You are on page 1of 10

JOURNAL OF BACTERIOLOGY, Mar. 2005, p. 1763–1772 0021-9193/05/$08.00ϩ0 doi:10.1128/JB.187.5.1763–1772.

2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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 Scientific 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. 184:4161–4167, 2002; N. Baranova and H. Nikaido, J. Bacteriol. 184:4168–4176, 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, flagellar 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 flagellar 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 deficiency 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 37°C 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 specific 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 Scientific 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.

1763 Funcion de los sis. 2 componentes

The signal density of each spot in an array was quantified by using the ImaGene software. and recombinant clones were isolated as chloramphenicol-resistant colonies. The chloramphenicol resistance gene (cat). Specific primer pairs were designed with the ABI PRISM Primer Express software (PE Applied Biosystems). In brief. we constructed high-copy-number plasmid pUCbaeR containing the baeR gene (Table 1). flanked by Flp recognition target sites. Bulk cDNA samples were synthesized from total RNA derived from E. pUC118 and pUCbaeR were used in the following microarray experiments.). and the structures of the deleted loci were confirmed by performing a series of PCRs with primers complementary to cat and to adjacent regions. pUCbaeR BamHI-PstI fragment containing baeR with 100-bp upstream flanking 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. Chromosomal DNA was isolated from the mutants obtained. BACTERIOL. 21). Plasmid construction. cat was eliminated by using plasmid pCP20 as described previously (11). delivered with a multipoint inoculator (Sakuma Seisakusyo. We used the E. which resulted in the baeR gene being arranged in the same orientation as the lactose promoter of the pUC118 vector.1764 NISHINO ET AL. and data analysis were performed as described previously (46). Japan). Inc.6. This fragment contained baeR with a 100-bp upstream sequence. The MICs of novobiocin for NKE41 (KAM3/pUC118) and NKE42 (KAM3/ pUCbaeR) were 2 and 16 ␮g/ml. 43. real-time PCR following reverse transcription. The resulting PCR product was used to transform the recipient W3110 strain expressing Red recombinase. The PCR fragment was cloned between the BamHI and PstI sites of vector pUC118 (Takara Bio Inc. Strain or plasmid Genotype Source or reference E.2 (BioDiscovery).000 ␮g/ml. RNA extraction. coli strain W3110. Shiga. Total RNA from strains NKE10 and NKE11 was extracted from mid-exponential-phase cultures as described above. rrsA of the 16S rRNA gene was chosen as the normalizing gene. Gene disruption was performed by the method of Datsenko and Wanner (11). This plasmid conferred novobiocin and deoxycholate resistance to acrB-deficient strain KAM3. a normalized relative Cy5/Cy3 ratio of 4 or above was considered a significant increase in expression.5% NaCl) plates containing novobiocin or sodium deoxycholate (Sigma) at various concentrations. The baeR gene was in the same orientation as the lactose promoter of the pUC118 vector. TABLE 1. coli strains W3110 KAM3 NKE41 NKE42 NKE10 NKE11 NKE52 NKE56 NKE57 NKE122 NKE124 Plasmids pUC118 pUCbaeR Wild type acrB-deficient 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. which contain most of the genomic open reading frames of E. Organisms were tested by using a final inoculum size of 104 CFU/spot. To determine the effect of overexpression of baeR on gene expression. Japan).25 or below was considered a significant decrease in expression.). NKE10 has a single copy of baeR in its chromosome and harbors the vector plasmid pUC118. Basically. respectively. 0. 0. coli W3110 strain as a host for microarray analysis. Takara Bio.5% yeast extract. coli gene expression. and during the reactions the fluorescence signal due to SYBR Green intercalation was monitored to quantify the double-stranded DNA product formed in each PCR cycle. The MICs of deoxycholate for these strains were 1. RESULTS Effect of overexpression of baeR on gene expression. while NKE11 bears high-copy-number plasmid pUCbaeR (Ta- . pUCbaeR conferred multidrug resistance to W3110 ⌬acrB and to KAM3 (data not shown). Inc. Strains and plasmids J. The MICs of compounds were defined as the lowest concentrations that severely inhibited bacterial cell growth. The RNA concentration was determined spectrophotometrically (56). Tokyo. The nucleotide sequence of the recombinant plasmid was determined with an ABI PRISM 3100-Avant genetic analyzer (Applied Biosystems). DNA microarray analysis. baeR was expected to be expressed from the lactose promoter. pUCbaeR W3110 ⌬BaeR binding motif for mdtA. which introduced BamHI and PstI sites at the ends of the amplified fragment.250 and 40. The baeR gene was amplified from W3110 genoimic DNA by using primers 5Ј-CGCGGATCCTTGAAGCACATAATGGTCGCA-3Ј and 5Ј-CGCCTGCAGCTAAACGATGCGGCAGGCGTC-3Ј. because the intrinsic multidrug exporter AcrB masks the effect of baeR overexpression. The cells were rapidly collected for total RNA extraction when the culture reached an optical density at 600 nm of 0. Labeled cDNA probes were purified and hybridized to glass slide microarrays (IntelliGene E. and a ratio of 0. DNA microarrays. version 4. coli cells by using TaqMan reverse transcription reagents (PE Applied Biosystems) and random hexamers as primers. Total RNA was isolated from bacterial cultures with an RNeasy Protect Bacteria mini kit (QIAGEN) and RNase-free DNase (QIAGEN) as described previously (40. Thus. The absence of genomic DNA in DNase-treated RNA samples was confirmed by both inspecting nondenaturing agarose electrophoresis gels and performing PCR with primers known to target the genomic DNA. coli (46). Real-time PCR was performed with each specific primer pair by using SYBR Green PCR master mixture (PE Applied Biosystems). 45). was amplified 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. This study in the absence of isopropyl-␤-D-thiogalactopyranoside (56). allow comprehensive studies of BaeR-controlled E. The reactions were performed with an ABI PRISM 7000 sequence detection system (PE Applied Biosystems). and were incubated at 37°C for 18 h in air. Plasmid pKD3 was used as a template. pUCbaeR did not confer drug resistance to wild-type E. Inc. Construction of gene deletion mutants. because the DNA microarrays were made from DNA fragments of W3110 (Takara Bio.. Determination of specific transcript levels by quantitative. The slides were scanned for fluorescence intensity by using a 428 array scanner (Affymetrix). The antibacterial activities of agents were determined on L agar (1% tryptone. Agar plates were prepared by the twofold agar dilution technique recommended by the Japan Society of Chemotherapy (20. microarray hybridization. respectively. Susceptibility testing.0. cDNA labeled with Cy3-dUTP (NKE10) and Cy5-dUTP (NKE11) was synthesized from each lot of total RNA by random priming. Preparation of fluorescently labeled cDNA. version 2. coli CHIP.

chemotaxis. was significantly increased (at least fourfold as determined by microarray analysis) (Fig. These 11 gene clusters are located at different positions on the E. one of which contains the amn and yeeN genes. flhD and flhC. Also regulated by flhDC are genes associated with chemotaxis and aerotaxis. 1). and spy expression was found (Table 2) (Fig. acrD. 2.1-. was increased 12-fold.9-fold as determined by microarray analysis and 12fold as determined by qRT-PCR). It is thought that both mdtABCD and baeSR are regulated by BaeR. flagellar biosynthesis. 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). We chose target genes whose expression levels were enhanced by baeR amplification. 1) (6. 35). which encodes a nonessential maltodextrin-metabolizing enzyme.0-fold increase) or flhC expression (1. and motAB. Effect of the baeSR deletion on gene expression. which encodes maltodextrin phosphorylase. middle. which encodes a cognate sensor of the BaeR response regulator (37) and is located downstream of mdtABCD (42). and 6. The degree of induction determined by microarray analysis was usually lower than that determined by qRT-PCR. and yieI was observed by both microarray and qRT-PCR analyses (Table 2). Elevated expression of genes related to flagellar synthesis and chemotaxis due to baeR overexpression. These genes comprise both middle and late genes required for the synthesis and assembly of flagella. Both in the DNA microarray analysis and in the qRT-PCR analysis. which encode the translocation complex. Maltose and maltodextrin are transported through a pore consisting of maltoporin encoded by lamB. Flagellar genes are expressed in three stages. Maltose and maltodextrins are present at high concentrations in the intestinal tracts of animals as by-products of starch metabolism. chemotactic responses. 187. malS. periplasmic ␣-amylase. 2005 GENE REGULATION BY BaeSR 1765 ble 1). early. Known genes in the BaeR regulon. the expression of genes regulated by flhDC. the level of enhancement of expression of creB was 2. Transcription of these genes was elevated 13-. as described above.7). However. creD. while the expression of two genes (ibpA and ibpB) was repressed more than fourfold (Table 2). Enhanced expression of gene clusters due to BaeR overproduction. malE. We found that BaeR increased the expression of the outer membrane channel tolC gene (7. Microarray analysis showed no significant effect of overexpression of baeR on flhD expression (1. The expression of lamB. mdtB. The two early genes. 1). Raffa and Raivio have reported that envelope stress induces the expression of spy via a BaeSR signal transduction pathway (49). however. 2. The PhoBR two-component system controls the genes of the phosphate (Pho) regulon for assimilation of alternative P sources (65. the specific pore for maltodextirns and the receptor for phage ␭ in E. including cheAMRWZ. significantly increased (approximately 5. These genes are related to D-galactarate metabolism (18. as follows: malP. The genes used for maltose and maltodextrin metabolism also showed increased expression. BaeR overproduction increased the expression of baeS. tsr. respectively (Fig. probably because the dynamic range of the former analysis is narrower than that of the latter (Table 2) (40). 1). because they form an operon and are transcribed from the same promoter (Fig. and malFGK. 1). 66). Expression of most of these genes was also increased more than fourfold in the baeR-overexpressing strain (Fig. The microarray and qRT-PCR results showed that the expression levels of phoB and phoR were increased by baeR amplification (Fig. enhancement of mdtA. 2. 1). the expression of 15 genes . As shown in Table 3. fliACDST. and multidrug transport. 6. a periplasmic protein with an unknown function. it was found that overproduction of BaeR increases the expression of mdtABCD (yegMNOB) and acrD (5. coli. mdtD. as well as transcriptional regulators of flagellar gene expression (flgM and fliA).1-. In E.to 20-fold) (Table 2 and Fig. 1). maltose transport. In previous studies. 1). yidS. The CreBC system appears to be connected to carbon and energy metabolism (67). To elucidate BaeSR regulation further. BaeR also increased the expression of the creB response regulator gene of the creBC two-component system. Increased expression of the CreB regulon members (4).2-fold increase). coli. one of which contains the gsp and yghU genes. form an operon through which environmental control of flagellar synthesis is coordinated. Elevated transcription of the gar operon (garPLRK) and garD was observed in the strain bearing the baeR amplification. The increased baeR gene dosage in NKE11 resulted in a 24fold increase in the expression of cognate baeR transcripts. 36).0-. including flgL. and one of which contains the yieI and yieJ genes (Fig. and aer. 2. tap.6-fold.9-fold. the exception was aer (3. these clusters contain genes related to two-component signal transduction. 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. and aerotaxis are coordinately regulated by flhDC (10). which serves as a channel for sugar migration across the outer membrane. BaeR increased the expression of malE. Mainly.2-fold. we investigated the effects of baeSR deletion on the gene expression levels. 1). Also. BaeR increased the expression of three other clusters. All changes in gene expression greater than fourfold were also observed by qRT-PCR. The expression of 11 gene clusters was increased by BaeR overproduction. and late. The expression of genes under the control of MalT was.VOL. Increased expression of the maltose operon due to baeR overexpression. which is required for the function of the MdtABC and AcrD multidrug export systems (Table 2) (41. 42). encoding maltoporin. and the expression of 58 other genes (open reading frames) was elevated more than fourfold. coli chromosome (Fig. Enhanced expression of other operons. We reinvestigated the BaeR-dependent induction of these genes by qRT-PCR analysis. and the gene expression levels were compared by the qRT-PCR method (Table 3). Total RNA from exponential-phase cells of W3110 and NKE52 (W3110 ⌬baeSR) was collected. 1). a regulator required for transcription at mal promoters (9).5-fold. Both the transport and the utilization of these compounds are regulated by MalT. 12fold. The functions of the other genes are unknown (Table 2).1-fold increase as determined by microarray analysis). flagellar biosynthesis. According to the microarray analysis results. Genomic analysis demonstrated that there was increased expression of the maltose system in the baeR-overexpressing strain (Fig. Elevated expression of two-component regulatory system genes. 36). 15. The comprehensive transcript profiles of these two strains prepared from exponential-phase cells were compared. and malM. mdtC. which encodes a maltose-binding protein.

21 35.1 4.2 9.maiami.4 4.9 7.med.17 0.3 5. Based on information from the E. membrane) Suppressor of lon.0 5.000 37 21 79 76 17 73 86 29 35 26 18 50 190 14 120 12 48 18 15 14 6.4 32 9. filament capping protein. coli Genome Project database (http://www. hook-filament junction protein Maltose transport protein (ABC superfamily. chemotactic response regulator Putative oxidoreductase with FAD/NAD(P)-binding domaind Conserved protein Phosphonate transport protein (ABC superfamily.4 18 7. torque generator Tagatoso-6-phosphate aldolase I.genome.1 4.9 4.7 6.9 8.9 4.000 640 490 280 15.0 6.4 5. putative exisionase Proton conductor component of motor.4 5.5 4. glycerol diffusion Flagellar biosynthesis.4 19 6. aspartate sensor receptor Flagellar biosynthesis (D)-Galactarate dehydrogenase Methyl-accepting chemotaxis protein IV.8 5.1 31 15 7. glutathionylspermidine synthetase (C terminal) Periplasmic protein of mal regulon Maltoporin. flavin adenine dinucleotide.4 5.6 6.0 7.edu/) (6).6 23 6. specific for NADPϩ Sensory histidine kinase in two-component regulatory system with PhoB Qin prophage Flagellar biosynthesis. 1766 .0 4.7 4.3 7.8 35 110 0. Blattner number. Based on information from the Gene ProtEC database (http://genprotec. membrane) Flagellar biosynthesis.edu/ecogene/ecoweb/) (55).mbl. linking torque machinery to cell wall e14 prophage. 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). regulation Chemotactic response.8 7.1 8.3 6.6 5. receptor of phage T6 and colicin K Aminoglycoside/multidrug efflux 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 9.9 19 8.TABLE 2.1 130 9.9 39 8. serine sensor receptor Sensory histidine kinase in two-component regulatory system wtih BaeR Outer membrane channel. d FAD.1 4.2 5. E. CheY protein phophatase Putative dehydrogenase Nucleoside channel.4 4. subunit together with AgaY Purine-binding chemotaxis protein.7 5.0 5.0 5. 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.2 6.3 5. high-affinity receptor for maltose and maltose oligosaccharides NAD kinase Putative membrane protein Multidrug transport protein (RND family) Methyl-accepting chemotaxis protein 1. 53). isocitrate dehydrogenase. b Known or predicted function c 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) fliC garD (yhaG) tap garL (yhaF) cheR yidS ypfH phnD malF sulA aroF flgL malG fliD glpF fliT phnJ ycaC cheA icdA phoR flxA fliS 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.1 10 13 60 7.5 6. peri_bind) Maltose transport protein (ABC superfamily. repressor of class 3a and 3b operons (RflA activity) Enables flagellar motor rotation.0 5. enables filament assembly MIP channel.1 6.7 4. inhibitor of cell division and FtsZ ring formation upon DNA damage or inhibition 3-Deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHP synthetase). 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.5 5.3 8. coli genes whose relative expression levels were increased or decreased by baeR amplification Effect of BaeR on gene expression (fold change) Microarray Real-time PCR Gene a b no.8 7.2 7.25 0.5 4.16 Based on information from the EcoGene database (http://bmb.wisc.edu/) (52.1 140 28 5. peptide sensor receptor Alpha-dehydro-beta-deoxy-D-glucarate aldolase Glutamate methyltransferase. tyrosine repressible Flagellar biosynthesis.5 5.3 4.6 5.3 9.1 4.2 5.0 0.

Pasteur. qRT-PCR revealed that expression of mdtA. Gene clusters whose expression was increased by BaeR overproduction. 2005 GENE REGULATION BY BaeSR 1767 FIG.0. cheR. and 2. mdtD. phnD.0. 8. cheZ. b1141. yidS. 2. 1. 3. coli is grown in the presence of indole (13).9. 2. Effect of addition of indole on gene expression. respectively. not determined. was decreased by baeSR deletion by more than a factor of two.0. ycaC.VOL.6. baeR was overexpressed from the high-copy-number plasmid pUCbaeR. 2. 2.fr /Colibri/). acrD. 2.0. Also. spy.D. 2. The expression changes for genes detected by real-time qRT-PCR are indicated under the gene names. 2. 3. Raffa and Raivio found that the BaeSR system controls the expression of spy in response to the addition of indole (49). 187. Previously. and tap decreased by factors of 9. mdtC.0. The kb (kilobase pair) data indicate the positions on E. found that the Spy protein level is elevated when E. 3.3.8. coli chromosomal DNA. 2.0. phnJ.1.. lamB. mdtB. The numbers in parentheses indicate the expression level changes detected by microarray analysis. To help us . N. as annotated on the Colibri website (http://genolist.2.8. 2. Garbe et al.3.0.

..0 1..... fliD ...........1 0..............................................................................................................5 1..............4 1.........2 0...................2 2...........................................................3 1.. acrD......1 0.0 1..........................9 1... ansB ............................4 1...............................4 1....... 2......................... baeS ............... ybiC ............. malK ............. respectively............................4 1..........1 0......... mdtB (yegN)......4 0.....................6 1...................................................4 NE 1.........2 1........................3.......................................................... Expression levels of E....... flgL......... cheR ..........6 0.........7 1... creD.......... yieJ ............................... 2................ flxA...................... cheM (tar) ..6 1...... acrD .....................4 1....3 0....2 0.....................4 1.....0 1.....0 13 0........................... phnJ ................................................................... fliC .... motA .......................... 2.....................................9 1............0 NE 1..................... 5........................................................ BACTERIOL...3 0.......5 0......7 1.....6 1...3 1..........9 0.2 0.........................4 1........................ mdtC (yegO) .........8 0.....4 0.......7 0.............................................. understand the role of BaeSR regulation in response to indole........ 2................8 3...................1...................................................3 0.................................................... 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 ......................................0 3.........3 0..........................5 0..... gsp ................................7 1.... W3110 cells were grown in L-broth with or without 2 mM indole..1 0......................3 0..7 0........8 0...... no expression........................... malM ....... malF ....................1 1........5 0..................................2 14 2........5 0............ yeeN ...........................1 0........... yfjB ..6 0.... tsr ............................................................8 1...............................4 0................ elaA .4................................................ spy..4 0............1 0..................2 2.................................1 0................................0 0.........................................................1 0....3..........3 1...................1 1.......................................................................0 0... agaZ ..........................6 0...........................................4 0............. yghU.........9 0.......1 1............................................... malG....................................................................1 0............................8 1..........0 1................................................ coli was grown in the presence of indole (Table 4).....................4..................... cheZ ...9 1...........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) fliC garD (yhaG) tap garL (yhaF) cheR yidS ypfH phnD malF sulA aroF flgL malG fliD glpF fliT phnJ ycaC cheA icdA phoR flxA fliS motB b1141 motA agaZ cheW cheZ ybiC tsx acrD mdtD (yegB) 5..............................5 1.................................................................2 0.........................................8 0.......................2 0...........................2 0.....................3 0............... yiaD ...............1 1....................................................2 0..........................................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......0 1......3 2...............2 9.................................................... motB ........................................2.......1 0..................9 0. aroF ......3 1...................6 0.1 1........2 0..............................................6 0....8 0.....1 1..........8 2.........................9 1. and icdA increased by factors of 20.........8 1............ NE........... We also ................... A total of 59 genes were examined....................4 2........................1 1... TABLE 4...... 2.................9 0..............................................................................................7 0...............2 0..................................9 2...2 1............. yghU .. 1..3 1........ mdtC.........4 1..................... malE ...........................7 0.............7 0..... fliT .......................................................................................6 2..... icdA ................1 0..8 0......... garD (yhaG)......................................................... NE. mdtD........................5 1..................... tolC .........3 1............................................1 0...1 0...........3 1.. mdtA (yegM) ....... 14.....3...... ypfH ................5 0........ cheA ...................0 1........... after which total RNA was collected for qRT-PCR............................1 0............. lamB ......1 2..................1 0.............9 1........................... phnD ... qRT-PCR showed that expression of glpF......................... sulA .....................1 1.................................................................................3 0....................3 3...........................2 0..................................................0 1.....2..... garL (yhaF)................................. garP (yhaU).........0 0.................... mdtD (yegB).................. phoB ............. glpF ....................8 0........ b1141 ..................................................................... coli genes in the baeSR deletion mutant J.............................................1 0..5 2................... fliS ..4 1.......... amn ................................... tsx ..........7 0.........................................1 1. cheW .............2 0................. 2..........................1 1................. baeR ............. 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.4 2.....................................8 NE 0.........1 NE 0....................... ycaC . mdtA...................6 0..4 1........................................................................1 0......... mdtB..........................2 1.............7 1.......................7 0................... tap .......................... spy ............................................................3 2........... and 2..........2 2..........................8 2. phoR ..................................................2 0........9 0.......... ycaC...............................1 1....................4 1.....................................2 0........................5 0.......................................0 2.7 2............ yieI .........0 2.................... no expression.........................................4 2...........................4...............................8 0....................................................7 0......3 1.................0 1................1768 NISHINO ET AL..................1 0..........................1 20 0......... 2... yidS .... TABLE 3.....7 1............................. and the expression of 11 of these genes was increased more than twofold when E...................4 0..............1 8...

acrD. 2. . The expression levels of 15 of the 59 genes were decreased by deletion of baeSR.000 1. Analysis with the motif-finding program Align ACE (54) revealed a highly conserved 18-bp sequence in the upstream regions of spy. 3).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). Our microarray analysis identified 59 genes whose expression was increased by baeR overexpression. Consensus sequence in the upstream regions of BaeR-regulated genes. the consensus sequence was found in the promoter region of spy. Also. 187. 3).000-fold). but the level of identity was low (50%) (Fig. GC-3Ј (where D is G. Effect of deletion of the BaeR-binding site sequence Fold increasea Gene Strain NKE11 (wild type. spy. and BaeSR two-component systems. Overexpression of BaeR did not increase the expression levels of these genes without the BaeRbinding motifs (Table 5). we suggest that expression of spy. Consensus sequences were found in the upstream regions of mdtA. we identified seven genes as members of the BaeSR regulon (Fig. These genes are most likely regulated by BaeR directly. mdtA (yegM) mdtB (yegN) mdtC (yegO) mdtD (yegB) baeS acrD tolC spy baeR 490 280 190 110 120 35 12 640 15. CreBC. Most of the expression levels (10 genes.6 38 12 590 11. and acrD is directly regulated by the BaeSR two-component system. To test this hypothesis. we analyzed upstream regions of spy. acrD. The expression of 15 genes was decreased by ⌬baeSR.1 0. The amplification of baeR in NKE56 (8. expression of phoBR twocomponent system genes was increased by baeR amplification (Table 2). NKE56 (W3110 ⌬creBC/pUCbaeR).400-fold) was lower than that in NKE57 (21. pUCbaeR) Strain NKE122 (⌬BaeR binding motif for acrD. 2).1 0. mdtA. and acrD (Fig. probably because the growth of NKE56 was slower than the growth of NKE57 (data not shown). 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. The program also found a similar sequence in the upstream region of ycaC. Baranova and Nikaido reported that BaeR binds to the upstream region of mdtA (5). and the expression of 11 genes was increased by indole. acrD. Overproduction of BaeR also increased expression of the CreB regulon (4). Recently. indole appears to induce the expression of these genes via the BaeSR two-component signal transduction system. and ycaC.VOL. We deleted the BaeR-binding motifs for the acrD gene and for the mdt operon. our group found that BaeR binds to the upstream regions of both mdtA and acrD (unpublished data). the mdtABCD operon. 3. we investigated the effect of deletion of the BaeR-binding motif on gene induction. and ycaC. and yieI (Table 2) and the creB response regulator gene (2. The seven genes in the overlap of all three data sets were identified as components of the BaeSR regulon. The 18-bp consensus sequence is 5Ј-TTTTTCTCCATDATTG FIG. This regulon contains the spy. and ycaC genes. mdtA.6 0. A. To put it differently. Thus. 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. pUCbaeR) FIG. Effect of deletion of phoBR or creBC on BaeR-induced gene expression. As described above. These data indicate that there is a possibility of cross-regulation among the PhoBR. We summarize the expression data sets in Fig. mdtABCD. or T). and NKE10 (W3110/pUC118) for qRTPCR analysis. To test whether this sequence is required for gene induction by BaeR. pUCbaeR) Strain NKE124 (⌬BaeR binding motif for mdtA. Total RNA was collected from exponential-phase cells of NKE57 (W3110 ⌬phoBR/pUCbaeR). Intersections of genomic data sets.4 1. The numbering is relative to the start codon of the genes. and 11 genes were identified as indole-regulated genes. To identify the BaeR-binding motif. yidS. BaeR-binding site sequence motifs. Using these three expression data sets. 2. we investigated the effects of deletion of phoBR and creBC on gene induction by baeR amplification.7 13 600 17. including expression of creD. The expression levels of genes in NKE57 and NKE56 were compared to those in NKE10 (Table 6). 2005 GENE REGULATION BY BaeSR 1769 TABLE 5. as described in the text. mdtA.6-fold increase as determined by microarray analysis). 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 profiles of the baeSR mutant and wild type with the addition of indole. acrD. Thus.000 510 300 190 120 130 1. and ycaC. the exception was glpF) were not increased in the ⌬baeSR strain following growth in the presence of indole. investigated whether this gene induction is dependent on the BaeSR system. malE.

41).8 2. and multidrug transport.4 6. Also. flagellar biosynthesis. and fliCDST/flgL (related to flagellar biosynthesis). found that spy is a component of a regulon of the CpxAR two-component system that responds to envelope stresses (51). in order to understand the whole picture of BaeSR regulation in response to signals. like all the expression profile data.000 0. or T).4 6.9 4.2 8.9 0.3 1.1 3. Analysis of upstream regions of the BaeSR regulon with the motif-finding program revealed an 18-bp consensus sequence.7 0. probably because of cross talk between the . Seventeen of the genes overlapped.3 2.9 6.1 6.400 0. we combined the three expression data sets to define the members of the BaeSR regulon.3 75 94 31 240 310 53 8. 5Ј-TTTTTC TCCATDATTGGC-3Ј (where D is G. cheAM RWZ-tap (related to chemotactic response).6 0. This situation is very similar to the expression control of the mdtEF (yhiUV) multidrug efflux system and tolC by the EvgAS two-component system (40. Effect of deletion of phoBR and creBC on expression levels of genes that were increased by baeR amplification 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) fliC garD (yhaG) tap garL (yhaF) cheR yidS ypfH phnD malF sulA aroF flgL malG fliD glpF fliT phnJ ycaC cheA icdA phoR flxA fliS motB b1141 motA agaZ cheW cheZ ybiC tsx acrD mdtD (yegB) 1.1 8. 24.1 0.3 2. and CreBC two-component systems. and the mdtABCDbaeSR operon. 39). like some other drug transporters of E.2 7.2 7.9 0.0 0. BACTERIOL.3 70 0. PhoBR.8 0. We discovered that overproduction of BaeR affects the expression of gene clusters related to two-component signal transduction. In this study.8 9.2 0. a critical distinction when regulatory networks are mapped.0 16 3. BaeR controls the expression of acrD and mdtABC multidrug efflux system genes. AcrD and MdtABC drug exporters need outer membrane protein TolC in order to function (15. 38.3 3.7 5. and glpF was not increased by baeR overexpression only in the ⌬creBC strain.7 2.1 18 10 0.400 650 380 54 21.1 4.2 2. A. we also investigated the effect of deletion of baeSR on gene expression levels by using qRT-PCR methods. a The values in boldface type indicate increases of more than twofold compared to the expression levels of the genes in NKE10 (W3110/pUC118).0 11 20 100 11 50 6. we found a lot of genes whose BaeR dependence was not known previously. qRT-PCR analysis with the ⌬phoBR ⌬creBC strain revealed novel cross-regulation among the BaeSR. 23.0 18 14 0.7 0. Since a large amount of BaeR may cause indirect regulation and this may not occur under the normal growth conditions.6 0.5 28 3. Furthermore. However.0 47 4.4 300 0. 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 (30.3 0. DISCUSSION In this work we examined the utility of microarray analysis for determining the global effects of baeR gene dosage amplification.4 8.4 1. PhoBR.5 0. 43.4 4.2 0. J.0 0. the data obtained. coli (12. 36). the acrD gene.5 6. cannot be used to distinguish direct targets from indirect targets.6 0. These data indicate that there is a novel interaction among the BaeSR. yidS.8 3. 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 W3110.1 2. In this study. 64). Raivio et al. we found that BaeR overproduction increased the expression of tolC. 44).0 NE 3. Spy (spheroplast protein Y). and CreBC two-component systems.4 5.9 31 11 1.8 3. and it revealed that this consensus sequence is required for gene induction by BaeR.6 0.1 1. but the function of Spy remains unknown.8 11 0. In this study. chemotatic responses.0 0. We concluded that only these three regulons are directly regulated by the BaeSR system.1 4.1 0.8 3. was initially identified as a periplasmic protein whose expression is induced by spheroplast formation (14). These 17 genes are malEFGKM-lamB (related to maltose transport).9 0. one of the components of the BaeSR regulon.0 22 42 PhoBR system and the CreBC system (63.0 1.1 0. TABLE 6.1770 NISHINO ET AL. maltose transport. we investigated the effect of addition of indole on gene expression by qRT-PCR analysis.9 1.9 0.0 16 23 44 8.6 4.0 14 NE 6.6 1.5 0. no expression. The expression of yieIJ. NE.7 5.6 0. The consensus sequence is present in the upstream regions of the spy gene.8 23 27 4.8 3.3 1.2 2.0 3.

68). Nishino. Shao. 22. A. and induction of multidrug transporters. Goeden. FEMS Microbiol. M. Hoch. and Yoshihiko Inazumi for communicating unpublished results. L. and (iii) quorum sensing. Junko Yamada for providing plasmid pUCbaeR. G. G. Chemother.. H. Y. K. N. Kobayashi. 276:26955–26961. Rev. Davis. Glutathionylspermidine metabolism in Escherichia coli. 10. ASM Press. and A. B. FEBS Lett. Wanner and Tomofusa Tsuchiya for providing strains and plasmids. 52:576–582. 12. Nakamura. 33. K. A. J. Chem. Microbiol. Science and Technology of Japan. A. Plunkett. Takahiro Hirata. 1998. In this study. 269:3479–3484. K. 19. Bock. J.. The complete genome sequence of Escherichia coli K-12. Glasner. D. Fischer. is needed in order to understand the biological significance of the regulatory networks for them and may provide further insights into the role of multidrug transporters in the physiology of the cell. Forestry and Fisheries of Japan. we found that the BaeSR system controls acrD and mdtABC in response to indole and probably other signals.. J. USA 97:6640–6645. R. Proc. Rev. N. 1992.. Bacteriol. B. F. T. Lomovskaya et al. Ma et al. K.VOL. Two-component signal transduction. T. Blattner. Japan Society of Chemotherapy. A. 15. Indole is a toxic compound that disrupts the bacterial envelope (49). Evolution of enzymatic activities in the enolase superfamily: characterization of the D-glucarate/galactarate catabolic pathway in Escherichia coli. Imamura. Y. J. 6. (ii) the growth phase. L. A. and R. R. N. A. L. Gerlt. W. 57). Indole can also act as an extracellular signal in E. the structural gene for the sigma S subunit of RNA polymerase in Escherichia coli. Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli. J. 2002. M. Sports. K. Also. and members of our labs for helpful discussions. T. Iimuma. Nikaido. S. Method for the determination of minimum inhibitory concentrations (MICs) by the micro-broth dilution method. Y. A new periplasmic protein of Escherichia coli which is synthesized in spheroplasts but not in intact cells. Biol.. Kobayashi. and B. Bloch. Silhavy. 546:241–246. 1997. Biochem. 14. Plant Cell Physiol. T. N. A. 62: 204–229. 64:694–708.. A. Hirata. and Y. 185:1851–1856. M. T. Shibayama. 16. T. 58). Huisman. 1995. Sugiyama. ␤-Lactam resistance modulated by the overexpression of response regulators of two-component signal transduction systems in Escherichia coli. Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar Typhimurium and Escherichia coli. Yukawa. Yamaguchi. Bacteriol. Hughes. 11. 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).. A. C. Chem. Membrane topology of ABC-type macrolide antibiotic exporter MacB in Escherichia coli. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. These data suggest that regulation of multidrug transporters has a strong relationship to (i) stress responses. Thus. 2003. and G. 3rd. such as inside hosts. A. T. and H. 2000. R. J. G. Role of multiple efflux pumps in Escherichia coli in indole expulsion. 1998. Mol. Washington. Bacteriol. Japan Society of Chemotherapy.. and R. 3. Kawamura-Sato. 2001.. Cell-to-cell signalling in Escherichia coli and Salmonella enterica. Stierhof. Babbitt. 2002. Riley. Novel macrolide-specific ABC-type efflux transporter in Escherichia coli. Antimicrob. by a grant from the COE Program in the 21st Century of the Japan Society for the Promotion of Science. Kolter. 44).. 2. Palmer. 173:78– 82. M. A. This work was supported by grants-in-aid from the Ministry of Education. and regulation. 61). and characterization of a bifunctional glutathionylspermidine synthetase/amidase. T. J.. an alternative sigma factor. 23. Baranova. T. 179:345–352. Science 277:1453–1474. J. Lange.. Mizuno. K. Hirakawa. D. M.. The unorthodox histidine kinases BvgS and EvgS are responsive to the oxidation status of a quinone electron carrier. Chilcott. 2000. Hagenmaier. Some information about the regulation of multidrug transporters has been reported previously. 1995. Shuman. and A. and H. 1999. our group found that the CpxAR two-component system also regulates the expression of acrD (15). Mol. and J. J. Yamaguchi. and acrEF (50. D.. we found that histone-like protein H-NS represses the expression of acrEF and mdtEF (45). C. Horton. 17. 270:14031–14041. H. Lett. R. R. Avison. the CpxAR system plays an important role in response to envelope stresses (49). H-NS represses the expression of many genes that are expressed in the stationary phase.. coli to survive stresses (25. Mayhew. Biochemistry 37:14369–14375. 1981. Rose.. E. N. Maltose/maltodextrin system of Escherichia coli: transport. A small. Wanner. D. overproduction. 10:133–138. 1996. Biol. M. there is a strong relationship among envelope stresses.C. Bollinger. D. 179:2073–2076. S. and T. and A. Rode. 2000. It has been reported that the expression of mdtEF (yhiUV) is controlled by RpoS (3. W. Garbe. Identification of transcriptional start sites and the role of ppGpp in the expression of rpoS. Chemotherapy (Tokyo) 29:76–79. Escherichia coli CreBC is a global regulator of gene expression that responds to growth in minimal media. Bacteriol. Trends Genet. 9. Recently.4-dinitrophenol (28). 20. and H. K. ACKNOWLEDGMENTS We thank Barry L. cloning. Zhang. reported that the expression of acrAB is induced by fatty acids. S. J. 24.. which is needed for E. Also. 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. metabolism. R. 7. Hubbard. acrD. M. C. Taniguchi. 2004. 18. Storz. The EvgAS two-component system regulates the expression of mdtEF (yhiUV) and emrKY (43. Microbiol. S. Koch. Kobayashi. coli (1. Yamaguchi. G. Natl. Gross.. Purification. Mau. 187. Datsenko. Indole-inducible proteins in bacteria suggest membrane and oxidant toxicity. Biol. reported that the emrAB drug exporter genes are induced by salicylic acid and 2. stable RNA induced by oxidative stress: role as a pleiotropic regulator and antimutator. the quorum-sensing regulator SdiA controls the expression of acrAB. by a grant-in-aid from the Zoonosis Control Project of the Ministry of Agriculture. Nishino. Kiba. M. F. Hirata. A. B. J. Recently. 25. Altuvia. Horii. coli and found that 20 genes encode transporters of some drugs and/or toxic compounds (42). M. and it can be exported by the AcrEF multidrug transporter (22). K. Bacteriol. 1. and M. Boos. Biol. Ohta. Method for the determination of minimum inhibitory concentrations (MICs) of aerobic bacteria by the agar dilution method. Eur. Mol. Simon. P. J. Hengge-Aronis. Yamaguchi. Hirakawa. 1997. 2003. Perna. and U. 1999. Henning. Collado-Vides. and K. Nishino. 52:933–945. and C. Walsh. I. Chemotherapy (Tokyo) 40:184–189. The key to understanding how bacteria use these multiple transporters lies in analysis of the regulation of transporter expression. J. K. 1997. Nishino. Bennett. 1995. and P. 2003. D. Fralick. Microbiol. 8. Nishino was supported by a research fellowship from the Japan Society for the Promotion of Science for Young Scientists. 1994. Comprehensive studies on the drug resistance mediated by the overexpression of response regulators of two-component signal transduction systems in Escherichia coli. Hanaki. . Kirkpatrick. Arch.. and by a grant from Core Research Evolutional Science and Technology (CREST) of the Japan Science and Technology Corporation. 183:5639– 5644. B.. Yamada. Hirata. J. Hidetada Hirakawa. J. Where are these regulatory networks and multidrug transporters needed in bacterial cells? Further investigation of the regulation of multidrug transporters in several natural environments. Suzuki. Burland. Ueguchi. Sci. 2005 GENE REGULATION BY BaeSR REFERENCES 1771 We previously cloned all of the gene clusters encoding putative and known drug transporters of E. V. Acad. Protein histidine kinases and signal transduction in prokaryotes and eukaryotes.. R. Jr. H. D. and M. N. Postow. Arakawa. J. T. RpoS is also required for the expression of many genes in stationary-phase cells. Microbiol. 5. Cell 90:43–53. 177:4676–4680. Weinstein-Fischer. Kwon. W. T. Compilation and characterization of Arabidopsis thaliana response regulators implicated in His-Asp phosphorelay signal transduction. J. K. Also. 178:5803–5805. Gregor. C. sodium chloride. J.. and ethanol (29). J. and T. 40:733–742. D. 21. and A. 13. Culture. J. Walsh. 4. cell-tocell signaling. Alex. 2001. T. Bacteriol. 184:4168–4176. Ahmer.

Masuda. Biol. 2004. C. Magasanik. DNA Res. 1997. Maeda. 184:4161–4167. and E. J. X. J. 42. J. Edwards. M. Jiang. 177:2328–2334. 2003. Schramm. Bacteriol. Rahmati. S. T. Trends Microbiol. 62. G. 297–315. A two-component multidrug efflux pump. MdtABC. Roth. Nishino. 32. S. Reznikoff. L. The phoBR operon in Escherichia coli K-12. Kofoid. T. 2nd ed. Nat. B. 186:1423–1429. F. C. Curtiss III. Shaulsky. Riley. 36. I.. 1998. Hirakawa. Signal transduction schemes of bacteria. 28. and T. J. R. and T. Nikaido. Y. Swanson. 43:677–685. and overproduction of the enzyme. S. Mizuno. Masaoka. and W. Biol. K. R. K. Yang. J. H.. Chang. M. and A. Mol. Magasanik. Identification of conserved. 26. 1989. 183:5803– 5812. Role of histone-like protein H-NS in multidrug resistance of Escherichia coli. Roles of TolC-dependent multidrug transporters of Escherichia coli in resistance to ␤-lactams. J.. Schaechter. and A. 57. K. Escherichia coli and Salmonella typhimurium: cellular and molecular biology. M. p. 175:2143–2149. in Bacillus subtilis.. N. 55. J. M. Riley. Audia. S.. 2002.. E. J. and H. J. Microbiol. 37:1186–1197. S. P. 49. L. H. 47. cloning. E. C. In F. Maeda. Ding. 39. E.. Molecular cloning: a laboratory manual. Curtiss III. Bacteriol. J. J. B. Schellhorn. Raffa. 59. and H. Y. Oshima. 2003. 185:2667–2672. Nikaido. J. O. Loomis. D. 2002. EvgA of the two-component signal transduction system modulates production of the yhiUV multidrug transporter in Escherichia coli. Schramm. the module. A twocomponent histidine kinase gene that functions in Dictyostelium development. 43. 47:3030–3033. Wang. Fritsch. S. Reznikoff. J. S. LaRossa.. 34. J. 52. 1997. Sammartano.. 182:2307–2310. and J. 40. Protein evolution viewed through Escherichia coli protein sequences: introducing the notion of a structural segment of homology. 68. L. C. Structure and regulation of the AMP nucleosidase gene (amn) from Escherichia coli. Ishige. Bacteriol. Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli. Mizushima. A. 45. Tethering of CpxP to the inner membrane prevents spheroplast induction of the cpx envelope stress response. Overexpression of the response regulator evgA of the two-component signal transduction system modulates multidrug resistance conferred by multidrug resistance transporters. Laird. 169:5569–5574. Neidhardt. C. 2000. H. Fraley. 56. deRiel. 50.. B. Y. Nishino. G. Church. and L. EMBO J. M. M. 2002. A two-component system that regulates an osmosensing MAP kinase cascade in yeast. W. C. T. C. and T. 54. 4:161–168. and V. Bacteriol. 27. P. Global impact of sdiA amplification revealed by comprehensive gene expression profiling of Escherichia coli. Yamaguchi. B. K. 2000. Bacteriol. W. 180:6283–6291. and H. 2000. Washington. C. F.C. J. E. Gene regulation by phosphate in enteric bacteria. J.. 2:489–493. H. Prevention of drug access to bacterial targets: permeability barriers and active efflux. 1989. M. I. Bacteriol. 38. Y. M. 168:13–21. Yamaguchi. 51. Nishino. Alex.). P. L. W. ASM Press. Bacteriol. R. M. Bacteriol. Bacteriol. J. Parkinson. and H. Bacteriol. L. 2:14. Control of the AcrAB multidrug efflux pump by quorum-sensing regulator SdiA. Wanner. M. 184:2319–2323. Ingraham. C. C. Bacteriol.-K. Monterrubio. Hiraga. 45:1599–1611. Yamaguchi. Cook. C. and M. Wanner.. L. Biochemistry 28:8726–8733.). K. A. and R. Hearst. 268:857–868. 1996. Inazumi. N. Are the multiple signal transduction pathways of the Pho regulon due to cross talk or cross regulation?. Y.. Riley. Mol. Wang. Yamaguchi. and A. Simon. Trends Biochem. Raivio. D. D. 1996. W. K. L. Leung. 1998. D.. Escherichia coli gene products: physiological functions and common ancestries. and D-glycerate utilization in Escherichia coli. Baldoma. Kuroda. C. Maniatis. 1992. Hughes. 2001. Tracy. G. 26:71–112. 174: 2053–2058. 183:2265–2272. P. 61. J. J.. L. Rudd. D. and R.. Tsuchiya. Obradors. Y. Bacteriol. E. J. BMC Evol. Raivio. M. J. 1996. J. Yamagata. Joly. B. Mori. Landes Co. Microbiol. and J. Hearst. L. L. Bacteriol. Wanner. E. Mol.1772 NISHINO ET AL. and T. Lin. K. K. 1994. J. Nishino. Histidine and aspartate phosphorylation: two-component systems and the limits of homology. 1986. and A. Global analysis of genes regulated by EvgA of the two-component regulatory system in Escherichia coli. B. Microbiol. Ma. R. L. Carbon and nitrogen substrate utilization by archival Salmonella typhimurium LT2 cells. 1994. Biotechnol.. T. 1992. and A. 183:1455–1458. J. 65. Nagasawa. Lin. Chem. Kim. RpoS-dependent stationary-phase genes of Escherichia coli. V. G. Communication modules in bacterial signaling proteins. and V. Labedan. Rev. 44. T. Analysis of a complete library of putative drug transporter genes in Escherichia coli. In E. S. T. Silhavy. 2000. Yamaguchi. S. B. Mizuno.. Multidrug efflux pumps of gram-negative bacteria. L. K. J. 60. Ma. Lomovskaya. Meyer. and H. Nikaido. 41. (Tokyo) 114:350–357. A common regulator for the operons encoding the enzymes involved in D-galactarate. N. J. S. T. Nucleic Acids Res. 35. L. A third envelope stress signal transduction pathway in Escherichia coli. L. Yamada. Ara. A. Neidhardt.. Davenport.. Wurgler-Murphy. Aguilar. 2nd ed. Wei. N. The structural gene for AMP nucleosidase. J. J. Escherichia coli and Salmonella: cellular and molecular biology. Kvalnes-Krick. Genet.. Indole can act as an extracellular signal in Escherichia coli. and B. 33. D.. Bacteriol. 1987.. Science 264:382–388. B. Daniels. L. BACTERIOL. Saito. 63. and T. Antimicrob. Microbiol. R. 48. Compilation of all genes encoding two-component phosphotransfer signal transducers in the genome of Escherichia coli. Labedan. Tuveson. p. J. Estep. Microbiol. E. S. E. 2001. Regulation of gene expression in Escherichia coli. 2001. Bacteriol. Sambrook.. EmrR is a negative regulator of the Escherichia coli multidrug resistance pump EmrAB. Lee. 45:673–695. ASM Press. Austin. McCann. Low. L. K. Low. Hirata. L. and E. 1993. J. N. EbrAB. Ingraham. and A. 1357–1381. B. 15:3890–3898. p. The putative sigma factor KatF is regulated posttranscriptionally during carbon starvation. Leung. Haldimann. T. 178:5853–5859.. T. Lynch (ed. Cold Spring Harbor. D. J. and A. L. Lewis. Zechiedrich. K. Annu. Eisenstark. Mol. 1984. N. 182:2672–2674. L. R. Wanner. Sci. Wada. S. D. Wanner. Hirata.. S. B.C. H. Nature 369:242–245. Nishino. W. S. 183:4210–4216. 2118–2202. 31. D.. 46. Umbarger (ed. Novel members of the twocomponent signal transduction genes in Escherichia coli. K. 67. A. 30. Phosphorus assimilation and control of the phosphate regulon. Morita.. 2001. Nishino. Cook. and A. 2002. Is cross regulation by phosphorylation of two-component response regulator proteins important in bacteria? J. L. and B. Mol. Escalante.. Schaechter. Matin. Nishino. 1994. Badia.. 1996. J. 66. H. 29. D. W. J. 58. Pon.Y. B. S. N. 1994. Control of sensitivity to inactivation by H2O2 and broad-spectrum near-UV radiation by the Escherichia coli katF locus.. Smulski. 19:485–490. and A. Washington. Wei. and A. Rather. R. 16:45–55. Agents Chemother. B. Y.). Lin and A. 2002. 2002. J.. 37. Efflux pumps and drug resistance in gram-negative bacteria. Riley.. Tex. M. Chang. 53. . In F. Cold Spring Harbor Laboratory. Nikaido. Parkinson. K. Cell Biochem. Ueno. 1995. and G. Matin. H. Bacteriol. Biochem. Mol.. Mapping. and L. K. Yamaguchi. Bacteriol. Yamaguchi. 28:60–64. 64. EcoGene: a genome sequence database for Escherichia coli K-12. D-glucarate. K. Cell 73:857– 871.. 1993. Nagakubo. Finding DNA regulatory motifs within unaligned noncoding sequences clustered by whole-genome mRNA quantitation. and P. R. K. E. J. and B. 1995. 1993. 16:939–945. Biol. D. F. Alberti. Davidson. Genome-wide analysis of deoxyadenosine methyltransferase-mediated control of gene expression in Escherichia coli. 1993.. The putative response regulator BaeR stimulates multidrug resistance of Escherichia coli via a novel multidrug exporter system. 51:47–54. J. 1996. Umbarger (ed. 259:6972–6978. Kawagoe. R. M.