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PubMed2001

Molecular characterization of a chromosomal locus in Staphylococcus aureus that contributes to oxidative defence and is highly induced by the cell-wall-active antibiotic oxacillin.

Previous studies employing two-dimensional gel electrophoresis and N-terminal protein sequencing have shown elevated synthesis of the enzyme methionine sulfoxide reductase (MsrA) in Staphylococcus aureus in response to cell-wall-active antibiotics. In the present study, the S. aureus msrA gene was cloned, overexpressed, purified as His-tagged MsrA and shown to have methionine sulfoxide reductase activity. The transcription of msrA was studied by assaying beta-galactosidase activity in an msrA promoter::lacZ fusion strain and by Northern blot analysis. Transcription of msrA was increased by oxacillin; but not by a variety of other stresses including H2O2. Northern blot analysis revealed that the size of the msrA transcript was 2.3 kb, considerably larger than the 531 nt msrA ORF. The msrA transcription start site was mapped 25 nt upstream of the msrA start codon. Computer analysis from database sequences indicated at least three additional ORFs downstream of msrA. The deduced amino acid sequences of two of these three ORFs showed significant sequence homologies to PilB, and enzyme IIA of the phosphotransferase system, respectively. The third ORF could not be identified by homology searches. Northern blot hybridization with probes specific to the msrA downstream region indicated that the S. aureus msrA was transcribed as part of a polycistronic message. Interestingly, purified S. aureus PilB was shown to possess approximately approximately 28-fold higher methionine sulfoxide reductase activity than the MsrA. An insertional knockout mutation in the first gene of this operon resulted in increased susceptibility of the mutant to H2O2 compared to the parent strain, but not to oxacillin.

Bacterial Proteins↗
PubMed2004

Cloning and characterization of the MAL11 gene encoding a high-affinity maltose transporter from Torulaspora delbrueckii.

The transport and regulation of maltose utilization by Torulaspora delbrueckii, one of the most abundant non-Saccharomyces species present in home-made corn and rye bread dough, has been investigated. A DNA fragment containing the MAL11 gene from T. delbrueckii (TdMAL11) was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an open reading frame (ORF) of 1884 bp, encoding a 627-amino acid membrane protein, which displays high homology to other yeast maltose transporters. Upstream of TdMAL11, the DNA insert contained a partial ORF (TdMAL12) on the opposite strand, which showed high similarity to the S. cerevisiae MAL12 gene. Sequence analysis, Northern blot and transport measurements indicated that TdMAL11 expression is regulated by the carbon source. Attempts to disrupt TdMAL11 revealed the presence of two functional MAL loci. Disruption of a single copy decreased the V(max) of maltose transport, but not the K(m), whereas the double disruption abolished the uptake of this sugar in T. delbrueckii.

Amino Acid Sequence↗
PubMed1994

Cloning and sequencing of sakP encoding sakacin P, the bacteriocin produced by Lactobacillus sake LTH 673.

Sakacin P is a heat-stable, unmodified peptide bacteriocin produced by Lactobacillus sake LTH 673. The strain was isolated from fermented dry sausages and is well adapted to this habitat. The bacteriocin inhibits the growth of the opportunistic food pathogens Enterococcus faecalis and Listeria monocytogenes and therefore, it may improve the hygienic status of fermented food, i.e. meat products. Oligonucleotide probes were designed from the N-terminal amino acid sequence of sakacin P and used to identify sakP, the structural gene of sakacin P, on the chromosome of L. sake LTH 673. SakP was cloned into Escherichia coli NM554 and the nucleotide sequence of the gene and its adjacent regions were determined. Sakacin P appears to be synthesized as a prepeptide of 61 amino acids which is proteolytically processed to the mature bacteriocin consisting of 43 amino acids. Sequencing of the cloned fragment also revealed the presence of two other open reading frames orfX and orfY, which are located upstream and downstream of sakP, respectively, putatively encoding proteins of 52 and 98 amino acids, respectively. The functions of both ORFs remain unknown. Primer extension analysis revealed a promoter upstream of sakP. Two transcripts of approximately 0.35 and 1.0 kb were detected by Northern hybridization encoding either only sakP, or both sakP and orfY, respectively.

Amino Acid Sequence↗
PubMed1992

On the variability of the 3' terminal sequence of the turnip mosaic virus genome.

The sequence of the 3'-terminal 1223 nucleotides (nts) of a Japanese isolate of turnip mosaic virus (TuMV-Jap) RNA has been determined. The sequence reveals a single open reading frame (ORF) which terminates at a position 212 nts upstream of the 3' poly(A)-tract. Determination of the N-terminal amino acids of TuMV-Jap coat protein (CP) mapped the CP cistron within this ORF and revealed a Glu-Ala dipeptide sequence as the putative cleavage site by which the CP is released from the viral polyprotein. The predicted amino acid sequence of the TuMV-Jap CP shows 97.2% identity with that of a Canadian isolate of TuMV (TuMV-Can) and 99% with a second, Chinese, isolate (TuMV-Chi). However, the 3'-terminal non-translated region (NTR) of TuMV-Jap RNA is significantly shorter (212 nts) than the 3'-NTR of TuMV-Can RNA (668 nts), but of equal length as the 3'-NTR of the TuMV-Chi isolate which also measures 212 nts. The 3'-NTRs of both the TuMV-Jap and TuMV-Chi RNAs show homology with the first 201 nucleotides of the TuMV-Can RNA 3'-NTR. A search in the EMBL nucleotide sequence database revealed that the 467 nt-long unique extension of the 3'-NTR of TuMV-Can RNA has 89.8% homology to a part of the chloroplast ribosomal protein 12 gene (rsp12-gene). Irrespective of the origin of this extra sequence in the reported TuMV-Can sequence, which may have been introduced by a genuine RNA recombination event, it is concluded that the standard TuMV genome has a CP gene of 864 nts and an conserved 3'-NTR of approximately 212 nucleotides in length.

Amino Acid Sequence↗
PubMed1994

Inhibition of Agrobacterium tumefaciens oncogenicity by the osa gene of pSa.

The IncW plasmid pSa originally derived from Shigella flexneri completely inhibits the tumor-inducing ability of Agrobacterium tumefaciens when it is resident in this organism. Oncogenic inhibition is mediated through the expression of the osa gene on pSa. This gene is part of a 3.1-kb DNA segment of pSa that contains four open reading frames revealed by sequencing. Specific deletions and TnCAT insertions within this segment localized the oncogenic inhibitory activity to the last open reading frame, orf-4, designated osa (for oncogenic suppression activity). No promoter exists immediately upstream of the coding sequence of osa since TnCAT insertions or deletions into orf-3 caused the loss of oncogenic inhibition. Deletion analysis showed that the promoter of orf-1 is required for osa transcription. The first three orfs have no role in oncogenic inhibition, since osa alone placed under the control of a constitutive Pkm promoter completely inhibited A. tumefaciens oncogenicity. This inhibition of oncogenicity by osa is not limited to a specific host plant but appears to show broad host specificity. Because the osa-encoded product has close homologies to the fiwA-encoded product of the IncP plasmid RP1, osa may be involved in fertility inhibition that would prevent or reduce the formation of stable mating pairs and T-DNA transfer between A. tumefaciens and plants.

Agrobacterium tumefaciens↗
PubMed1985

Identification of hepatitis B virus polypeptides encoded by the entire pre-s open reading frame.

The open reading frame (ORF) that encodes the 226-amino-acid coat protein (hepatitis B virus surface antigen [HBsAg]) of hepatitis B virus has the potential to encode a 400-amino-acid polypeptide. The entire ORF would direct the synthesis of a polypeptide whose C-terminal amino acids represent HBsAg with an additional 174 amino acids at the N terminus (pre-s). Recently, virus particles have been shown to contain a polypeptide that corresponds to HBsAg with an additional 55 amino acids at the N terminus encoded by the DNA sequence immediately upstream of the HBsAg gene. A novel ORF expression vector containing the TAC promoter, the first eight codons of the gene for beta-galactosidase, and the entire coding sequence for chloramphenicol acetyltransferase was used in bacteria to express determinants of the 174 amino acids predicted from the pre-s portion of the ORF. The resulting tribrid protein containing 108 amino acids encoded by pre-s was expressed as one of the major proteins of bacteria harboring the recombinant plasmid. Single-step purification of the tribrid fusion protein was achieved by fractionation on a chloramphenicol affinity resin. Polyclonal antiserum generated to the fusion protein was capable of detecting 42- and 46-kilodalton polypeptides from virus particles; both polypeptides were also shown to contain HBsAg determinants. The ability of the polyclonal antiserum to identify polypeptides with these characteristics from virus particles presents compelling evidence that the DNA sequence of the entire ORF is expressed as a contiguous polypeptide containing HBsAg. The presence of multiple promoters and primary translation products from this single ORF argues that the function and potential interaction of the encoded polypeptides play a crucial role in the life cycle of the virus. Furthermore, the procedure and vector described in this report can be applied to other systems to facilitate the generation of antibodies to defined determinants and should allow the characterization of the epitope specificity of existing antibodies.

DNA, Recombinant↗
PubMed2002

Macropodid herpesvirus 1 encodes genes for both thymidylate synthase and ICP34.5.

Macropodid herpesvirus 1 (MaHV-1) is an unclassified alphaherpesvirus linked with the fatal infections of kangaroos and other marsupials. During the characterisation of the internal repeat region of MaHV-1, an open reading frame (ORF) encoding for thymidylate synthase (TS) gene was identified and completely sequenced. Southern blot analysis confirmed the presence of two copies of the TS gene in the MaHV-1 genome as expected. Computer analysis of the TS ORF showed it was 948 nucleotides in length. A putative polyadenylation signal was identified 17-22 bp inside the ORF implying a minimal or absent 3' untranslated region. The predicted polypeptide was 316 amino acid residues in length and contained the highly conserved motifs for folate binding and F-dUMP binding, typical of all TS enzymes. Interestingly, MaHV-1 TS polypeptide had highest similarity to the human TS polypeptide (81%) compared to the TS polypeptides of other herpesviruses (72-75%). Immediately upstream of the TS gene, a second ORF of 510 bp, encoding a polypeptide with 170 amino acid residues, was identified. The carboxyl domain of this MaHV-1 polypeptide shared 68% similarity to a 59 amino acid motif of human herpesvirus 1 ICP34.5, identifying it as the MaHV-1 ICP34.5 homologue. This is the first report of a herpesvirus that encodes for both TS and ICP34.5.

Alphaherpesvirinae↗
PubMed1995

Genomic organization of the Klebsiella pneumoniae cps region responsible for serotype K2 capsular polysaccharide synthesis in the virulent strain Chedid.

The genomic organization of the chromosomal cps region that is responsible for capsular polysaccharide synthesis in Klebsiella pneumoniae Chedid (O1:K2) was investigated. Deletion analyses and Southern hybridization studies suggested that the central region of the cloned 29-kb BamHI fragment is indispensable for K2 capsular polysaccharide synthesis. The 24,329-bp nucleotide sequence of the Klebsiella cps region was determined and deposited in the EMBL and GenBank databases through DDBJ and assigned accession number D21242. Nineteen possible open reading frames (ORFs) were identified in the sequenced area. Among them, 13 ORFs are very close to each other. Six of the 19 ORFs show considerable nucleotide sequence similarities to Salmonella typhimurium cpsG, cpsB, rfbP, and orf2.8, Escherichia coli gnd, and Haemophilus influenzae bexD, respectively. Moreover, the deduced amino acid sequence of the ORF10 product demonstrated a highly hydrophobic profile and showed putative membrane topology similarity to Rickettsia prowazekii ATP/ADP translocase. Nucleotide sequence similar to the sigma 54-dependent promoter, as well as the usual -35 and -10 sequences, were identified just upstream of ORF3, which is the first ORF in the polycistronic structure. Furthermore, a sequence (GGGCGGTAGCGT) found just downstream of the sigma 54-dependent promoter-like sequence was generally conserved among gene clusters implicated in cell surface polysaccharide synthesis, such as Salmonella rfb and viaB and E. coli kpsMT and rfaQPG. A possible transcriptional terminator with a hairpin loop structure found just downstream of ORF15 that is a homolog of E. coli gnd. K2 capsular polsaccharide biosynthesis in E. coli K-12 depends on cpsB (mannose-1-phosphate guanyltransferase gene), and Klebsiella cpsB, found in the downstream region of the polycistronic structure, was able to complement cpsB of E. coli. Results of transposon insertion and promoter-cloning analyses were consistent with the results of nucleotide sequence analysis.

Amino Acid Sequence↗
PubMed1986

Sequence and transcripts of the bacteriophage T4 DNA repair gene uvsY.

We have cloned, sequenced and analyzed transcription of the phage T4 uvsY gene. This gene is transcribed from a single gp MotA-dependent middle promoter to give a major transcript of approximately 930 nucleotides and a minor transcript of approximately 620 nucleotides. All in vivo and in vitro uvsY transcripts show anomalous migration in agarose gels. The uvsY transcript contains an open reading frame coding for an 137 amino acid [15.8 kilodaltons (kD)] UvsY protein and two unidentified open reading frames, ORF UvsY.-1 (9.0 kD) and ORF UvsY.-2 (6.0 kD). Our DNA sequence differs in only three places from that published by TAKAHASHI et al. However, one of these changes alters the predicted carboxy terminus of the UvsY protein. Marker rescue experiments map gene 25 to the region upstream of uvsY. Gene 25 is likely, although not certain, to correspond to an ORF that is found upstream from uvsY and is translated in the same direction.

Amino Acid Sequence↗
PubMed2006

Genomic cloning and characterization of a PPA gene encoding a mannose-binding lectin from Pinellia pedatisecta.

A gene encoding a mannose-binding lectin, Pinellia pedatisecta agglutinin (PPA), was isolated from leaves of Pinellia pedatisecta using genomic walker technology. The ppa contained an 1140-bp 5'-upstream region, a 771-bp open reading frame (ORF) and an 829-bp 3'-downstream region. The ORF encoded a precursor polypeptide of 256 amino acid residues with a 24-amino acid signal peptide. There were one putative TATA box and six possible CAAT boxes lying in the 5'-upstream region of ppa. The ppa showed significant similarity at the nucleic acid level with genes encoding mannose-binding lectins from other Araceae species such as Pinellia ternata, Arisaema hererophyllum, Colocasia esculenta and Arum maculatum. At the amino acid level, PPA also shared varying homology (ranging from 40% to 85%) with mannose-binding lectins from other plant species, such as those from Araceae, Alliaceae, Iridaceae, Lillaceae, Amaryllidaceae and Bromeliaceae. The cloning of the ppa gene not only provides a basis for further investigation of PPA's structure, expression and regulation mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into tobacco and rice in the future.

Amino Acid Sequence↗
PubMed1994

DNA sequence and transcriptional analysis of the simian varicella virus glycoprotein B gene.

The varicella-zoster virus (VZV) glycoprotein B (gB) is a major viral antigen which elicits immunity and neutralizing antibodies. In this study, the genomic map position and DNA sequence of a simian varicella virus (SVV) homologue of the VZV gB gene was identified and the transcript analysed. A 32P-labelled VZV gB DNA probe hybridized to a subclone of the SVV BamHI B restriction endonuclease fragment indicating the fine map position of SVV DNA sequences homologous to the VZV gB gene. The SVV gB DNA sequence was determined and analysis revealed a 2751 base pair open reading frame (ORF) with 71.1% identity to the VZV gB gene and 53.8% identity to the herpes simplex type 1 gB gene. The SVV gB ORF encodes a 916 amino acid polypeptide with a predicted molecular mass of 104K. The deduced SVV and VZV gB polypeptides share 78.9% amino acid identity and predicted N-linked glycosylation sites, cleavage sites and transmembrane regions. 32P-labelled SVV gB DNA and RNA probes hybridized to a 3.5 kilobase SVV polyadenylated transcript. Primer extension experiments identified transcript start sites for the SVV and VZV gB genes and permitted a comparison of the sequences upstream of the SVV and VZV gB ORFs. The SVV and VZV gB promoter elements are similar in content and align closely. The VZV gB transcript start site suggests a gB polypeptide initiation site which is inconsistent with the previously reported ATG start codon.

Amino Acid Sequence↗
PubMed1987

Immunological analysis of 140-kDa adenovirus-encoded DNA polymerase in adenovirus type 2-infected HeLa cells using antibodies raised against the protein expressed in Escherichia coli.

The E2B region of adenovirus genome contains a long open reading frame (ORF) extending from 24 to 14.2 map units which encodes most of the 140-kDa DNA polymerase. It was cloned at the polylinker region of pUC18 vector with Escherichia coli JM109 as the host. A clone was serendipitously isolated that expressed in E. coli a protein of approximately 120 kDa in size at high levels. DNA sequence analysis of this clone showed the presence of an in-frame fusion of a region, encoding 13 amino acids located upstream, to the first ATG of the ORF. Polyclonal antibodies raised against this protein purified from E. coli were used for immunological analysis. The antibodies were able to detect a 140- and a 66-kDa polypeptide from the adenovirus type 2-infected HeLa cells on Western blots. In addition, the antibodies showed evidence of cross-reactivity with partially purified DNA polymerase alpha from uninfected HeLa cells. The subcellular localization of the viral polymerase in the infected HeLa cells by using indirect immunofluorescence showed that the viral protein is associated with globular structures in the nucleus. The replicating viral DNA and the polymerase were colocalized in these globular sites. Furthermore, HeLa cells infected with Ad5ts149, a temperature-sensitive mutant defective in DNA replication, showed the presence of these globular sites only at the permissive temperature, suggesting that these sites are probably involved in viral DNA replication.

Adenoviruses, Human↗
PubMed1997

Identification of two sequence elements associated with the gene encoding the 24-kDa crystalline component in Bacillus thuringiensis ssp. fukuokaensis: an example of transposable element archaeology.

A 6.5-kb fragment of plasmid DNA from Bacillus thuringiensis (Bt) ssp. fukuokaensis that encodes a 24-kDa crystalline component was analyzed to identify additional open reading frames (orfs). A novel Bt IS240-like element was found upstream of this gene and is considered to be a vestige of a once active insertion sequence due to a stop codon that interrupts the long orf encoding the putative transposase. This element was bounded by 17-bp terminal inverted repeats that defined the length of the insertion sequence as 802 bp. Further upstream of this element two tandem overlapping and out of phase open reading frames (orfX and orfY) were identified which represent the first example of an IS150-like element in Bt containing both orfs. orfX and orfY are not bounded by terminal inverted repeats but are associated with a gene encoding a putative site-specific recombinase of a type found in Staphylococcus aureus Class II transposons but not previously in Bt.

Amino Acid Sequence↗
PubMed2006

Simian varicella virus gene 28 and 29 promoters share a common upstream stimulatory factor-binding site and are induced by IE62 transactivation.

Simian varicella virus (SVV) is a neurotropic alphaherpesvirus that causes a natural, varicella-like disease in non-human primates. After resolution of the primary disease, SVV, like its human counterpart, varicella-zoster virus (VZV), establishes latent infection in the neural ganglia of the host. In this study, gene expression of SVV open reading frames (ORFs) 28 and 29, which encode the viral DNA polymerase and DNA-binding protein, respectively, was characterized during lytic infection of Vero cells. The results indicate that the intergenic region controlling gene 28 and 29 expression includes overlapping, divergent promoters. The ORF 28 and 29 promoters are active in SVV-infected Vero cells, but not in uninfected cells. The SVV immediate-early gene 62 (IE62) product transactivates ORF 28 and 29 expression, and a cellular upstream stimulatory factor-binding site is important for efficient IE62 induction of genes 28 and 29. DNA sequence analysis of the 185 bp intergenic region identified putative cellular transcription factor-binding sites. Transcriptional analysis mapped ORF 28 and 29 RNA start sites. A recombinant SVV was employed to demonstrate that the ORF 29 promoter can express a heterologous gene (green fluorescent protein) when inserted into a novel site (the ORF 12/13 intergenic region) within the SVV genome. The findings demonstrate similarities between SVV and VZV ORF 28/29 expression and indicate that the simian varicella model may be useful to investigate the differential regulation of viral genes during lytic and latent infection.

Amino Acid Sequence↗
PubMed2002

Characterization of the I-Spom I endonuclease from fission yeast: insights into the evolution of a group I intron-encoded homing endonuclease.

The first group I intron in the cox1 gene (cox1I1b ) of the mitochondrial genome of the fission yeast Schizosaccharomyces pombe is a mobile DNA element. The mobility is dependent on an endonuclease protein that is encoded by an intronic open reading frame (ORF). The intron-encoded endonuclease is a typical member of the LAGLIDADG protein family of endonucleases with two consensus motifs. In addition to this, analysis of several intron mutants revealed that this protein is required for intron splicing. However, this protein is one of the few group I intron-encoded proteins that functions in RNA splicing simultaneously with its DNA endonuclease activity. We report here on the biochemical characterization of the endonuclease activity of this protein artificially expressed in Escherichia coli. Although the intronic ORF is expressed as a fusion protein with the upstream exon in vivo, the experiments showed that a truncated translation product consisting of the C-terminal 304 codons of the cox1I1b ORF restricted to loop 8 of the intron RNA secondary structure is sufficient for the specific endonuclease activity in vitro. Based on the results, we speculate on the evolution of site-specific homing endonucleases encoded by group I introns in eukaryotes.

Cloning, Molecular↗
PubMed1994

Analysis of duplicated gene sequences associated with tfdR and tfdS in Alcaligenes eutrophus JMP134.

Plasmid pJP4 of Alcaligenes eutrophus JMP134 encodes the degradation of 2,4-dichlorophenoxyacetic acid. A 1.2-kb BamHI-XhoI region of the restriction fragment BamHI-E has been proposed to contain the regulatory gene tfdR (A. R. Harker, R. H. Olsen, and R. J. Seidler, J. Bacteriol. 171:314-320, 1989; B. Kaphammer, J. J. Kukor, and R. H. Olsen, J. Bacteriol. 172:2280-2286, 1990). When sequenced and analyzed, the region is shown to contain two incomplete open reading frames (ORFs) positioned divergently. The complete DNA sequence for one of the two ORFs was obtained by sequencing the adjacent restriction fragment BamHI-F. The DNA sequence reveals 100% identify with the regulatory gene tfdS of pJP4. An XbaI-PstI fragment, containing the complete ORF, encodes a 32,000-Da protein which binds to the promoter regions upstream from tfdA and tfdDII. The deduced amino acid sequence of the complete ORF shows similarity with sequences of activator proteins TcbR, CatM, and CatR of the LysR family. The complete ORF represents the regulatory gene tfdR. The deduced amino acid sequence of the incomplete ORF, situated divergently from tfdR, indicates similarity to chloromuconate cycloisomerases produced by genes tfdD and tcbD of plasmids pJP4 and pP51, respectively. This ORF is identified as part of a putative isofunctional gene, tfdDII.

Alcaligenes↗
PubMed1998

Gene arrangement in the upstream region of Clostridium botulinum type E and Clostridium butyricum BL6340 progenitor toxin genes is different from that of other types.

The cluster of genes encoding the botulinum progenitor toxin and the upstream region including p21 and p47 were divided into three different gene arrangements (class I-III). To determine the gene similarity of the type E neurotoxin (BoNT/E) complex to other types, the gene organization in the upstream region of the nontoxic-nonhemagglutinin gene (ntnh) was investigated in chromosomal DNA from Clostridium botulinum type E strain Iwanai and C. butyricum strain BL6340. The gene cluster of type E progenitor toxin (Iwanai and BL6340) was similar to those of type F and type A (from infant botulism in Japan), but not to those of types A, B, and C. Though genes for the hemagglutinin component and P21 were not discovered, genes encoding P47, NTNH, and BoNT were found in type E strain Iwanai and C. butyricum strain BL6340. However, the genes of ORF-X1 (435 bp) and ORF-X2 (partially sequenced) were present just upstream of that of P47. The orientation of these genes was in inverted direction to that of p47. The gene cluster of type E progenitor toxin (Iwanai and BL6340) is, therefore, a specific arrangement (class IV) among the genes encoding components of the BoNT complex.

Botulinum Toxins↗
PubMed2004

A novel outer-membrane anion channel (porin) as part of a putatively two-component transport system for 4-toluenesulphonate in Comamonas testosteroni T-2.

Inducible mineralization of TSA (4-toluenesulphonate) by Comamonas testosteroni T-2 is initiated by a secondary transport system, followed by oxygenation and oxidation by TsaMBCD to 4-sulphobenzoate under the regulation of TsaR and TsaQ. Evidence is presented for a novel, presumably two-component transport system (TsaST). It is proposed that TsaT, an outer-membrane porin, formed an anion-selective channel that works in co-operation with the putative secondary transporter, TsaS, located in the inner membrane. tsaT was identified as a 1017-bp ORF (open reading frame) on plasmid pTSA upstream of the TSA-catabolic genes in the tsa operon. Expression of tsaT was regulated by TsaR, the transcriptional activator of the tsa regulon. The presence of tsaT was concomitant with the presence of the tsa operon in different TSA-degrading isolates. tsaT was expressed in Escherichia coli and was detected in the outer membrane. A 22-amino-acid leader peptide was identified. Purified protein reconstituted in lipid bilayer membranes formed anion-selective channels with a single-channel conductance of 3.5 nS in 1 M KCl. Downstream of tsaT, a constitutively expressed 720-bp ORF (tsaS) was identified. tsaS coded for a hydrophobic protein predicted to have six transmembrane helices and which is most likely localized in the cytoplasmic membrane. tsaS is adjacent to tsaT, but showed a different transcriptional profile.

Benzenesulfonates↗