Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444A, PO Box 446, 11001 Belgrade, Serbia and Montenegro
Correspondence
Goran Jovanovic
mgjovano{at}eunet.yu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: Laboratory of Structural Microbiology, RU Box 52, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
Present address: Department of Microbiology and Immunology, Health Sciences Center, University of Oklahoma, PO Box 26901, Oklahoma City, OK 73190, USA.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The CysB protein consists of several functional domains (Lochowska et al., 2001). The N-terminal region comprises the helixturnhelix (HTH) DNA-binding motif flanked by other amino acids responsible for specificity of binding to DNA sequences. The central region is important for ligand (e.g. NAS) recognition and binding inside the cavity formed by the homodimer (Tyrell et al., 1997
; Verschueren et al., 2001
). The C-terminal domain is proposed to be involved in homo-oligomerization of this protein as well as in DNA binding of the HTH motif (Lochowska et al., 2001
). Transcription factors that contain the HTH motif bind DNA as dimers with the binding motifs positioned in parallel (Pabo & Sauer, 1992
; Perez-Rueda & Collado-Vides, 2000
). The CysB crystal structure predicts HTH motifs being positioned perpendicularly in the dimer, suggesting an unusual mechanism of binding to DNA (Tyrell et al., 1997
). Although there is no strict binding consensus sequence on DNA according to data collected to date, the CysB tetramer recognizes AT-rich sequences in the context of the features of the LysR motif with the characteristic sequence T-N11-A as the core of an inverted repeat (IR) found to be the binding site for most of the LTTR proteins (Goethals et al., 1992
; Lochowska et al., 2001
). The specific structures of the CysB binding sites have been recognized as wide DNA regions covered with several divergently or convergently oriented half-sites separated either by a few nucleotides (activator sites) or by one or two turns of DNA helix (repressor sites) (Hryniewicz & Kredich, 1994
, 1995
; Kredich, 1996
). Finally, DNA-bending sensitive sites were defined in some regulatory regions characteristic for the cys regulon (Kredich, 1996
).
Besides being implicated in sulphur utilization and the synthesis of cysteine, CysB activates the expression of the adi and lysU genes (Shi & Bennett, 1994; Rowbury, 1997
). Recently, we found a novel CysB-regulated gene, hslJ, involved in displaying a novobiocin resistance (NovR) phenotype in E. coli shown to be independent of previously characterized genes (e.g. gyrB, cls, nov) implicated in this phenomenon (Lilic et al., 2003
). The hslJ gene encodes a putative outer-membrane protein (OMP) but the role of HslJ in the mechanism that enables cells to display novobiocin resistance is unknown. Originally, it was shown that in E. coli C600 cysB (SY380), AB1157 cysB(Ts) (SY381) and MC4100 cysB (SY602) mutants the novobiocin resistance was increased 615-fold in comparison to cysB wild-type (WT) isogenic strains (Rakonjac et al., 1991
; Lilic et al., 2003
). Using the random insertion of the lacZ reporter gene we discovered the hslJ gene, whose expression is negatively regulated by CysB (Lilic et al., 2003
). Expression of the hslJ : : lacZ gene fusion is elevated in cysB mutants and the five- to sixfold overproduction of HslJ in either cysB+ or cysB mutant strains increases the novobiocin resistance. A cysB hslJ double mutant does not exhibit the resistance to novobiocin. The hslJ gene expression is found to be negatively autogenously controlled in trans. However, according to analysis of the deduced amino acid sequence of HslJ this protein resides in the outer membrane. This prediction is strengthened by the fact that the hslJ : :
Kan mutation induces the psp operon (Lilic et al., 2003
). The psp operon is specifically induced by overexpression of WT and mutant OMPs (e.g. pIV, and a number of other secretins) as well as by expression of mutant envelope proteins (Model et al., 1997
). Hence, it is very likely that HslJ is localized in the outer membrane, implying the existence of an additional factor involved in negative regulation of hslJ expression.
The results presented by Lilic et al. (2003) did not answer the question whether CysB plays the role of a repressor acting directly and regulating the transcription of hslJ. In this study, we defined the hslJ promoter region and determined the mechanism of negative regulation imposed by CysB. Also, we addressed the question whether transcription of the hslJ is subject to double negative control by both CysB repression and the HslJ feedback autoregulation. We characterized the MC4100 cysB mutated allele in strain SY602 and tested it and other cysB alleles in the context of either hslJ regulation or control of the cys regulon.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To characterize the cysB mutation in strain SY602 (MC4100 cysB) we isolated the chromosomal DNA of SY602 and performed the PCR using the primers (IMGGE, Belgrade) CYSB-fw1 (5'-TTTAGCATGCAATTACAACAAC-3'), carrying the SphI restriction site (underlined), and CYSB-rev1 (5'-GAAGATCTTTTTTCCGGCAGTTT-3'), carrying the BglII restriction site (underlined). A 1 kb DNA fragment from PCR carrying the mutated cysB allele was then digested by SphI and BglII and cloned into vector pQE70, creating plasmid pVGM9. The entire cysB allele was sequenced.
RNA manipulations.
Total RNA was isolated according to Aiba et al. (1981) and Gerendasy & Ito (1990)
. Briefly, strains MC4100 and SY602 (MC4100 cysB) were cultured in 10 ml minimal medium A supplemented with cysteine overnight at 37 °C. The cells were harvested and resuspended in 10 ml protoplast buffer (15 mM Tris/HCl pH 8, 0·45 M sucrose, 8 mM EDTA) and then 80 µl lysozyme (1 mg ml-1) was added. Protoplasts were centrifuged and the pellet was resuspended in 0·5 ml lysis buffer (20 mM Tris/HCl pH 8, 10 mM NaCl, 1 mM NaCl, 1 mM sodium citrate, 1·5 % SDS), then 15 µl diethyl pyrocarbonate (DEPC) was added and the suspension was incubated for 5 min at 37 °C and then put on ice. After treatment with 250 µl NaCl (40 %, w/v) and incubation on ice for 10 min, the suspension was centrifuged for 1 h. The RNA was extracted with phenol/chloroform (1: :1, v/v) at room temperature and precipitated by adding 1 ml ethanol (at -70 °C). The RNA pellet was washed with 70 % ethanol and dissolved in water. We added 3 vols 4 M sodium acetate to concentrate the RNA solution and then reprecipitated. The RNA quality and concentrations were determined by measurement of A260 and A280. RNA with a A260/A280 ratio of 1·52·0 was used for further experiments.
Primer extension analysis was performed according to Sambrook et al. (1989) using primer HSLJ-rev3 (5'-GCCATCAGCAGGCTTAGC-3') (Genosys), 4326 nt downstream from the translational start, and 100 µg of total RNA isolated from strain MC4100 or SY602 (MC4100 cysB). RNA samples were resuspended in RNase-free water and incubated at 37 °C for 1 h in the presence of RNase-free DNase (Promega). Primer was labelled at its 5' end by using [
-32P]dATP and T4 polynucleotide kinase (New England Biolabs). The cDNA was extended at 37 °C for 40 min with MMLv Reverse Transcriptase (Pharmacia). The products were loaded on a 5 % polyacrylamide gel together with a nucleotide sequence (Sanger et al., 1977
) generated with the same (unlabelled) primer and plasmid pHV3002 as a template.
-Galactosidase assay.
E. coli strains carrying the plasmid hslJlacZ operon fusions were grown overnight at 37 °C in LB broth containing the appropriate antibiotic and diluted 100-fold into the same medium. Following growth to mid-exponential phase (OD600 0·4), cultures were assayed for -galactosidase activity by the method of Miller (1992)
.
Overproduction and purification of CysB-His6 protein.
Plasmid pVGM1 was used for overexpression and purification of the CysB-His6 fusion protein following the protocol in the Qiagen manual. The E. coli cysB gene was amplified by PCR using plasmid pJOH1 as a template and the pair of primers CYSB-fw1 and CYSB-rev1. The PCR product was digested with SphI and BglII and cloned into the SphI/BglII sites of vector pQE70 to generate the plasmid pVGM1. This construct was checked by sequencing (Sanger et al., 1977) using primers Type III/IV (Qiagen) and Reverse sequencing (Qiagen). Plasmid pVGM1 was used to transform E. coli strains SY602 and SY380 previously transformed with plasmid pREP4. The recombinant strains were grown in LB medium at 37 °C to mid-exponential phase (OD600 0·4). IPTG (1 mM) was then added and incubation continued for 3 h. Purification of the overproduced protein CysB-His6 (strain SY380/pREP4/pVGM1) was carried out at room temperature using the Ni-NTA agarose matrix of the QIAexpressionizt kit (Qiagen). Briefly, the cells were centrifuged, resuspended in 1/25 of the culture volume of sonication buffer, disrupted by freezing (in dry ice/ethanol, with thawing in cold water), and sonication, and the cell debris was removed by centrifugation. E. coli crude extracts were loaded on a 1 ml Ni-NTA agarose column previously equilibrated with sonication buffer and the CysB-His6-tagged protein was eluted with an imidazole gradient (100500 mM). The eluted fractions were subjected to SDS-PAGE as described by Laemmli (1970)
. The protein eluate was later dialysed overnight against a storage buffer (50 mM Tris/HCl pH 8·0, 1 mM EDTA and 20 % glycerol). The concentration of CysB-His6 was determined using the Bradford protein assay (Bio-Rad) (Bradford, 1976
). The purified protein was stored at -80 °C.
Nondenatured protein molecular mass determination.
Native molecular masses of the fusion protein CysB-His6 were determined by the method of Bryan (1977). Purified CysB-His6 (10 µg), and molecular size protein markers (20 µg) (nondenatured protein molecular mass marker kit, Sigma) in 20 mM Tris/HCl (pH 7·4), 1 mM EDTA and 50 mM NaCl buffer were subjected to electrophoresis on a set of native protein gels that contained various concentrations of acrylamide (6, 7, 8·5 and 10 %), and stained with Coomassie blue. Considering the pI (around 7) of CysB-His6, besides using gels of pH 8·8, we used native gels of pH 9·5. Both sets of gels worked equally well. The relative mobilities (RF), construction of the Ferguson plots and the coefficients of retardations (Kr; negative slope) of each protein species deduced from the slopes were determined. The logarithm of the Kr of the markers was then plotted versus the logarithm of their molecular masses. The plots obtained were used to determine the molecular sizes [molecular mass (Da)x1000] of CysB-His6 by extrapolation of their respective Kr.
Electrophoretic mobility-shift experiments.
DNA mobility-shift experiments were performed essentially as described by Prentki et al. (1987). DNA probes carrying different fragments of the hslJ promoter region were obtained by PCR amplification using pHV3002 as a template and different sets of primers: probe 301, HSLJ-fw3 (5'-GCAAAACTTAAGCAATCTGGAAAAAGGCG-3') (Genosys) plus HSLJ-rev2 (5'-GTCACGGGCTTACCG-3') (Genosys); probe 135, HSLJ-fw4 (5'-TGAAGAAAGTAGCCGCG-3') (Genosys) plus HSLJ-rev2; and probe C, HSLJ-fw2 plus HSLJ-rev1. DNA probes 301 and 135 were labelled at the 5' end by using [
-32P]dATP and T4 polynucleotide kinase (New England Biolabs). Probe C was used as an unlabelled specific competitor in 50-fold molar excess. Briefly, CysB protein was pre-incubated in binding buffer (10 mM Tris/HCl, pH 7·5; 70 mM KCl; 5 mM MgCl2; 1 mM DTT; 1 mM EDTA; 12·5 %, v/v, glycerol; 0·1 % Triton X-100; 200 µg ml-1 BSA) for 5 min at 4 °C before the labelled DNA (12 ng) and in some cases NAS (1, 3 or 5 mM) and/or unlabelled specific probe were added. All reaction mixtures contained 1000-fold weight excess of poly(dI-dC) (Pharmacia). Binding reaction mixtures (total volume 20 µl) were incubated for 20 min at 37 °C. After pre-electrophoresis (2 h), the samples were loaded onto 5 % native polyacrylamide gels in 0·5x TBE and run at a voltage of 12 V cm-1. The gels were then dried and exposed to X-ray film.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
We searched for the sequence proposed to be the consensus for binding of LTTRs such as the CysB protein. Nucleotide sequence analysis for direct and inverted repeats was done using the PCgene program. Inspection of the hslJ regulatory region revealed the IR sequence CTATTcttaaAATAG positioned 32 nucleotides upstream of the hslJ ORF (Fig. 1b). This AT-rich sequence overlaps the -10 hexamer of the putative
70 hslJ promoter as well the hslJ TSP and resembles the conserved base pairs of a generic T-N11-A LysR motif present in the binding sites for CysB and most LTTRs (Goethals et al., 1992
; Lochowska et al., 2001
). In addition, a second putative CysB binding site aTaATccccaATgAc was found 10 bp upstream of the first one (Fig. 1b
). This site is not AT-rich but it contains the core of the LysR motif and the imperfect IR, overlaps the -35 hexamer of the putative hslJ promoter, and in concert with the first site resembles the organization of the CysB repressor sites CBS-B and CBS-K2 found in the cys regulon promoters (Hryniewicz & Kredich, 1995
).
Definition of the hslJ promoter region
Different hslJlacZ operon fusions were constructed to dissect the regulatory region and to determine a DNA fragment that carries an active hslJ promoter. The constructs carried either the largest portion of the hslJ regulatory region A (pVGM3) or 5' deletion derivatives resulting in regions B (pVGM4) and C (pVGM5) (Fig. 2). All constructs contained the putative
70 hslJ promoter. Since hslJ was proposed to be a CysB-regulated gene, the activities of the hslJlacZ operon fusions were measured in the WT MC4100 and in a cysB mutant strains. As shown in Fig. 2
, the deletion analysis showed that all constructs, including one with region C carrying the putative
70 hslJ promoter with the rest of 5' region deleted, were active. The
-galactosidase activities were at the same level in all constructs and slightly higher in the cysB mutant strain. This result defined region C to be the hslJ promoter region containing both the determined putative
70 hslJ promoter and the potential CysB repressor site (two LysR motifs).
|
|
However, a CysB-dependent negative regulation of the hslJ transcription should be direct. Since all results were obtained with the construct carrying the hslJ promoter region C, and since this region contains a putative CysB repressor site composed of two LysR motifs' found to be the consensus sequences for binding of the LTTRs, CysB could bind the hslJ regulatory region and act as a direct repressor of the hslJ transcription.
Purification of the active CysB regulator
We wanted to analyse the CysB binding properties of the hslJ regulatory region in vitro. In order to approach this issue, by adding His tag at the C-terminal end of CysB (36·3 kDa), we constructed the fusion protein CysB-His6 (37·3 kDa) encoded from the plasmid pVGM1. The in vivo activity of this protein is proved by complementation of the cysteine auxotrophy. The cysB cysteine auxotrophs SY602/pREP4 and SY380/pREP4 were transformed with pVGM1. The transformants grew on minimal medium plates in the absence of cysteine (data not shown). Hence, we used strain SY380/pREP4 harbouring plasmid pVGM1 to overexpress and purify the fusion protein CysB-His6 placed under the IPTG inducible promoter (see Methods). After 3 h induction in the presence of 1 mM IPTG, we went through the protein purification procedure and eluted >90 % pure CysB-His6 fusion protein (37·3 kDa) in the fraction obtained by 0·250·3 M imidazole (Fig. 3). Only 10 % of the induced protein was soluble (90 % left in the pellet after lysis) (data not shown).
|
CysB binding in the hslJ regulatory region
We used the purified CysB fusion protein to analyse binding of this transcription factor in the hslJ regulatory region. It has been shown for the cys regulon target genes that CysB binds DNA in both promoter and coding regions (-110 to +30) (Kredich, 1996). The protein in different concentrations, probe 301 comprising the 166 nt of the hslJ regulatory region (region B, 150 nt, is inside this portion of probe 301) and the 135 nt of the 5' region of the hslJ gene, and probe 135 covering only the 5' region of the hslJ gene were used (Fig. 4
a). Gel retardation assay revealed that the CysB fusion protein binds only a DNA fragment containing the hslJ regulatory region (166 nt), making distinct DNAprotein complexes Cpx1 and Cpx2 (Fig. 4b
, lanes 13). The appearance of these complexes was dependent on the protein concentration. Using region C as an unlabelled probe (Fig. 4c
, lane 5) diminished the binding of CysB to probe 301. Formation of two complexes, such as Cpx1 and Cpx2, usually indicates two binding sites. As shown above, region C is the hslJ promoter region that carries the putative hslJ promoter and two LysR motifs' (the putative CysB repressor site composed of two binding sites) (Fig. 1b
, Fig. 2
). Therefore, CysB most likely binds the sequence predicted to be the CysB repressor site in an hslJ regulatory region, and upon binding acts directly as a repressor of hslJ transcription.
|
In all CysB-dependent regulations described to date, NAS plays the role of an inducer when used in concentrations like those used in this study (Kredich, 1996). We showed that CysB negatively regulates hslJ transcription, where it is proposed to bind the LysR motifs' that resemble the topology of the CysB repressor site that overlaps the hslJ promoter and the hslJ TSP. However, in the control of hslJ transcription where CysB acts as a repressor, NAS enhances the binding of this regulator to DNA. Therefore, it seems that the term inducer cannot be applied to NAS as regards CysB-mediated regulation of hslJ transcription.
cysB mutations
The CysB regulator is a multi-domain protein that contains the functional domains responsible for DNA binding (N-terminus), interaction with ligands (e.g. NAS) (central domain), and oligomerization and DNA binding (C-terminus) (Schell, 1993; Lochowska et al., 2001
). The different MC4100 cysB mutants used in this work carry cysB alleles with specific mutations affecting the function of the entire protein or the specific functional domain.
cysB is a null mutation with the major portion of the cysB gene deleted and hence strain EC2549 entirely lacks the CysB product (Lochowska et al., 2001
). However, the CysB I33N mutant protein produced by strain EC2275 carries a missense mutation in the recognition helix of the HTH motif in the CysB DNA binding domain (Lochowska et al., 2001
) (Fig. 5
b). This mutation has been shown to diminish the binding of CysB to both the activator and the repressor sites in the regulatory regions of the cys regulon genes and consequently the active regulation failed (Colyer & Kredich, 1994
). The interallelic complementation of this allele with the other specific cysB allele encoding CysB T149Ter restored the functionality of the CysB regulator (Colyer & Kredich, 1994
). In the previous and in this work we used the spontaneous MC4100 cysB mutant SY602 obtained by selection of NovR colonies on plates supplemented with 400 µg novobiocin ml-1 (Lilic et al., 2003
). This mutant, besides exhibiting resistance to novobiocin, was a cysteine auxotroph. We further characterized this mutant and found that the phenotypes obtained were due to mutation in the cysB gene (Lilic et al., 2003
). The previous work by Rakonjac et al. (1991)
and Lilic et al. (2003)
and this study established that the NovR phenotype related to the cysB mutations is the consequence of elevated HslJ expression upon lack of hslJ transcription repression otherwise imposed by CysB. Hence, the repression of hslJ transcription, the NovS phenotype and the biosynthesis of the cysteine are the outcomes of the WT CysB activities.
|
The insertion of IS1 element interrupts the region that encodes the CysB domain responsible for the oligomerization and binding of this regulator to DNA (Fig. 5b). In order to answer whether the CysB mutant regulator encoded by the cysB831 : : IS1 allele is produced as a CysB S277Ter protein (Fig. 5a, b
) and whether its functionality can be restored by complementation with the other, different, CysB mutant(s), we performed an interalellic complementation experiment. We followed the resistance to novobiocin as a measure of hslJ expression and the biosynthesis of cysteine as a functionality of the cys regulon control enabled by CysB. The results are presented in Table 3
. Plasmid pVGM9 carrying the cysB831 : : IS1 allele complemented the chromosomal copy of the EC2275 cysB allele encoding the CysB I33N protein, decreasing the novobiocin resistance, but it failed to complement the cysteine auxotrophy of strain EC2275. As a control, neither the cysB831 : : IS1 allele nor the EC2275 cysB allele complemented a
cysB mutation in strain EC2549, while the WT cysB+ efficiently restored the NovS and the cysteine prototrophy phenotypes. Hence, we showed that the functional restoration of CysB regarding the repression of the hslJ transcription and the consequent NovS phenotype can be obtained to a certain extent by complementation of the alleles that encode the CysB I33N and CysB S277Ter proteins. However, the combination of these mutated CysB proteins did not complement the cysteine auxotrophy. This result suggests that the restored oligomer of the CysB regulator partially represses the hslJ transcription while not functioning in regulating the transcription of the cys regulon genes. This result is different from that obtained by Colyer & Kredich (1994)
, where the interallelic complementation of the CysB I33N and the CysB T149Ter mutant regulators restored the functional regulation of the cys regulon genes and complemented the cysteine auxotrophy. One may suspect that the DNA-binding property of the LTTR CysB slightly differs in the hslJ system in comparison to the cys regulon target promoters. It could be that binding is correct but the transcription activation (CysB as an activator) important for the cys regulon genes but not for the hslJ transcription regulation (CysB as a repressor) failed.
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 12, 248254.[CrossRef]
Bryan, J. K. (1977). Molecular weights of protein multimers from polyacrylamide gel electrophoresis. Anal Biochem 78, 513519.[Medline]
Bullock, W. O., Fernandez, M. & Shotr, J. M. (1987). XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. BioTechniques 5, 376378.
Casadaban, M. J. (1976). Transposition and fusion of the lac genes in the selected phenotypes in E. coli using bacteriophage lambda and Mu. J Mol Biol 104, 541555.[Medline]
Colyer, T. E. & Kredich, N. M. (1994). Residue threonine-149 of the Salmonella typhimurium CysB transcription activator: mutations causing constitutive expression of positively regulated genes of the cysteine regulon. Mol Microbiol 13, 797805.[Medline]
Galas, D. J. & Chandler, M. (1991). Bacterial insertion sequences. In Mobile DNA, pp. 109162. Edited by D. E. Berg & and M. M. Howe. Washington, DC: American Society for Microbiology.
Gerendasy, D. & Ito, J. (1990). Nucleotide sequence and transcription of the right early region of bacteriophage PRD1. J Bacteriol 172, 18891898.[Medline]
Goethals, K., Van Montagu, M. & Holsters, M. (1992). Conserved motifs in a divergent nod box of Azorhizobium caulinodans ORS571 reveal a common structure in promoters regulated by LysR-type proteins. Proc Natl Acad Sci U S A 89, 16461650.[Abstract]
Grana, D., Gardella, T. & Susskind, M. M. (1988). The effect of mutations in the ant promoter of phage P22 depend on context. Genetics 120, 319327.
Gross, A. C. (1996). Function and regulation of the heat shock proteins. In Escherichia coli and Salmonella: Cellular and Molecular Biology, vol. 2, pp. 13821399. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Hopwood, D. A., Bibb, J. M., Chater, K. F. & 7 other authors (1985). Genetic Manipulation of Streptomyces, a Laboratory Manual. Norwich, UK: John Innes Foundation.
Hryniewicz, M. M. & Kredich, N. M. (1994). Stoichiometry of binding of CysB to the cysJIH, cysK, and cysP promoter regions of Salmonella typhimurium. J Bacteriol 176, 36733682.[Abstract]
Hryniewicz, M. M. & Kredich, N. M. (1995). Hydroxyl radical footprints and half-site arrangements of binding sites for the CysB transcriptional activator of Salmonella typhimurium. J Bacteriol 177, 23432353.[Abstract]
Kredich, N. M. (1996). Biosynthesis of cysteine. In Escherichia coli and Salmonella: Cellular and Molecular Biology, vol. 1, pp. 514527. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680690.[Medline]
Lange, R. & Hengge-Aronis, R. (1991). Growth phase-regulated expression of bolA and morphology of stationary-phase Escherichia coli cells is controlled by the novel sigma factor S (rpoS). J Bacteriol 173, 44744481.[Medline]
Lilic, M., Jovanovic, M., Jovanovic, G. & Savic, D. J. (2003). Identification of the CysB-regulated gene, hslJ, related to the Escherichia coli novobiocin resistance phenotype. FEMS Microbiol Lett 224, 239246.[CrossRef][Medline]
Lochowska, A., Iwanicka-Nowicka, R., Plochocka, D. & Hryniewicz, M. M. (2001). Functional dissection of the LysR-type CysB transcriptional regulator. J Biol Chem 276, 20982107.
Miller, J. H. (1992). A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Model, P., Jovanovic, G. & Dworkin, J. (1997). The Escherichia coli phage-shock-protein (psp) operon. Mol Microbiol 24, 255261.[Medline]
Ostrowski, J., Jagura-Burdzy, G. & Kredich, N. M. (1987). DNA sequences of the cysB regions of Salmonella typhimurium and Escherichia coli. J Biol Chem 262, 59996005.
Pabo, C. O. & Sauer, R. T. (1992). Transcription factors: structural families and principles of DNA recognition. Annu Rev Biochem 61, 10531095.[CrossRef][Medline]
Parry, J. & Clark, D. P. (2002). Identification of a CysB-regulated gene involved in glutathione transport in Escherichia coli. FEMS Microbiol Lett 209, 8185.[Medline]
Perez-Rueda, E. & Collado-Vides, J. (2000). The repertoire of DNA-binding transcriptional regulators in Escherichia coli K-12. Nucleic Acids Res 28, 18381847.
Prentki, P., Chandler, M. & Galas, D. J. (1987). Escherichia coli integration host factor bends the DNA at the ends of IS1 and in an insertion hotspot with multiple IHF binding sites. EMBO J 6, 24792487.[Abstract]
Rakonjac, J., Milic, M. & Savic, D. J. (1991). cysB and cysE mutants of Escherichia coli K12 show increased resistance to novobiocin. Mol Gen Genet 228, 307311.[Medline]
Rowbury, R. J. (1997). Regulatory components, including integration host factor, CysB and H-NS, that influence pH responses in Escherichia coli. Lett Appl Microbiol 24, 319328.[CrossRef][Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74, 54635467.[Abstract]
Schell, M. A. (1993). Molecular biology of the LysR family of transcriptional activators. Annu Rev Microbiol 47, 597626.[CrossRef][Medline]
Shi, X. & Bennett, G. N. (1994). Effects of rpoA and cysB mutations on acid induction of biodegradative arginine decarboxylase in Escherichia coli. J Bacteriol 176, 70177023.[Abstract]
Simons, R. W., Houman, F. & Kleckner, N. (1987). Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53, 8596.[CrossRef][Medline]
Tyrell, R., Verschueren, H. K., Dodson, E. J., Murshudov, G. N., Addy, C. & Wilkinson, A. J. (1997). The structure of the cofactor-binding fragment of the LysR family member, CysB: a familiar fold with a surprising subunit arrangement. Structure 5, 10171032.[Medline]
van der Ploeg, J. R., Eichhorn, E. & Leisinger, T. (2001). Sulfonate-sulfur metabolism and its regulation in Escherichia coli. Arch Microbiol 176, 18.[CrossRef][Medline]
Verschueren, K. H., Addy, C., Dodson, E. J. & Wilkinson, A. J. (2001). Crystallization of full-length CysB of Klebsiella aerogenes, a LysR-type transcriptional regulator. Acta Crystallogr D Biol Crystallogr 57, 260262.[CrossRef][Medline]
Received 30 June 2003;
revised 1 September 2003;
accepted 16 September 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |