regulon with the motif-nding program revealed an 18-bp consensus sequence, 5-TTTTTC TCCATDATTGGC-3 (where D is G, A, or T), and it revealed that this consensus sequence is required for gene induction by BaeR. The consensus sequence is present in the upstream regions of the spy gene, the acrD gene, and the mdtABCDbaeSR operon. We concluded that only these three regulons are directly regulated by the BaeSR system. Furthermore, qRT-PCR analysis with the phoBR creBC strain revealed novel cross-regulation among the BaeSR, PhoBR, and CreBC two-component systems. Spy (spheroplast protein Y), one of the components of the BaeSR regulon, was initially identied as a periplasmic protein whose expression is induced by spheroplast formation (14). Raivio et al. found that spy is a component of a regulon of the CpxAR two-component system that responds to envelope stresses (51), but the function of Spy remains unknown. BaeR controls the expression of acrD and mdtABC multidrug efux system genes. Both AcrD and MdtABC belong to the resistance-nodulation-cell division (RND) transporter family that plays a major role in producing both intrinsic and elevated levels of resistance to a very wide range of noxious compounds in gram-negative bacteria (30, 38, 39). AcrD and MdtABC drug exporters need outer membrane protein TolC in order to function (15, 36), like some other drug transporters of E. coli (12, 23, 24, 41). In this study, we found that BaeR overproduction increased the expression of tolC. This situation is very similar to the expression control of the mdtEF (yhiUV) multidrug efux system and tolC by the EvgAS two-component system (40, 43, 44).

a The values in boldface type indicate increases of more than twofold compared to the expression levels of the genes in NKE10 (W3110/pUC118). NE, no expression.

BaeR did not increase the expression of 17 genes in the phoBR strain or the expression of 21 genes in the creBC strain that were induced by BaeR in W3110. Seventeen of the genes overlapped, probably because of cross talk between the

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REFERENCES

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We previously cloned all of the gene clusters encoding putative and known drug transporters of E. coli and found that 20 genes encode transporters of some drugs and/or toxic compounds (42). The key to understanding how bacteria use these multiple transporters lies in analysis of the regulation of transporter expression. In this study, we found that the BaeSR system controls acrD and mdtABC in response to indole and probably other signals. Indole is a toxic compound that disrupts the bacterial envelope (49). Indole can also act as an extracellular signal in E. coli (1, 61), and it can be exported by the AcrEF multidrug transporter (22). Recently, our group found that the CpxAR two-component system also regulates the expression of acrD (15). Also, the CpxAR system plays an important role in response to envelope stresses (49). Thus, there is a strong relationship among envelope stresses, cell-tocell signaling, and induction of multidrug transporters. Some information about the regulation of multidrug transporters has been reported previously. Ma et al. reported that the expression of acrAB is induced by fatty acids, sodium chloride, and ethanol (29). Lomovskaya et al. reported that the emrAB drug exporter genes are induced by salicylic acid and 2,4-dinitrophenol (28). The EvgAS two-component system regulates the expression of mdtEF (yhiUV) and emrKY (43, 44). Also, Bock and Gross suggested that the EvgS sensor is connected to the oxidation status of the cell via a link to the ubiquinone pool (7). It has been reported that the expression of mdtEF (yhiUV) is controlled by RpoS (3, 58), an alternative sigma factor, which is needed for E. coli to survive stresses (25, 33, 57). RpoS is also required for the expression of many genes in stationary-phase cells. Recently, we found that histone-like protein H-NS represses the expression of acrEF and mdtEF (45). H-NS represses the expression of many genes that are expressed in the stationary phase. Also, the quorum-sensing regulator SdiA controls the expression of acrAB, acrD, and acrEF (50, 68). These data suggest that regulation of multidrug transporters has a strong relationship to (i) stress responses, (ii) the growth phase, and (iii) quorum sensing. Where are these regulatory networks and multidrug transporters needed in bacterial cells? Further investigation of the regulation of multidrug transporters in several natural environments, such as inside hosts, is needed in order to understand the biological signicance of the regulatory networks for them and may provide further insights into the role of multidrug transporters in the physiology of the cell.

ACKNOWLEDGMENTS We thank Barry L. Wanner and Tomofusa Tsuchiya for providing strains and plasmids; Junko Yamada for providing plasmid pUCbaeR; Takahiro Hirata, Hidetada Hirakawa, and Yoshihiko Inazumi for communicating unpublished results; and members of our labs for helpful discussions. K. Nishino was supported by a research fellowship from the Japan Society for the Promotion of Science for Young Scientists. This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by a grant-in-aid from the Zoonosis Control Project of the Ministry of Agriculture, Forestry and Fisheries of Japan, by a grant from the COE Program in the 21st Century of the Japan Society for the Promotion of Science, and by a grant from Core Research Evolutional Science and Technology (CREST) of the Japan Science and Technology Corporation.

1. Ahmer, B. M. 2004. Cell-to-cell signalling in Escherichia coli and Salmonella enterica. Mol. Microbiol. 52:933945. 2. Alex, L. A., and M. I. Simon. 1994. Protein histidine kinases and signal transduction in prokaryotes and eukaryotes. Trends Genet. 10:133138. 3. Altuvia, S., D. Weinstein-Fischer, A. Zhang, L. Postow, and G. Storz. 1997. A small, stable RNA induced by oxidative stress: role as a pleiotropic regulator and antimutator. Cell 90:4353. 4. Avison, M. B., R. E. Horton, T. R. Walsh, and P. M. Bennett. 2001. Escherichia coli CreBC is a global regulator of gene expression that responds to growth in minimal media. J. Biol. Chem. 276:2695526961. 5. Baranova, N., and H. Nikaido. 2002. The baeSR two-component regulatory system activates transcription of the yegMNOB (mdtABCD) transporter gene cluster in Escherichia coli and increases its resistance to novobiocin and deoxycholate. J. Bacteriol. 184:41684176. 6. Blattner, F. R., G. Plunkett, 3rd, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y. Shao. 1997. The complete genome sequence of Escherichia coli K-12. Science 277:14531474. 7. Bock, A., and R. Gross. 2002. The unorthodox histidine kinases BvgS and EvgS are responsive to the oxidation status of a quinone electron carrier. Eur. J. Biochem. 269:34793484. 8. Bollinger, J. M., Jr., D. S. Kwon, G. W. Huisman, R. Kolter, and C. T. Walsh. 1995. Glutathionylspermidine metabolism in Escherichia coli. Purication, cloning, overproduction, and characterization of a bifunctional glutathionylspermidine synthetase/amidase. J. Biol. Chem. 270:1403114041. 9. Boos, W., and H. Shuman. 1998. Maltose/maltodextrin system of Escherichia coli: transport, metabolism, and regulation. Microbiol. Mol. Biol. Rev. 62: 204229. 10. Chilcott, G. S., and K. T. Hughes. 2000. Coupling of agellar gene expression to agellar assembly in Salmonella enterica serovar Typhimurium and Escherichia coli. Microbiol. Mol. Biol. Rev. 64:694708. 11. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:66406645. 12. Fralick, J. A. 1996. Evidence that TolC is required for functioning of the Mar/AcrAB efux pump of Escherichia coli. J. Bacteriol. 178:58035805. 13. Garbe, T. R., M. Kobayashi, and H. Yukawa. 2000. Indole-inducible proteins in bacteria suggest membrane and oxidant toxicity. Arch. Microbiol. 173:78 82. 14. Hagenmaier, S., Y. D. Stierhof, and U. Henning. 1997. A new periplasmic protein of Escherichia coli which is synthesized in spheroplasts but not in intact cells. J. Bacteriol. 179:20732076. 15. Hirakawa, H., K. Nishino, T. Hirata, and A. Yamaguchi. 2003. Comprehensive studies on the drug resistance mediated by the overexpression of response regulators of two-component signal transduction systems in Escherichia coli. J. Bacteriol. 185:18511856. 16. Hirakawa, H., K. Nishino, J. Yamada, T. Hirata, and A. Yamaguchi. 2003. -Lactam resistance modulated by the overexpression of response regulators of two-component signal transduction systems in Escherichia coli. J. Antimicrob. Chemother. 52:576582. 17. Hoch, J. A., and T. J. Silhavy. 1995. Two-component signal transduction. ASM Press, Washington, D.C. 18. Hubbard, K. B., M. Koch, D. R. J. Palmer, P. C. Babbitt, and J. A. Gerlt. 1998. Evolution of enzymatic activities in the enolase superfamily: characterization of the D-glucarate/galactarate catabolic pathway in Escherichia coli. Biochemistry 37:1436914375. 19. Imamura, A., N. Hanaki, A. Nakamura, T. Suzuki, M. Taniguchi, T. Kiba, C. Ueguchi, T. Sugiyama, and T. Mizuno. 1999. Compilation and characterization of Arabidopsis thaliana response regulators implicated in His-Asp phosphorelay signal transduction. Plant Cell Physiol. 40:733742. 20. Japan Society of Chemotherapy. 1981. Method for the determination of minimum inhibitory concentrations (MICs) of aerobic bacteria by the agar dilution method. Chemotherapy (Tokyo) 29:7679. 21. Japan Society of Chemotherapy. 1992. Method for the determination of minimum inhibitory concentrations (MICs) by the micro-broth dilution method. Chemotherapy (Tokyo) 40:184189. 22. Kawamura-Sato, K., K. Shibayama, T. Horii, Y. Iimuma, Y. Arakawa, and M. Ohta. 1999. Role of multiple efux pumps in Escherichia coli in indole expulsion. FEMS Microbiol. Lett. 179:345352. 23. Kobayashi, N., K. Nishino, T. Hirata, and A. Yamaguchi. 2003. Membrane topology of ABC-type macrolide antibiotic exporter MacB in Escherichia coli. FEBS Lett. 546:241246. 24. Kobayashi, N., K. Nishino, and A. Yamaguchi. 2001. Novel macrolide-specic ABC-type efux transporter in Escherichia coli. J. Bacteriol. 183:5639 5644. 25. Lange, R., D. Fischer, and R. Hengge-Aronis. 1995. Identication of transcriptional start sites and the role of ppGpp in the expression of rpoS, the structural gene for the sigma S subunit of RNA polymerase in Escherichia coli. J. Bacteriol. 177:46764680.

1772

NISHINO ET AL.

J. BACTERIOL.
48. Parkinson, J. S., and E. C. Kofoid. 1992. Communication modules in bacterial signaling proteins. Annu. Rev. Genet. 26:71112. 49. Raffa, R. G., and T. L. Raivio. 2002. A third envelope stress signal transduction pathway in Escherichia coli. Mol. Microbiol. 45:15991611. 50. Rahmati, S., S. Yang, A. L. Davidson, and E. L. Zechiedrich. 2002. Control of the AcrAB multidrug efux pump by quorum-sensing regulator SdiA. Mol. Microbiol. 43:677685. 51. Raivio, T. L., M. W. Laird, J. C. Joly, and T. J. Silhavy. 2000. Tethering of CpxP to the inner membrane prevents spheroplast induction of the cpx envelope stress response. Mol. Microbiol. 37:11861197. 52. Riley, M., and B. Labedan. 1996. Escherichia coli gene products: physiological functions and common ancestries, p. 21182202. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C. 53. Riley, M., and B. Labedan. 1997. Protein evolution viewed through Escherichia coli protein sequences: introducing the notion of a structural segment of homology, the module. J. Mol. Biol. 268:857868. 54. Roth, F. P., J. D. Hughes, P. W. Estep, and G. M. Church. 1998. Finding DNA regulatory motifs within unaligned noncoding sequences clustered by whole-genome mRNA quantitation. Nat. Biotechnol. 16:939945. 55. Rudd, K. E. 2000. EcoGene: a genome sequence database for Escherichia coli K-12. Nucleic Acids Res. 28:6064. 56. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 57. Sammartano, L. J., R. W. Tuveson, and R. Davenport. 1986. Control of sensitivity to inactivation by H2O2 and broad-spectrum near-UV radiation by the Escherichia coli katF locus. J. Bacteriol. 168:1321. 58. Schellhorn, H. E., J. P. Audia, L. I. Wei, and L. Chang. 1998. Identication of conserved, RpoS-dependent stationary-phase genes of Escherichia coli. J. Bacteriol. 180:62836291. 59. Swanson, R. V., L. A. Alex, and M. I. Simon. 1994. Histidine and aspartate phosphorylation: two-component systems and the limits of homology. Trends Biochem. Sci. 19:485490. 60. Tracy, B. S., K. K. Edwards, and A. Eisenstark. 2002. Carbon and nitrogen substrate utilization by archival Salmonella typhimurium LT2 cells. BMC Evol. Biol. 2:14. 61. Wang, D., X. Ding, and P. N. Rather. 2001. Indole can act as an extracellular signal in Escherichia coli. J. Bacteriol. 183:42104216. 62. Wang, N., G. Shaulsky, R. Escalante, and W. F. Loomis. 1996. A twocomponent histidine kinase gene that functions in Dictyostelium development. EMBO J. 15:38903898. 63. Wanner, B. L. 1993. Gene regulation by phosphate in enteric bacteria. J. Cell Biochem. 51:4754. 64. Wanner, B. L. 1992. Is cross regulation by phosphorylation of two-component response regulator proteins important in bacteria? J. Bacteriol. 174: 20532058. 65. Wanner, B. L. 1996. Phosphorus assimilation and control of the phosphate regulon, p. 13571381. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. ASM Press, Washington, D.C. 66. Wanner, B. L., and B. D. Chang. 1987. The phoBR operon in Escherichia coli K-12. J. Bacteriol. 169:55695574. 67. Wanner, B. L., W. Jiang, S.-K. Kim, S. Yamagata, A. Haldimann, and L. L. Daniels. 1996. Are the multiple signal transduction pathways of the Pho regulon due to cross talk or cross regulation?, p. 297315. In E. C. C. Lin and A. S. Lynch (ed.), Regulation of gene expression in Escherichia coli. R. G. Landes Co., Austin, Tex. 68. Wei, Y., J. M. Lee, D. R. Smulski, and R. A. LaRossa. 2001. Global impact of sdiA amplication revealed by comprehensive gene expression proling of Escherichia coli. J. Bacteriol. 183:22652272.

26. Leung, H. B., K. L. Kvalnes-Krick, S. L. Meyer, J. K. deRiel, and V. L. Schramm. 1989. Structure and regulation of the AMP nucleosidase gene (amn) from Escherichia coli. Biochemistry 28:87268733. 27. Leung, H. B., and V. L. Schramm. 1984. The structural gene for AMP nucleosidase. Mapping, cloning, and overproduction of the enzyme. J. Biol. Chem. 259:69726978. 28. Lomovskaya, O., K. Lewis, and A. Matin. 1995. EmrR is a negative regulator of the Escherichia coli multidrug resistance pump EmrAB. J. Bacteriol. 177:23282334. 29. Ma, D., D. N. Cook, M. Alberti, N. G. Pon, H. Nikaido, and J. E. Hearst. 1995. Genes acrA and acrB encode a stress-induced efux system of Escherichia coli. Mol. Microbiol. 16:4555. 30. Ma, D., D. N. Cook, J. E. Hearst, and H. Nikaido. 1994. Efux pumps and drug resistance in gram-negative bacteria. Trends Microbiol. 2:489493. 31. Maeda, T., S. M. Wurgler-Murphy, and H. Saito. 1994. A two-component system that regulates an osmosensing MAP kinase cascade in yeast. Nature 369:242245. 32. Masaoka, Y., Y. Ueno, Y. Morita, T. Kuroda, T. Mizushima, and T. Tsuchiya. 2000. A two-component multidrug efux pump, EbrAB, in Bacillus subtilis. J. Bacteriol. 182:23072310. 33. McCann, M. P., C. D. Fraley, and A. Matin. 1993. The putative sigma factor KatF is regulated posttranscriptionally during carbon starvation. J. Bacteriol. 175:21432149. 34. Mizuno, T. 1997. Compilation of all genes encoding two-component phosphotransfer signal transducers in the genome of Escherichia coli. DNA Res. 4:161168. 35. Monterrubio, R., L. Baldoma, N. Obradors, J. Aguilar, and J. Badia. 2000. A common regulator for the operons encoding the enzymes involved in D-galactarate, D-glucarate, and D-glycerate utilization in Escherichia coli. J. Bacteriol. 182:26722674. 36. Nagakubo, S., K. Nishino, T. Hirata, and A. Yamaguchi. 2002. The putative response regulator BaeR stimulates multidrug resistance of Escherichia coli via a novel multidrug exporter system, MdtABC. J. Bacteriol. 184:41614167. 37. Nagasawa, S., K. Ishige, and T. Mizuno. 1993. Novel members of the twocomponent signal transduction genes in Escherichia coli. J. Biochem. (Tokyo) 114:350357. 38. Nikaido, H. 1996. Multidrug efux pumps of gram-negative bacteria. J. Bacteriol. 178:58535859. 39. Nikaido, H. 1994. Prevention of drug access to bacterial targets: permeability barriers and active efux. Science 264:382388. 40. Nishino, K., Y. Inazumi, and A. Yamaguchi. 2003. Global analysis of genes regulated by EvgA of the two-component regulatory system in Escherichia coli. J. Bacteriol. 185:26672672. 41. Nishino, K., J. Yamada, H. Hirakawa, T. Hirata, and A. Yamaguchi. 2003. Roles of TolC-dependent multidrug transporters of Escherichia coli in resistance to -lactams. Antimicrob. Agents Chemother. 47:30303033. 42. Nishino, K., and A. Yamaguchi. 2001. Analysis of a complete library of putative drug transporter genes in Escherichia coli. J. Bacteriol. 183:5803 5812. 43. Nishino, K., and A. Yamaguchi. 2002. EvgA of the two-component signal transduction system modulates production of the yhiUV multidrug transporter in Escherichia coli. J. Bacteriol. 184:23192323. 44. Nishino, K., and A. Yamaguchi. 2001. Overexpression of the response regulator evgA of the two-component signal transduction system modulates multidrug resistance conferred by multidrug resistance transporters. J. Bacteriol. 183:14551458. 45. Nishino, K., and A. Yamaguchi. 2004. Role of histone-like protein H-NS in multidrug resistance of Escherichia coli. J. Bacteriol. 186:14231429. 46. Oshima, T., C. Wada, Y. Kawagoe, T. Ara, M. Maeda, Y. Masuda, S. Hiraga, and H. Mori. 2002. Genome-wide analysis of deoxyadenosine methyltransferase-mediated control of gene expression in Escherichia coli. Mol. Microbiol. 45:673695. 47. Parkinson, J. S. 1993. Signal transduction schemes of bacteria. Cell 73:857 871.