Functional dissection of the LysR-type CysB transcriptional regulator. Regions important for DNA binding, inducer response, oligomerization, and positive control.

CysB is a tetrameric LysR-type transcriptional regulator that acts as an activator of cys regulon genes and as an autorepressor. Positive control of cys genes requires the presence of the inducer N-acetylserine. Following random and site-directed mutagenesis of the cysB gene, 20 CysB variants were isolated. Six single amino acid substitutions within the N terminus of CysB abolished the DNA-binding ability of the protein. Seven mutations in the central region of CysB affected its response to the inducer. Four of these CysB mutants retained repressing activity, but lost their activating function in vivo. Their DNA binding characteristics were consistent with an inability to respond to acetylserine by a qualitative change in the DNA-protein interaction. Three of the single residue substitutions resulted in constitutive activity of CysB. The electrophoretic mobility of the complex formed by one of the CysBc variants with the cysP promoter suggested a dimeric state of this protein. Characteristics of six truncated CysB variants lacking 5-30 C-terminal residues indicated the involvement of the C terminus in the DNA binding, oligomerization, and stability of CysB. The single substitution Y27G resulted in the CysBpc variant, able to bind DNA and to respond to the inducer by a qualitative change in the DNA-protein complex, but defective in the positive control of the cysP promoter.

The Escherichia coli ssuEADCB gene cluster is required for the utilization of sulfur from aliphatic sulfonates and is regulated by the transcriptional activator Cbl.

The growth properties of an Escherichia coli strain carrying a chromosomal deletion of the ssuEADCB genes (formerly designated ycbPONME) indicated that the products of this gene cluster are required for the utilization of sulfur from aliphatic sulfonates. Sequence similarity searches indicated that the proteins encoded by ssuA, ssuB, and ssuC are likely to constitute an ABC type transport system, whereas ssuD and ssuE encode an FMNH(2)-dependent monooxygenase and an NAD(P)H-dependent FMN reductase, respectively (Eichhorn, E., van der Ploeg, J. R., and Leisinger, T. (1999) J. Biol. Chem. 274, 26639-26646). Synthesis of beta-galactosidase from a transcriptional chromosomal ssuE'-lacZ fusion was repressed by sulfate or cystine and depended on the presence of a functional cbl gene, which encodes a LysR-type transcriptional regulator. Electrophoretic mobility shift assays with the ssu promoter region and measurements of beta-galactosidase from plasmid-encoded ssuE'-'lacZ fusions showed that full expression of the ssu operon required the presence of a Cbl-binding site upstream of the -35 region. CysB, the LysR transcriptional regulator for the cys genes, was not required for expression of a chromosomal ssuE'-lacZ fusion although the ssu promoter region contained three CysB-binding sites. Integration host factor could also occupy three binding sites in the ssu promoter region but had no influence on expression of a chromosomal ssuE'-lacZ fusion.

M.(phi)BssHII, a novel cytosine-C5-DNA-methyltransferase with target-recognizing domains at separated locations of the enzyme.

In all cytosine-C5-DNA-methyltransferases (MTases) from prokaryotes and eukaryotes, remarkably conserved amino acid sequence elements responsible for general enzymatic functions are arranged in the same canonical order. In addition, one variable region, which includes the target-recognizing domain(s) (TRDs) characteristic for each enzyme, has been localized in one region between the same blocks of these conserved elements. This conservation in the order of conserved and variable sequences suggests stringent structural constraints in the primary structure to obtain the correct folding of the enzymes. Here we report the characterization of a new type of a multispecific MTase, M.(phiphi)BssHII, which is expressed as two isoforms. Isoform I is an entirely novel type of MTase which has, in addition to the TRDs at the conventional location, one TRD located at a non-canonical position at its N-terminus. Isoform II is represented by the same MTase, but without the N-terminal TRD. The N-terminal TRD provides HaeII methylation specificity to isoform I. The TRD is fully functional when engineered into either the conventional variable region of M.(phiphi)BssHII or the related monospecific M.phi3TII MTase. The implications of this structural plasticity with respect to the evolution of MTases are discussed.

Involvement of CysB and Cbl regulatory proteins in expression of the tauABCD operon and other sulfate starvation-inducible genes in Escherichia coli.

Starvation for sulfate results in increased synthesis of several proteins in Escherichia coli. Among these Ssi (sulfate starvation-induced) proteins are the products of the tauABCD genes, which are required for utilization of taurine as sulfur source for growth. In this study, the role of the cbl gene in expression of tauABCD and other ssi genes was investigated. The protein encoded by cbl shows high sequence similarity to CysB, the LysR-type transcriptional activator of the genes involved in cysteine biosynthesis. Strain EC2541, which contains an internal deletion in cbl, was unable to utilize taurine and other aliphatic sulfonates as sulfur sources. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that many of the Ssi proteins were not synthesized in EC2541. Expression of a translational tauD'-'lacZ fusion required the presence of both cbl and cysB. The interactions of CysB and Cbl with the promoter region of tauABCD were studied by using gel mobility shift experiments and DNase I footprinting. CysB occupied multiple binding sites, whereas Cbl occupied only one site from 112 to 68 bp upstream of the transcription start site. Acetylserine, the inducer of transcription of CysB-regulated genes, stimulated binding of CysB but not of Cbl. Sulfate had no effect on binding of both proteins to the tauABCD promoter region. These results indicate that Cbl is a transcription factor for genes required for sulfonate-sulfur utilization and maybe for other genes whose expression is induced by sulfate starvation.

A new gene, cbl, encoding a member of the LysR family of transcriptional regulators belongs to Escherichia coli cys regulon.

The cbl (cysB-like) gene has been identified in Escherichia coli. The analysis of the cloned cbl sequence revealed strict homology to an ORF of unknown function found initially in Klebsiella aerogenes [Schwacha and Bender, J. Bacteriol. 175 (1993) 2107-2115]. The predicted Cbl protein has structural features of the LysR family of transcriptional activators. It is also strongly similar to the CysB protein, the activator of the cys regulon. The position of cbl on the Ec physical map has been established at a 2070-kb (43.5 min) region between asnU and asnV. The gene is expressed in vivo as a 1-kb monocistronic transcript starting from one major transcription start point. Unexpectedly, the in vivo expression of cbl has shown dependence on CysB, belonging to the same family of proteins. The promoter region of cbl binds purified CysB protein in a manner similar to other CysB-responsive promoters. A cbl disruption mutant was constructed by insertion of a KmR gene cartridge into the ORF on the chromosome. Phenotypes related to cbl expression suggest the involvement of the gene in an accessory regulatory circuit within the cys regulon engaging, in the last step, the function of the cysM gene encoding O-acetylserine (thiol)-lyase B.

[Appearance of antibodies against hepatitis type A virus (HAV) in alcoholics]. / Wystepowanie przeciwcial przeciwko wirusowi zapalenia watroby typu A (HAV) u alkoholików.

In 144 alcoholics--patients of alcoholic outpatient clinic anti-HAV antibodies were studied using the commercially available ELISA test. Anti-HCV antibodies were found in 86% of the alcoholics, and this prevalence was not higher than in the control group. No differences were found in the titre of antibodies against cytomegaly and herpes virus between the subjects with and without anti-HAV antibodies which suggests the specificity f the determined anti-HAV antibodies.

Prevalence of markers of hepatotropic viruses among drug addicts in Warsaw, Poland.

We studied 100 unselected parenteral drug abusers for infection with hepatitis C, B, A and D virus (HCV, HBV, HAV and HDV). Seventy-six percent had serological evidence of HCV infection. 12% were positive for HBsAg and at least one marker of HBV infection was present in 69%. These results were significantly higher than in a matched control population. Compared to controls, the prevalence of anti-HAV (65%) was not significantly increased in drug addicts. Of the anti-HCV-positive drug addicts, 80.3% had at least one marker of HBV infection compared to 33.3% of anti-HCV-negative cases (p less than 0.001). No such correlation was found between the prevalence of HCV or HBV infection markers and the presence of anti-HAV. Antibodies against HDV were detected in 16 (16%) of the samples from drug addicts. No significant association was found between antibodies to HCV and gender, age and duration of drug abuse. The risk of HBV infection increased significantly with years of drug abuse but was not associated with age and sex. The presence of anti-HAV was related to age only. Sixteen (16%) of the subjects were definitely positive for anti-HIV-1, but at the time of the study they were asymptomatic. No significant association was found between the presence of anti-HIV and the prevalence of serological markers of HBV, HCV, HAV and HDV infection.

[Occurrence of antibodies against hepatitis C virus (HCV) among alcoholics]. / Wystepowanie przeciwcial przeciwko wirusowi zapalenia watroby typu C (HCV) u alkoholików.

144 consecutive alcoholics attending an outpatient clinic were tested for the prevalence of anti-HCV with commercially available ELISA test (Abbott HCV EIA). Positive sera were further tested with neutralization assay (Abbott HCV Neutralization Assay). Anti-HCV were found in 35 (24%). There was no difference in the prevalence or mean titre of anti-CMV and anti-HSV between patients with and without anti-HCV what points to the specificity of tested antibodies.

[Delta virus infection among drug addicts]. / Zakazenie wirusem zapalenia watroby typu delta wsród narkomanów.

Altogether 240 unselected intravenous drug addicts were tested for the presence of infection with delta virus (HDV). Anti-delta were found in 16 (16%) out of 100 drug addicts tested in the years 1988-1989 and in only one drug addict (0.7%) out of 140 tested in the years 1985-1986. It seems that delta virus has been introduced to Warsaw drug community in the mid-eighties and has spread enormously since that time.

[Antibodies against Cytomegalovirus and Herpes simplex virus in alcoholics and drug addicts]. / Przeciwciala przeciwko wirusowi cytomegalii i wirusowi herpes simplex u alkoholików i narkomanów.

Anti-CMV and anti-HSV type 1 of the IgG class were tested in 123 consecutive outpatient alcoholics and 95 consecutive drug addicts admitted to a detoxification ward. The prevalence of both viral markers in these groups was not different from the prevalence found among controls, however, antibody titres were significantly higher. Only weak correlation was found between titres of anti-CMV and anti-HSV in tested subjects. The reasons responsible for high titres of anti-CMV and anti-HSV remain to be determined.