High-resolution S1 Nuclease Mapping Research Articles

Identification of the σ⁷⁰-Dependent Promoter Controlling Expression of the ansPAB Operon of the Nitrogen-Fixing Bacterium Rhizobium etli.

The aim of the present work was to examine the putative promoter region of the operon ansPAB and to determine the general elements required for the regulation of transcriptional activity. The transcriptional start site of the ansPAB promoter was determined by using highresolution S1-nuclease mapping. Sequence analysis of this region showed -10 and -35 elements, which were consistent with consensus sequences for R. etli promoters that are recognized by the major form of RNA polymerase containing the σ(70) transcription factor. Mutation studies affecting several regions located upstream of the transcriptional start site confirmed the importance of these elements on transcriptional expression.

SabR enhances nikkomycin production via regulating the transcriptional level of sanG, a pathway-specific regulatory gene in Streptomyces ansochromogenes.

BackgroundsabR is a pleiotropic regulatory gene which has been shown to positively regulate the nikkomycin biosynthesis and negatively affect the sporulation of Streptomyces ansochromogenes. In this study, we investigate the mechanism of SabR on modulating nikkomycin production in Streptomyces ansochromogenes.ResultsThe transcription start point of sabR was determined by high-resolution S1 nuclease mapping and localized at the nucleotide T at position 37 bp upstream of the potential sabR translation start codon (GTG). Disruption of sabR enhanced its own transcription, but retarded the nikkomycin production. Over-expression of sabR enhanced nikkomycin biosynthesis in Streptomyces ansochromogenes. EMSA analysis showed that SabR bound to the upstream region of sanG, but it did not bind to the upstream region of its encoding gene (sabR), sanF and the intergenic region between sanN and sanO. DNase 1 footprinting assays showed that the SabR-binding site upstream of sanG was 5'-CTTTAAGTCACCTGGCTCATTCGCGTTCGCCCAGCT-3' which was designated as SARE. Deletion of SARE resulted in the delay of nikkomycin production that was similar to that of sabR disruption mutant.ConclusionsThese results indicated that SabR modulated nikkomycin biosynthesis as an enhancer via interaction with the promoter region of sanG, and expanded our understanding about regulatory cascade in nikkomycin biosynthesis.

Identification of promoters recognized by RNA polymerase containing Mycobacterium tuberculosis stress-response sigma factor σF

A previously optimized Escherichia coli two-plasmid system was used to identify Mycobacterium tuberculosis promoters recognized by RNA polymerase containing the M. tuberculosis stress response sigma factor sigma(F). The method allowed the identification of five new sigma(F)-dependent promoters. Transcriptional start points of the promoters were determined by high-resolution S1-nuclease mapping using RNA prepared from E. coli containing the two-plasmid system. The promoters were confirmed by an in vitro transcription assay. The Mycobacterium smegmatis and Mycobacterium tuberculosis core RNA polymerases, after complementation with sigma(F), were able to recognize all the five promoters. All the promoters contained sequences highly similar to the sequence of the previously identified M. tuberculosis sigma(F)-dependent promoter, usfXp1. Comparison of the promoters revealed a sigma(F) consensus sequence GtTtga-N(14-18)-GGGTAT. The sigma(F)-dependent promoters may govern expression of genes encoding a transcription regulator homologous to the response regulators of bacterial two-component signal transduction systems and proteins with unknown function.

Expression ofccaR, Encoding the Positive Activator of Cephamycin C and Clavulanic Acid Production inStreptomyces clavuligerus, Is Dependent onbldG

In Streptomyces coelicolor, bldG encodes a putative anti-anti-sigma factor that regulates both aerial hypha formation and antibiotic production, and a downstream transcriptionally linked open reading frame (orf3) encodes a putative anti-sigma factor protein. A cloned DNA fragment from Streptomyces clavuligerus contained an open reading frame that encoded a protein showing 92% identity to the S. coelicolor BldG protein and 91% identity to the BldG ortholog in Streptomyces avermitilis. Sequencing of the region downstream of bldG in S. clavuligerus revealed the presence of an open reading frame encoding a protein showing 72 and 69% identity to the ORF3 proteins in S. coelicolor and S. avermitilis, respectively. Northern analysis indicated that, as in S. coelicolor, the S. clavuligerus bldG gene is expressed as both a monocistronic and a polycistronic transcript, the latter including the downstream orf3 gene. High-resolution S1 nuclease mapping of S. clavuligerus bldG transcripts revealed the presence of three bldG-specific promoters, and analysis of expression of a bldGp-egfp reporter indicated that the bldG promoter is active at various stages of development and in both substrate and aerial hyphae. A bldG null mutant was defective in both morphological differentiation and in the production of secondary metabolites, such as cephamycin C, clavulanic acid, and the 5S clavams. This inability to produce cephamycin C and clavulanic acid was due to the absence of the CcaR transcriptional regulator, which controls the expression of biosynthetic genes for both secondary metabolites as well as the expression of a second regulator of clavulanic acid biosynthesis, ClaR. This makes bldG the first regulatory protein identified in S. clavuligerus that functions upstream of CcaR and ClaR in a regulatory cascade to control secondary metabolite production.

Characterization of the alternative sigma factor σG inStreptomyces coelicolor A3(2)

Using the previously established two-plasmid system for the identification of promoters recognized by a particular sigma factor, we identified two positive DNA fragments that were active only after induced sigG, encoding sigma factor sigmaG of Streptomyces coelicolor A3(2). High-resolution S1-nuclease mapping in the Escherichia coli two-plasmid system identified potential promoters, PG45 and PG54, whose sequences were similar to the consensus sequence of Bacillus subtilis promoters recognized by the general stress-response sigma factor sigmaB. However, both putative sigmaG-dependent promoters were not active in S. coelicolor. Sequence analysis of the regions potentially governed by the promoters revealed a gene encoding a hypothetical protein SCO5555 and the rrnE gene encoding rRNA operon. To confirm that sigG encodes sigma factor, the sigmaG protein was overproduced in E. coli and purified. In an in vitro transcription assay, sigmaG, after complementation with S. coelicolor core RNA polymerase, was able to recognize both sigmaG-dependent promoters and initiate transcription.

Response regulator ChiR regulates expression of chitinase gene, chiC, in Streptomyces coelicolor.

Transcription from the chiC promoter, directing expression of the chitinase gene, chiC, in Streptomyces coelicolor, was analyzed using xylE reporter gene and high-resolution S1-nuclease mapping. The transcription from the chiC promoter was induced by chitin, and this induction was dramatically reduced in the S. coelicolor chiR-disrupted strain. This indicated a dependence of chiC expression upon the chiR gene encoding a response regulator protein. To investigate this relationship, the S. coelicolor ChiR was overproduced using Escherichia coli T7 RNA polymerase expression system. However, gel mobility shift-assay with such a purified ChiR showed no binding in the chiC promoter region, which indicates a lack of specific phosphorylation of E. coli overproduced ChiR that is necessary for DNA-binding activity of response regulators.

AfsS is a target of AfsR, a transcriptional factor with ATPase activity that globally controls secondary metabolism in Streptomyces coelicolor A3(2).

AfsR is a pleiotropic, global regulator that controls the production of actinorhodin, undecylprodigiosin and calcium-dependent antibiotic in Streptomyces coelicolor A3(2). AfsR, with 993 amino acids, is phosphorylated on serine and threonine residues by a protein serine/threonine kinase AfsK and contains an OmpR-like DNA-binding fold at its N-terminal portion and A- and B-type nucleotide-binding motifs in the middle of the protein. The DNA-binding domain, in-dependently of the nucleotide-binding domain, contributed the binding of AfsR to the upstream region of afsS that locates immediately 3' to afsR and encodes a 63-amino-acid protein. No transcription of afsS in the DeltaafsR background and restoration of afsS transcription by afsR on a plasmid in the same genetic background indicated that afsR served as a transcriptional activator for afsS. Interestingly, the AfsR binding site overlapped the promoter of afsS, as determined by DNase I protection assay and high-resolution S1 nuclease mapping. The nucleotide-binding domain contributed distinct ATPase and GTPase activity. The phosphorylation of AfsR by AfsK greatly enhanced the DNA-binding activity and modulated the ATPase activity. The DNA-binding ability of AfsR was independent of the ATPase activity. However, the ATPase activity was essential for transcriptional activation of afsS, probably because the energy available from ATP hydrolysis is required for the isomerization of the closed complex between AfsR and RNA polymerase to a transcriptionally competent open complex. Thus, AfsR turns out to be a unique transcriptional factor, in that it is modular, in which DNA-binding and ATPase activities are physically separable, and the two functions are modulated by phosphorylation on serine and threonine residues.

Cloning of a two-component regulatory system probably involved in the regulation of chitinase in Streptomyces coelicolor A3(2).

Using the method for the identification of promoters recognized by the sporulation specific sigma factor (sigma F), we identified a positive 950 bp Sau3AI DNA fragment in Streptomyces coelicolor A3(2). High-resolution S1-nuclease mapping identified a potential promoter, PF35, in the E. coli two-plasmid system similar to the consensus sequence of Bacillus subtilis promoters recognized by the general stress-response sigma factor (sigma B). However, the putative sigF-dependent promoter, PF35, was inactive in S. coelicolor in the course of differentiation, and it was located divergently in the promoter region directing expression of the chiC gene encoding chitinase. Sequence analysis of the region potentially governed by PF35 revealed two translationally coupled genes encoding proteins similar to bacterial two-component regulatory systems, and with the highest similarity to the two-component system chiS, chiR, regulating chitinase activity in Streptomyces thermoviolaceus. However, the genes had a divergent orientation with respect to the PF35 promoter. Disruption of the S. coelicolor chiR gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin. Moreover, the chiR disruption did not affect the overall chitinase activity.

In vitro analysis of the butyrolactone autoregulator receptor protein (FarA) of Streptomyces lavendulae FRI-5 reveals that FarA acts as a DNA-binding transcriptional regulator that controls its own synthesis.

FarA of Streptomyces lavendulae FRI-5 is a specific receptor protein for IM-2, a butyrolactone autoregulator that controls the production of a blue pigment and the nucleoside antibiotics showdomycin and minimycin. Gel shift assays demonstrated that FarA binds to the farA upstream region and that this binding is abolished in the presence of IM-2. The FarA binding sequence was localized by DNase I footprinting to a 28-bp sequence located approximately 70 bp upstream of the farA translational start site. High-resolution S1 nuclease mapping of farA transcripts revealed a putative transcription start site, located at an A residue positioned 64 bp upstream from the farA translation start codon and 4 bp downstream from an Escherichia coli sigma(70)-like -10 recognition region. The FarA-binding sequence overlaps this -10 region and contains the farA transcription initiation site, suggesting that FarA acts as a repressor that, in the absence of IM-2, represses transcription of farA.

Characterization of binding sequences for butyrolactone autoregulator receptors in streptomycetes.

BarA of Streptomyces virginiae is a specific receptor protein for a member of butyrolactone autoregulators which binds to an upstream region of target genes to control transcription, leading to the production of the antibiotic virginiamycin M(1) and S. BarA-binding DNA sequences (BarA-responsive elements [BAREs]), to which BarA binds for transcriptional control, were restricted to 26 to 29-nucleotide (nt) sequences on barA and barB upstream regions by the surface plasmon resonance technique, gel shift assay, and DNase I footprint analysis. Two BAREs (BARE-1 and BARE-2) on the barB upstream region were located 57 to 29 bp (BARE-1) and 268 to 241 bp (BARE-2) upstream from the barB translational start codon. The BARE located on the barA upstream region (BARE-3) was found 101 to 76 bp upstream of the barA start codon. High-resolution S1 nuclease mapping analysis revealed that BARE-1 covered the barB transcription start site and BARE-3 covered an autoregulator-dependent transcription start site of the barA gene. Deletion and mutation analysis of BARE-2 demonstrated that at least a 19-nt sequence was required for sufficient BarA binding, and A or T residues at the edge as well as internal conserved nucleotides were indispensable. The identified binding sequences for autoregulator receptor proteins were found to be highly conserved among Streptomyces species.

The Streptomyces aureofaciens homologue of the sporulation gene whiH is dependent on rpoZ-encoded σ factor

Using the method for the identification of promoters recognized by a sporulation specific σ factor RpoZ, we identified a promoter in Streptomyces aureofaciens, directing expression of a gene having high sequence similarity (83% amino acid identity) to sporulation transcription factor WhiH of Streptomyces coelicolor. High-resolution S1-nuclease mapping using RNA prepared from S. aureofaciens from various developmental stages showed high similarity of P whiH promoter to the consensus sequence of flagellar and chemotaxis promoters. The promoter was induced at the time of aerial mycelium formation, and was off in S. aureofaciens strain with rpoZ-disrupted gene. The results suggest that the P whiH promoter is recognized by σ factor RpoZ in S. aureofaciens.

The bldD gene of Streptomyces coelicolor A3(2): a regulatory gene involved in morphogenesis and antibiotic production.

The bld mutants of Streptomyces coelicolor A3(2) are blocked at the earliest stage of sporulation, the formation of aerial hyphae, and are pleiotropically defective in antibiotic production. Using a phage library of wild-type S. coelicolor DNA, we isolated a recombinant phage which restored both sporulation and antibiotic production to strains carrying the single known bldD mutation. Nucleotide sequence analysis of a 1.3-kb complementing subclone identified an open reading frame, designated bldD, encoding a translation product of 167 amino acid residues. Nucleotide sequence analysis of the bldD-containing fragment amplified from the chromosome of a bldD mutant strain revealed a point mutation changing a tyrosine residue at amino acid position 62 to a cysteine. Although a comparison of the BldD sequence to known proteins in the databases failed to show any strong similarities, analysis of the BldD sequence for secondary structural elements did reveal a putative helix-turn-helix, DNA recognition element near the C terminus of the protein. A comparison of bldD transcript levels in the bldD+ and bldD mutant strains using both Northern blot analysis and S1 nuclease protection studies showed vast overexpression of bldD transcripts in the mutant, suggesting that BldD negatively regulates its own synthesis. High-resolution S1 nuclease mapping identified the transcription start point as a G residue 63 nucleotides upstream from the bldD start codon and 7 nucleotides downstream from -10 and -35 sequences resembling E. coli-like streptomycete promoters.

A method for the identification of promoters recognized by RNA polymerase containing a particular sigma factor: cloning of a developmentally regulated promoter and corresponding gene directed by the Streptomyces aureofaciens sigma factor RpoZ.

We have developed a method for the identification of promoters recognized by a particular sigma factor of RNA polymerase, based on a two-compatible plasmid system in Escherichia coli (Ec). Using the method, a DNA fragment containing the promoter, PREN40, recognized by sporulation-specific Streptomyces aureofaciens (Sa) sigma factor RpoZ, was cloned. High-resolution S1 nuclease mapping using RNA prepared from Ec, and Sa from various developmental stages has shown a high degree of similarity of PREN40 to consensus sequence of flagellar and chemotaxis promoters. The promoter was induced at the time of aerial mycelium formation, and was off in the Sa strain with the rpoZ-disrupted gene. A promoter-bearing DNA fragment was inserted into the promoter-probe plasmid pARC1 to give expression patterns consistent with the results of direct RNA analysis. The region downstream of the promoter was cloned in Sa. Sequence analysis revealed an open reading frame (ORF) of 283 amino acids (Mr 30006), encoding a highly basic (pI 12.35) protein with high percentage of serine, threonine and alanine (41.8%).

Transcription of the principal sigma-factor genes, rpoD and rpoS, in Pseudomonas aeruginosa is controlled according to the growth phase.

The rpoS gene encodes the second principal sigma factor of RNA polymerase in stationary-phase cells in Escherichia coli. We examined the transcription of Pseudomonas aeruginosa rpoS as to the growth of cells. The results of quantitative S1 nuclease mapping of rpoS and rpoD, encoding the principal sigma factor, indicated that the transcription of rpoS is induced in stationary-phase cells, whereas that of rpoD is induced in exponential-phase cells. By high-resolution S1 nuclease mapping, the 5'- and 3'-ends of rpoS mRNA were determined. The results indicated that rpoS is transcribed as a monocistronic mRNA. The sequence preceding the 5' end of rpoS mRNA showed poor homology to the consensus sequences of the previously known promoters. P. aeruginosa rpoS was not transcribed in E. coli. By in vitro transcription assaying, P. aeruginosa rpoS was shown to be transcribed by the RNA polymerase fraction containing the principal sigma (sigma 70)-RNA polymerase of P. aeruginosa.

Transcriptional analysis and heterologous expression of the gene encoding β-lactamase inhibitor protein (BLIP) from Streptomyces clavuligerus

Transcription of bli, the gene encoding beta-lactamase (Bla) inhibitor protein (BLIP) of Streptomyces clavuligerus, was analyzed by promoter-probe studies, Northern hybridization and high-resolution S1 nuclease mapping. The 1-kb SalI DNA fragment immediately upstream from the bli open reading frame (ORF) showed promoter activity when tested using the xylE-based promoter-probe vector, pIJ4083. The promoter activity was approx. 36-fold higher in S. clavuligerus than in S. lividans. Northern hybridization analysis of S. clavuligerus RNA revealed that bli was expressed as a 0.7-kb monocistronic transcript. High-resolution S1 nuclease mapping identified the transcription start point as an A residue 47 bp upstream from the bli start codon. When the bli ORF, along with 111 bp of upstream sequence including the promoter, was introduced into S. lividans, the transformants produced BLIP, but in amounts approx. 12-fold lower than that produced by S. clavuligerus. Involvement of some additional regulatory element that is present in S. clavuligerus, but absent in S. lividans, could explain the difference in the promoter activities and therefore the difference in the overall expression of bli in the two hosts.

Transcriptional mapping of the genes encoding the early enzymes of the cephamycin biosynthetic pathway of Streptomyces clavuligerus

Isopenicillin-N synthase (IPNS) of Streptomyces clavuligerus is encoded by the pcbC gene which is found within the cephamycin biosynthetic gene cluster. pcbC is located directly downstream from lat and pcbAB, which encode the enzymes, lysine ε-amino transferase and δ-( l-α-aminoadipyl)- l-cysteinyl- d-valine synthetase, respectively. These enzymes act prior to IPNS in the biosynthetic pathway, and the three genes are transcribed in the same direction. Previous pcbC transcriptional studies involving recombinant promoter probe plasmids. Northern analysis and 5' primer extension indicated the presence of a monocistronic 1.2-kb transcript that initiated within pcbAB, 92 -bp upstream from the pcbC start codon. S1 nuclease mapping studies have now shown, not only the transcript initiating 92 bp upstream from pcbC, but also a transcript initiating further upstream, possibly including the entire pcbAB gene. Promoter probe analysis and S1 nuclease mapping failed to detect promoter activity or a transcription start point ( tsp) directly upstream from pcbAB, suggesting that pcbAB transcripts initiated within or upstream from lat. Northern analysis, to search for a pcbAB transcript, showed no distinct transcript and indicated severely degraded mRNA. Similar results were obtained when Northern analysis was used to search for lat transcripts. Promoter probe analysis to locate the lat promoter indicated that a sequence promoting transcription was present in a 330-bp DNA fragment that extended from 227 -bp upstream from the lat structural gene to 103 bp inside the gene. The promoter showed modest activity in Streptomyces lividans, but had strong activity in S. clavuligerus. High-resolution S1 nuclease mapping of the 5' end of the lat transcript identified a T residue 88 -bp upstream from the lat start codon as the tsp. Comparisons of the S1 nuclease-protected DNA fragments generated using RNA isolated at various stages of growth indicated a temporally similar transcription pattern for all three genes. These data suggest that pcbC is transcribed as part of an operon together with pcbAB and lat, and also as a small monocistronic message.

Complex transcription of an operon encoding the SalI restriction-modification system of Streptomyces albus G.

High-resolution S1 nuclease mapping of mRNA synthesised in vivo, in vitro run-off transcription with RNA polymerase from Streptomyces lividans and gene fusions were used to analyse the transcriptional organization of the SalI restriction-modification system of Streptomyces albus G. The salIR and salIM genes that encode the restriction endonuclease and its cognate methyltransferase constitute an operon which is mainly transcribed from sal-pR1, a promoter located immediately upstream of salIR, with two possible minor promoters further upstream. Another promoter, sal-pM, is within the 3' end of the salIR coding region, and allows expression of the modification gene in the absence of sal-pR1. The sal-pM promoter might be involved in the establishment of modification prior to restriction endonuclease activity. Sequences upstream of the apparent transcriptional start sites for sal-pR1 and sal-pM show similarity with the -10 region of typical vegetatively expressed eubacterial promoters, but appropriately centered -35 regions are absent.

Transcriptional organization and regulation of the nosiheptide resistance gene in Streptomyces actuosus.

The nosiheptide resistance gene (nshR) and a putative regulatory gene (nshA) are found together on a 2326 bp BamHI-PstI DNA fragment isolated from Streptomyces actuosus ATCC 25421. The putative regulatory gene, nshA, situated upstream from the nosiheptide resistance gene in the 2326 bp DNA fragment, contains apparent DNA-binding and RNA-binding domains. Interruption of nshA in the chromosome of S. actuosus alters nosiheptide production, suggesting that nshA is involved in regulation of nosiheptide biosynthesis. Two transcription initiation sites were found upstream of nshA as demonstrated by high-resolution S1 nuclease mapping. A weak transcription start site for nshR was found which initiated transcription from the first nucleotide of the open reading frame. Although a stem-loop structure with apparent termination activity was found between nshA and nshR, readthrough of transcription between nshA and nshR was demonstrated by S1 nuclease mapping of the 3' terminus of the nshA transcript. Time-course S1 experiments of the three promoters (nshA-pl, nshA-p2, nshR-p) indicated highly regulated differential expression of the promoters. nshA-p2 is a strong, constitutive promoter whereas 30% of the total nshA-p1/p2 transcript reads through the terminator and into the nshR gene, accounting for more than half of the total steady-state nshR transcript. The implications of the regulation of nshA and nshR gene expression, as well as the expression of two other linked genes, are presented.

Cloning, nucleotide sequence, and transcriptional analysis of the NAD(P)-dependent cholesterol dehydrogenase gene from a Nocardia sp. and its hyperexpression in Streptomyces spp

NAD(P)-dependent cholesterol dehydrogenases [NAD(P)-CDH], which allow easier quantification of cholesterol by means of directly measuring the A340 of NAD(P)H, are useful for clinical purposes. The amino acid sequences of the NH2 terminus and the fragments obtained by CNBr decomposition of the NAD(P)-CDH from a Nocardia sp. were determined for preparation of synthetic oligonucleotides as hybridization probes. A 4.4-kbp BamHI fragment hybridizing to these probes was cloned on pUC19 in Escherichia coli. The nucleotide sequence together with the determined amino acid sequences revealed that this enzyme consists of 364 amino acids (Mr, 39,792) and contains an NAD(P)-binding consensus sequence at its NH2-terminal portion. High-resolution S1 nuclease mapping suggested that in NAD(P)-CDH of both Nocardia and Streptomyces spp. transcription initiates at the adenine residue, which is the first position of the translational initiation triplet (AUG) of this protein. The S1 mapping experiments also showed that cholesterol-dependent regulation in the Nocardia sp. occurred at the level of transcription. In Streptomyces lividans containing the cloned fragment, however, this promoter was expressed constitutively. DNA manipulation of the cloned gene in E. coli, including the generation of a ribosome-binding sequence at an appropriate position by oligonucleotide-directed mutagenesis, led to production of this protein in a very large amount but in the enzymatically inactive form of inclusion bodies. On the other hand, a Streptomyces host-vector system was successfully used for producing 40 times as much enzymatically active NAD(P)-CDH as that produced by the original Nocardia sp.

Nucleotide sequence and transcriptional analysis of the nosiheptide-resistance gene from Streptomyces actuosus

The nucleotide (nt) sequence of a 2326-bp BamHI- PstI DNA fragment previously isolated from Streptomyces actuosus ATCC25421 that confers resistance to the thiopeptide antibiotics, nosiheptide (Nh) and thiostrepton (Ts) upon Streptomyces lividans 1326 was determined. Two open reading frames (ORFs) were found in this 2326-bp sequence; one containing 699 nt and another of 822 nt, both reading in the same direction. The Nh-resistance gene determinant ( nsh) is encoded by orf822, as determined by the 74% identity of the deduced amino acid sequence of its gene product to that of the 23S rRNA methylase encoded by the Ts-resistance gene ( tsr) of Streptomyces azureus. (The respective sequences had a 72% homology.) ORF699, encoded by a gene situated upstream from orf822, contained an apparent α-helix-ß-turn-α-helix configuration which is common to DNA-binding proteins and suggests that ORF699 may be a regulatory protein. Two transcription start points ( tsp) were found upstream from orf699 as demonstrated by high-resolution S1 nuclease mapping. There was also a weak tsp for the nsh gene at the first nt of ORF. Moreover, transcription was observed to read through a stem-loop structure separating the orf699 and nsh genes, as demonstrated by S1 nuclease mapping of the 3′ terminus of the orf699 gene, suggesting an antitermination mechanism for regulation of nsh transcription.