Journal of Bacteriology, March 2004, p . 1423-1429, Vol . 186, No . 5

Role of Histone-Like Protein H-NS in Multidrug Resistance of Escherichia coli

Kunihiko Nishino^1,2,3,4 and Akihito Yamaguchi^2,3,4*

Department of Bacterial Infections, Research Institute for Microbial Diseases,¹ Faculty of Pharmaceutical Science, Osaka University, Suita, Osaka 565-0871,⁴ Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki,² Core Research Evolutional Science and Technology [CREST], Japan Science and Technology Corporation, Osaka 567-0047, Japan³

Received 22 August 2003/ Accepted 14 November 2003

 
The histone-like protein H-NS is a major component of the bacterial nucleoid and plays a crucial role in global gene regulationof enteric bacteria . It is known that the expression of a varietyof genes is repressed by H-NS, and mutations in hns result in various phenotypes, but the role of H-NS in the drug resistanceof Escherichia coli has not been known . Here we present datashowing that H-NS contributes to multidrug resistance by regulatingthe expression of multidrug exporter genes . Deletion of thehns gene from the acrAB mutant increased levels of resistanceagainst antibiotics, antiseptics, dyes, and detergents . Decreasedaccumulation of ethidium bromide and rhodamine 6G in the hnsmutant compared to that in the parental strain was observed,suggesting the increased expression of some drug exporter[s]in this mutant . The increased drug resistance and decreaseddrug accumulation caused by the hns deletion were completelysuppressed by deletion of the multifunctional outer membranechannel gene tolC . At least eight drug exporter systems requireTolC for their functions . Among these, increased expressionof acrEF, mdtEF, and emrKY was observed in the hns strain byquantitative real-time reverse transcription-PCR analysis . The hns-mediated multidrug resistance pattern is quite similar tothat caused by overproduction of the AcrEF exporter . Deletionof the acrEF gene greatly suppressed the level of hns-mediated multidrug resistance . However, this strain still retained resistance to some compounds . The remainder of the multidrug resistance pattern was similar to that conferred by overproduction of theMdtEF exporter . Double deletion of the mdtEF and acrEF genes completely suppressed hns-mediated multidrug resistance, indicatingthat hns-mediated multidrug resistance is due to derepressionof the acrEF and mdtEF drug exporter genes.

 
The emergence of bacterial multidrug resistance has become an increasing problem in the treatment of infectious diseases.Multidrug resistance often results from the overexpression ofmultidrug efflux transporters . Recent genome sequence analysishas revealed that bacteria have many intrinsic putative andproven drug transporter genes . We previously cloned all of thegene clusters encoding putative and known drug transportersof Escherichia coli and revealed that 20 genes actually encodethe transporters of some drugs and/or toxic compounds [30].Since the substrate spectra of these multidrug transporterspartially overlap, we are intrigued by the question of why bacteria,with their economically organized genomes, harbor such largesets of multidrug efflux genes . The key to understanding howbacteria utilize these multiple transporters lies in analysisof the regulation of transporter expression . In the presentstudy, we analyzed the relationship between the regulation ofdrug transporters and the E . coli nucleoid-associated proteinH-NS [histone-like nucleoid structuring protein].

H-NS, one of the most abundant proteins in the E . coli nucleoid, is widely distributed within gram-negative bacteria [4] . H-NSwas initially described as a transcription factor [10] and waslater shown to play roles in the structure and function of chromosomalDNA [2, 40] . H-NS is involved in the condensation of the bacterialchromosome and regulates the expression of many genes [5% ofthe open reading frames of the E . coli genome] . Most of thesegenes are related to bacterial adaptation to environmental conditionsand/or virulence [9] . H-NS modulates transcription through theformation of large nucleoprotein structures [6, 13, 39] . Mutations in hns result in various phenotypes, because H-NS is involved in the regulation of a variety of genes . However, the role of H-NS in the drug resistance of E . coli is unknown . In this paper, we report that H-NS controls the multidrug resistance of E. coli by regulating the expression of drug exporter genes.

 
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 [LB]broth [34] . Cells were rapidly collected for total RNA extractionwhen the cultures reached an optical density at 600 nm of 0.6.

 
RNA extraction. Total RNA from bacterial cultures was isolated by using an RNeasyProtect bacterial minikit and RNase-free DNase [both from Qiagen]in accordance with the manufacturer's instructions . The absenceof genomic DNA in DNase-treated RNA samples was confirmed byinspecting nondenaturing agarose electrophoresis gels and alsoby performing PCR with primers known to target the genomic DNA.RNA concentrations were determined spectrophotometrically [35].

Determination of specific transcript levels by quantitative real-time reverse transcription-PCR [qRT-PCR]. Bulk cDNA samples were synthesized from total RNA derived fromE . coli cells by using TaqMan reverse transcription reagents[Perkin-Elmer [PE] Applied Biosystems] and random hexamers asprimers . Specific primer pairs were designed with ABI PRISMPrimer Express software [PE Applied Biosystems] . rrsA of the16S rRNA gene was chosen as the normalizing gene . Real-timePCR was performed with each specific primer pair by using SYBRGreen PCR Master Mix [PE Applied Biosystems] . Reactions were performed with an ABI PRISM 7000 sequence detection system [PE Applied Biosystems]; during the reactions, the fluorescencesignal due to SYBR Green intercalation was monitored to quantifythe double-stranded DNA product formed in each PCR cycle.

Susceptibility testing. The antibacterial activities of the agents were determined onL agar [1% tryptone, 0.5% yeast extract, and 0.5% NaCl] platescontaining various compounds [oxacillin, erythromycin, novobiocin,doxorubicin, acriflavine, crystal violet, ethidium bromide,methylene blue, rhodamine 6G, tetraphenylphosphonium bromide,benzalkonium chloride, sodium dodecyl sulfate, and sodium deoxycholate]at various concentrations, as indicated . Agar plates were madeby the twofold agar dilution technique recommended by the JapanSociety of Chemotherapy [11, 12] . Organisms were tested at afinal inoculum size of 10⁵ CFU/spot, with the use of a multipoint inoculator [Sakuma Seisakusyo, Tokyo, Japan], and were incubatedat 37°C for 18 h in air . MICs of drugs and toxic compoundswere determined as the concentrations that severely inhibitedbacterial cell growth.

Construction of in-frame deletion mutants. To construct gene deletion mutants from E . coli W3104 cells[41], precise in-frame deletions were generated by crossoverPCR . Four sets of oligonucleotide primers [designations endingin -No, -Ni, -Ci, and -Co [Table 2]] were used for each gene.The fragment containing the deletion was then cloned into theBamHI site of the pKO3 vector [18], a gene replacement vector that contains a temperature-sensitive origin of replicationand markers for positive and negative selection for chromosome integration and excision . The deletion was introduced into the chromosome by use of the pKO3 gene replacement protocol, asdescribed previously [18].

 
Observation of drug accumulation in E . coli cells. E . coli cells were spotted onto L-agar plates containing a low concentration of ethidium bromide [1 µg/ml] or rhodamine6G [0.5 µg/ml] at a final inoculum size of 10⁵ CFU/spot,by use of a multipoint inoculator [Sakuma Seisakusyo], and wereincubated at 37°C for 18 h in air . Drug accumulation inE . coli cells was observed as the fluorescence of ethidium bromideor rhodamine 6G in cells under UV light, by use of an ElectronicU.V . Transilluminator FAS-II [TOYOBO, Osaka, Japan].

 
Deletion of the hns gene increases resistance to multiple antibiotics and toxic compounds. Deletion of hns did not change the drug susceptibility of wild-typeE . coli W3104 [41] [Table 3], because the intrinsic multidrugefflux transporter AcrAB masks the effect of hns deletion [Table 3] . In addition, qRT-PCR analysis did not detect any changes in the expression levels of acrA and acrB in the hns deletionstrain relative to the wild-type strain [data not shown] . We therefore used a host strain lacking the acrAB gene [W3104acrAB] [15] . AcrAB is constitutively expressed in E . coli and is largelyresponsible for the intrinsic resistance of E . coli to dyes,detergents, and most lipophillic antibiotics [38]. E . coli W3104acrAB showed hypersensitivity to these compounds [Table 3] . Deletionof hns increased the drug resistance of the acrAB deletion strainto multiple structurally unrelated compounds such as antibiotics,antiseptics, dyes, and detergents, as shown in Table 3.

 
Effect of deletion of hns on drug accumulation in E . coli cells. One of the major mechanisms of bacterial multidrug resistance is active drug efflux . Therefore, we investigated drug efflux in the hns-deficient mutant . E . coli W3104acrAB and W3104acrABhns cells were spotted onto agar plates containing 1 µg ofethidium bromide/ml or 0.5 µg of rhodamine 6G/ml, andthe plates were then incubated at 37°C for 18 h . Since theconcentrations of the drugs were eightfold lower than theirMICs for W3104acrAB, these compounds did not inhibit cell growth[Fig . 1A and C] . Accumulation of these drugs in E . coli cellswas observed from the fluorescence of ethidium bromide [Fig. 1B] and rhodamine 6G [Fig . 1D] under UV light . As shown in Fig.1B and D, hns deletion resulted in a drastic decrease in fluorescence,clearly indicating the active efflux of these drugs from hns cells.

 
Effect of tolC deletion on the effect of hns deletion. The results described above indicate that the expression ofa multidrug exporter[s] may be increased by hns deletion . Ina previous study, we revealed that at least 20 intrinsic drugefflux transporters are encoded in the E . coli chromosome [30]. Among these, RND [resistance nodulation cell division]-family transporters play major roles in both intrinsic and elevated resistance of gram-negative bacteria to a wide range of noxious compounds [22, 26, 27] . RND transporters need two other proteinsfor their function: a membrane fusion protein [MFP] and an outermembrane channel . In E . coli, all of the five RND drug exportersystems [AcrAB, AcrD, AcrEF, MdtEF, and MdtABC] require thecommon outer membrane channel TolC for their functions [5, 7, 8, 25, 29-31] . [YhiUV has been renamed MdtEF according to thesystematic nomenclature available at the EcoGene website [33; http://bmb.med.miami.edu/ecogene/ecoweb/].] In addition, two major facilitator superfamily drug transporter systems [EmrABand EmrKY] and one ABC drug transporter system [MacAB] alsoneed TolC for their functions [15, 16, 19, 31].

In order to determine whether or not hns deletion-mediated multidrug resistance is due to the TolC-dependent drug exporter[s], we investigated the effect of tolC deletion on the drug resistance of the hns strain . Deletion of tolC from strain W3104acrAB increasedthe susceptibilities of cells to some compounds, particularlynovobiocin, sodium dodecyl sulfate, and sodium deoxycholate.This increase is probably due to prevention of the leaking ofcompounds through TolC or inactivation of some TolC-dependentdrug exporter[s] . tolC deletion completely inhibited hns deletion-mediatedmultidrug resistance . tolC deletion from W3104acrABhns increasedsusceptibilities to all the compounds listed in Table 3 . tolCdeletion restored the accumulation of ethidium bromide and rhodamine6G in the hns strain [Fig . 1B and D, lanes 3] . These results indicated that hns deletion-mediated multidrug resistance is due to increased expression of a TolC-dependent drug exporter[s] caused by hns deletion.

Determination of the amounts of TolC-dependent drug exporter transcripts by qRT-PCR. In order to determine which drug exporters' expression is increasedby hns deletion, we investigated hns deletion-dependent changesin the amounts of mRNAs of drug exporter genes by qRT-PCR . TotalRNAs from exponential-phase cultures of W3104acrAB and W3104acrABhns were isolated, and cDNA samples were then synthesized by using TaqMan reverse transcription reagents [PE Applied Biosystems]and random hexamers as primers . Then real-time PCR of the cDNAswas performed with each specific primer pair by using SYBR GreenPCR Master Mix [PE Applied Biosystems] . The expression levelsof TolC-dependent drug exporter genes [except for AcrAB], typical TolC-independent drug exporter genes [mdfA, emrE, and mdtK [ydhEhas been renamed mdtK according to the systematic nomenclatureavailable at the EcoGene website]], and the tolC gene in W3104acrABhns were compared with those in W3104acrAB . The results are shownin Table 4 . The expression levels of three exporter genes [acrE,mdtE, and emrK] were significantly increased [more than fourfoldin comparison with basal levels] by hns deletion: 4.1-, 12-,and 6.7-fold increases were observed for acrE, mdtE, and emrK, respectively . Deletion of hns did not increase the expression levels of other drug exporter genes or of the tolC gene [Table 4].

 
Effects of deletion of drug exporter genes on hns deletion-mediated multidrug resistance. In order to determine whether or not hns deletion-mediated multidrugresistance is due to increased expression of the acrEF, mdtEF,and/or emrKY drug exporter genes, we investigated the effectsof these gene deletions on drug resistance levels of W3104acrAB and W3104acrABhns [Table 3] . When the acrEF, mdtEF, and emrKY genes were deleted one by one or simultaneously from W3104acrAB, resistance levels did not change, suggesting that these genes are not expressed under normal conditions . Single deletion of emrKY or mdtEF did not change the increased multidrug resistance of W3104acrABhns . On the other hand, deletion of acrEF fromW3104acrABhns drastically decreased the levels of hns deletion-mediated multidrug resistance, except for resistance to erythromycin, doxorubicin, and rhodamine 6G, indicating that hns deletion-mediated drug resistance is mainly due to AcrEF . However, this strain still retained some resistance to several compounds . That is,strain W3104acrABhnsacrEF showed decreased but significant resistanceto oxacillin, erythromycin, doxorubicin, crystal violet, ethidiumbromide, methyl viologen, and rhodamine 6G . The remaining drugresistance pattern was similar to that conferred by overproductionof MdtEF [YhiUV] [30] . Double deletion of acrEF-mdtEF from W3104acrABhns completely prevented hns deletion-mediated multidrug resistance, clearly indicating that hns deletion-mediated multidrug resistance is due to increased expression of these two drug exporter genes. The reason why the single deletion of mdtEF from W3104acrABhns did not change hns deletion-mediated resistance levels may be that increased expression of AcrEF masks the effect of mdtEF deletion . Deletion of emrKY from W3104acrABhnsacrEF and W3104acrABhnsacrEFmdtEF did not affect the drug susceptibilities of these strains.

Effects of hns deletion on the expression levels of other genes located near emrKY, mdtEF, and acrEF. We investigated the effects of hns deletion on the expression levels of genes located near emrK, mdtE, and acrE by qRT-PCRanalysis . The results are shown in Fig . 2. hns deletion increasedthe expression of genes near emrK [Fig . 2A] . Expression of emrY,emrK, evgA, evgS, yfdE, yfdV, yfdU, yfdW, yfdX, and ypdI increasedby factors of 8.6, 6.7, 10, 11, 15, 12, 19, 11, 4.2, and 4.2,respectively . hns deletion also increased the expression ofgenes near mdtE [Fig. 2B] . Expression of slp, yhiF, yhiD, hdeB,hdeA, hdeD, yhiE, mdtF, yhiW, gadX, and gadA increased by factorsof 160, 45, 62, 110, 110, 44, 34, 2.1, 18, 38, and 50, respectively.The effects of hns deletion on the expression of genes aroundacrE were lower than those on the expression of genes aroundemrK and mdtE [Fig . 2C] . Deletion of hns increased the expressionof one gene upstream of acrE [envR] and two downstream genes[acrF and yhdV] by factors of 6.1, 3.2, and 2.4, respectively.It is thought that envR is a repressor of the acrEF operon [3, 14, 30] . However, although the expression level of envR wasincreased by hns deletion, the expression level of acrEF wasalso increased . This result indicates that the hns effect overcomesthe inhibitory effect of EnvR.

 
In a previous study, it was found that the gene clusters shownin Fig . 2A and B are positively regulated by the EvgA response regulator of the two-component signal transduction system [28]. The expression level of evgA was increased by hns deletion [Fig.2A] . In order to determine whether or not the increased expressionof drug exporter genes caused by hns deletion [Fig . 2A and B]is due to increased expression of the evgAS two-component system,we deleted evgAS from W3104acrABhns . Deletion of evgAS fromthe hns deletion strain affected neither the increased expressionlevels of these genes [Fig. 2] nor the hns deletion-mediatedmultidrug resistance levels, even in the hns-acrEF deletionstrain [Table 3].

Recently, it was reported that the gene cluster shown in Fig. 2B is positively regulated by ydeO [23] and that the level ofydeO expression is increased by hns deletion [Fig . 2D] . Therefore,we investigated the effect of ydeO deletion . Deletion of ydeOaffected neither the increased expression of genes shown inFig . 2B nor hns deletion-mediated multidrug resistance . These data, together with those for evgAS deletion, clearly indicate that the hns deletion-mediated increase in the expression of drug exporter genes is independent of EvgAS- and YdeO-mediated regulation.

 
In this study, we found that H-NS represses the expression ofsome TolC-dependent multidrug exporter genes and that, as aresult, deletion of the hns gene confers multidrug resistanceon the acrAB-deficient strain . In addition, qRT-PCR analysisrevealed that expression of genes located near the mdtEF andemrKY exporter systems was increased by hns deletion . This observation is in good agreement with the microarray data of Hommais etal . [9].

Previously, Sulavik et al . constructed E . coli strains with deletions of putative drug exporters and outer membrane channels[38] . They reported that deletion of acrAB increased the drug susceptibility of E . coli cells, whereas deletion of the other drug exporter genes increased E . coli drug susceptibility slightly or not at all, indicating that most drug exporter genes arenot expressed under normal conditions . Therefore, studies onthe regulation of these drug exporter genes are necessary togain further insights into the physiological roles of multidrugexporters.

We previously found that overexpression of evgA, which is a response regulator of the two-component regulatory system, conferred multidrug resistance on E . coli cells [31, 32] . Later, Masudaand Church reported that overexpression of the ydeO regulatorygene also conferred multidrug resistance on E . coli [23] . The hns deletion-mediated increase in expression of drug exporter genes is independent of such transcriptional regulator-mediated upregulation . hns deletion-mediated regulation is more global than two-component system-mediated regulation.

Ma et al . reported that the expression of acrAB is induced by fatty acids, sodium chloride, and ethanol [21] . Lomovskaya etal . reported that the emrAB drug exporter gene is induced bysalicylic acid and 2,4-dinitrophenol [20] . In addition, it hasbeen reported that the expression of mdtEF [yhiUV] is controlledby RpoS [1, 37], a conserved alternative sigma factor that is needed for E . coli to survive stresses such as heat shock [17, 24], oxidative stress [17, 24], osmotic challenge [24], and near-UV light [36] . Thus, the regulation of drug exporter genesis closely related to stress responses . Hommais et al . suggestedthat the control of gene expression by H-NS has a strong relationshipwith the maintenance of intracellular homeostasis [9] . In thisstudy, we found that H-NS represses the expression of acrEFand mdtEF . Thus, it was revealed that H-NS-mediated maintenanceof intracellular homeostasis has a close relationship with theexpression of drug exporter genes.

 
We thank George M . Church for plasmid pKO3.

K . Nishino was supported by a research fellowship from the Japan Society for the Promotion of Science for Young Scientists . Thiswork was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan [to K.N . andA.Y.], by a grant-in-aid from the Zoonosis Control Project ofthe Ministry of Agriculture, Forestry and Fisheries of Japan[to K.N.], by a grant from the COE Program in the 21st Centuryof the Japan Society for the Promotion of Science [to K.N.],and by a grant from the Core Research Evolutional Science andTechnology [CREST] program of the Japan Science and TechnologyCorporation [to A.Y.].

 
* 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 .

1. 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 90:43-53.
2. Atlung, T., and H . Ingmer. 1997 . H-NS: a modulator of environmentally regulated gene expression . Mol . Microbiol . 24:7-17.
3. Blattner, F . R., G . Plunkett III, 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 277:1453-1474 .
4. Bloch, V., Y . Yang, E . Margeat, A . Chavanieu, M . T . Auge, B . Robert, S . Arold, S . Rimsky, and M . Kochoyan. 2003 . The H-NS dimerization domain defines a new fold contributing to DNA recognition . Nat . Struct . Biol. 10:212-218.
5. Elkins, C . A., and H . Nikaido. 2002 . Substrate specificity of the RND-type multidrug efflux pumps AcrB and AcrD of Escherichia coli is determined predominantly by two large periplasmic loops . J . Bacteriol . 184:6490-6498 .
6. Falconi, M., B . Colonna, G . Prosseda, G . Micheli, and C . O . Gualerzi. 1998 . Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity . A temperature-dependent structural transition of DNA modulates the accessibility of virF promoter to transcriptional repressor H-NS . EMBO J . 17:7033-7043 .
7. Fralick, J . A. 1996 . Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli. J . Bacteriol . 178:5803-5805 .
8. 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 . 185:1851-1856 .
9. Hommais, F., E . Krin, C . Laurent-Winter, O . Soutourina, A . Malpertuy, J . P . Le Caer, A . Danchin, and P . Bertin. 2001 . Large-scale monitoring of pleiotropic regulation of gene expression by the prokaryotic nucleoid-associated protein, H-NS . Mol . Microbiol . 40:20-36.
10. Jacquet, M., R . Cukier-Kahn, J . Pla, and F . Gros. 1971 . A thermostable protein factor acting on in vitro DNA transcription . Biochem . Biophys . Res . Commun . 45:1597-1607.
11. Japan Society of Chemotherapy. 1981 . Method for the determination of minimum inhibitory concentrations [MICs] of aerobic bacteria by the agar dilution method . Chemotherapy [Tokyo] 29:76-79.
12. Japan Society of Chemotherapy. 1992 . Method for the determination of minimum inhibitory concentrations [MICs] by the micro-broth dilution method . Chemotherapy [Tokyo] 40:184-189.
13. Jordi, B . J., and C . F . Higgins. 2000 . The downstream regulatory element of the proU operon of Salmonella typhimurium inhibits open complex formation by RNA polymerase at a distance . J . Biol . Chem . 275:12123-12128 .
14. Kobayashi, K., N . Tsukagoshi, and R . Aono. 2001 . Suppression of the hypersensitivity of the Escherichia coli acrB mutant to organic solvents by integrational activation of the acrEF operon with the IS1 or IS2 element . J . Bacteriol . 183:2646-2653 .
15. Kobayashi, N., K . Nishino, T . Hirata, and A . Yamaguchi. 2003 . Membrane topology of ABC-type macrolide antibiotic exporter MacB in Escherichia coli. FEBS Lett . 546:241-246.
16. Kobayashi, N., K . Nishino, and A . Yamaguchi. 2001 . Novel macrolide-specific ABC-type efflux transporter in Escherichia coli. J . Bacteriol . 183:5639-5644 .
17. Lange, R., D . Fischer, and R . Hengge-Aronis. 1995 . Identification 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 . 177:4676-4680.
18. Link, A . J., D . Phillips, and G . M . Church. 1997 . Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization . J . Bacteriol . 179:6228-6237.
19. Lomovskaya, O., and K . Lewis. 1992 . Emr, an Escherichia coli locus for multidrug resistance . Proc . Natl . Acad . Sci . USA 89:8938-8942.
20. Lomovskaya, O., K . Lewis, and A . Matin. 1995 . EmrR is a negative regulator of the Escherichia coli multidrug resistance pump EmrAB . J . Bacteriol . 177:2328-2334.
21. 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 efflux system of Escherichia coli. Mol . Microbiol . 16:45-55.
22. Ma, D., D . N . Cook, J . E . Hearst, and H . Nikaido. 1994 . Efflux pumps and drug resistance in gram-negative bacteria . Trends Microbiol. 2:489-493.
23. Masuda, N., and G . M . Church. 2003 . Regulatory network of acid resistance genes in Escherichia coli. Mol . Microbiol . 48:699-712.
24. McCann, M . P., C . D . Fraley, and A . Matin. 1993 . The putative sigma factor KatF is regulated posttranscriptionally during carbon starvation . J . Bacteriol . 175:2143-2149.
25. 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 . 184:4161-4167 .
26. Nikaido, H. 1996 . Multidrug efflux pumps of gram-negative bacteria . J . Bacteriol . 178:5853-5859.
27. Nikaido, H. 1994 . Prevention of drug access to bacterial targets: permeability barriers and active efflux . Science 264:382-388.
28. 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 . 185:2667-2672 .
29. Nishino, K., J . Yamada, H . Hirakawa, T . Hirata, and A . Yamaguchi. 2003 . Roles of TolC-dependent multidrug transporters of Escherichia coli in resistance to ß-lactams . Antimicrob . Agents Chemother. 47:3030-3033 .
30. Nishino, K., and A . Yamaguchi. 2001 . Analysis of a complete library of putative drug transporter genes in Escherichia coli. J . Bacteriol . 183:5803-5812 .
31. 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 . 184:2319-2323 .
32. 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 . 183:1455-1458 .
33. Rudd, K . E. 2000 . EcoGene: a genome sequence database for Escherichia coli K-12 . Nucleic Acids Res . 28:60-64 .
34. 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.
35. Sambrook, J., and D . W . Russell. 2001 . Molecular cloning: a laboratory manual, 3rd ed . Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
36. 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 . 168:13-21.
37. Schellhorn, H . E., J . P . Audia, L . I . Wei, and L . Chang. 1998 . Identification of conserved, RpoS-dependent stationary-phase genes of Escherichia coli. J . Bacteriol . 180:6283-6291 .
38. Sulavik, M . C., C . Houseweart, C . Cramer, N . Jiwani, N . Murgolo, J . Greene, B . DiDomenico, K . J . Shaw, G . H . Miller, R . Hare, and G . Shimer. 2001 . Antibiotic susceptibility profiles of Escherichia coli strains lacking multidrug efflux pump genes . Antimicrob . Agents Chemother . 45:1126-1136 .
39. Tupper, A . E., T . A . Owen-Hughes, D . W . Ussery, D . S . Santos, D . J . Ferguson, J . M . Sidebotham, J . C . Hinton, and C . F . Higgins. 1994 . The chromatin-associated protein H-NS alters DNA topology in vitro . EMBO J . 13:258-268.
40. Williams, R . M., and S . Rimsky. 1997 . Molecular aspects of the E . coli nucleoid protein, H-NS: a central controller of gene regulatory networks . FEMS Microbiol . Lett . 156:175-185.
41. Yamamoto, T., M . Tanaka, C . Nohara, Y . Fukunaga, and S . Yamagishi. 1981 . Transposition of the oxacillin-hydrolyzing penicillinase gene . J . Bacteriol . 145:808-813.

Free Online Full-text Article

What Is Water Purification?, What Is Salmonella?, What Is Bioreactor?, What Is Environmental Microbiology?, What Is Activated Sludge?, o, Bacteria, c, Microbe, n, Microorganism, r, Microorganisms, i, Bacterium, n, Escherichia coli, r, Bacillus, i, Staphylococcus, i, Escherichia coli, o, Salmonella, s, Penicillin, o, Microbial, i, S. cerevisiae, a, S. cerevisiae, c, S. cerevisiae, c, Staphylococcus aureus, s, Microbiological, a, Candida albicans, e, Escherichia coli, s, Antibiotics, i, Aeromonades, n, Biodegradation, s, Fermentations, e, S. cerevisiae, n, S. cerevisiae, n, Nitrosomonas




 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005