Expression of the E.coli hemolysin secretion gene WyB involves transcript anti-termination within the hty operon

The genes hty A and hly B dictating synthesis and secretion of hemolytic toxin by Escherichia coll are transcribed as part of an operon hly C.hly A.hly B but are separated by an inverted repeat and poly-T sequence characteristic of a rho-independent terminator. The hly A-hly B intergenic sequence caused a reduction of In vivo transcripttonal read-through from the gal promoter Into gal K when Inserted In the pKG190O terminator-probe vector and also In vitro 3' to the bacteriophage T7 a10 promotor. Hybridisation of mRNA generated by the hly determinant of recomblnant DNA pBR202-312 to an antlsense RNA probe spanning the hly Intergenlc sequence revealed specific termination of 80% of In vivo hly transcripts within the poly-T sequence, immediately proceeding the hly B translation start. Extension of the hly determinant with 3.5kbp of hty promotor-proximal DNA sequence raised Intracellular and cell-free hemolytic activity 3-fold and 20-fold respectively and Increased markedly the secretion of the 107Kd hemolysin protein (HlyA). Parallel hybridisation of In vivo mRNA to hly A and hlyB antlsense probes revealed that levels of hly A and hlyB mRNA were Increased by approximately 3-fold and 90-fold. The dramatically enhanced level of hly B mRNA resulted primarily from specific suppression of transcription termination at the intergenlc rho-lndependent terminator from 80% to 1%, thus allowing virtually all hly A transcripts to elongate into hly B. INTRODUCTION The Escherichia coll hemotysln (hly) determinant encodes the synthesis of cytotoxlc hemolysin and its secretion across both bacterial membranes without the aid of an N-terminal translocation signal or peptide cleavage (1,2). The genes for the synthesis (hly A) and cytoplasmic posttranslational activation (hly C) of cytotoxlc hemolysin have been shown to be transcrfoed in an operon together with one of the 2 contiguous specific secretion genes, hly B (3,4,5). This gene encodes HlyB, a putative ATP-bindlng membrane protein analogue of the mammalian Pglycoprotein (6,7), which together with a second membrane protein HlyD directs the complete export of hemolysin Into the medium (8,9,10). Transcription of the conserved hemolysin genes occurs in the order hly C.hly A, hly B from a promotor region proceeding hly C (3,11,12, fig. 1) but transcription of the synthesis and secretion genes is subject to pronounced operon polarity; the HlyB membrane protein Is made at relatively low levels (11,13) and transcripttonal activity measured by 6-gal fusions is markedly lower within the hly B gene (4,5). In this report we demonstrate that strong operon polarity is caused by termination of hly transcription between the genes hly A and hly B, imposed specifically by an inverted repeat and poly-T sequence characteristic of a rho-lndependent terminator. We also show © IRL Prea Limited, Oxford, England. 4 7 8 9 Nucleic Acids Research that in the presence of DNA proximal to the hly operon promotor region, efficient expression of tho hly 8 secretion gene is effected preferentially by the specific and strong suppression of transcription termination at this Intergenic rho-independent terminator. MATERIALS AND METHODS Bacterial strains and plasmids. E.coll strains HB101 and 5KC (rec A1 hsd R hsd D) were used in this study. Plasmld pBR202-312 was constructed from pBR322 and pANN202-312, which contains the E.coll hly determinant of the wild-type plasmld pHly152 (10,14); pEK50 contains the same hly determinant on a Sal I fragment of pHly152 but contains an additional ca.3460bp of hly promotor-proximal DNA, analogous to the plasmld described by Zabala et al. (15). Plasmids pKG19-t(+) and pKG19-t(-) were constructed by cloning the pBR202-312 477bp Pvu Il-Eco RV restriction fragment, which contains the hly A-hly B Intergenic region, into the Sma I site of terminator-probe vector pKG1900 (16) between the E.coll gal promoter and the galK gene. The orientation of the inserted fragment is Indicated by (+) or (-). Recombinant plasmids used for the preparation of anti-sense RNA probes were derived from the Genescribe vectors pT7-1 and pT7-2 (US Biochem. Corp.), which utilise the specific In vitro activation of the strong class III T7 bacteriophage 010 promotor by T7 RNA polymerase (a kind gift of Bill Studier, Brookhaven). The 477bp Pvu Il-Eco RV fragment spanning the Intergenic region was cloned in the correct and reverse orientations Into the Sma I site of pT7-2 to give respectively pT7-t(+) and pT7-t(-). The hly A-internal 812bp Hln dill -Eco Rl fragment was cloned into the corresponding sites of pT7-1 to give pT7-hly A and the 799bp Eco RV-Ps/1 fragment was cloned Into the polylinker Sma \-Pst I sites of pT7-2 to give pT7-hly B. The control measurement of bla transcript levels was made using antlsense RNA generated from pT7-bla, pT7-2 carrying the bla -Internal 296bp Hlnc\\-Pst I fragment. Assay o( hamorysln SBcrntlon. (a) Hemolytlc activity was measured during batch growth of E.coll 5KC transformants at 37°C, shaking In Brain Heart Infusion broth containing 50jig/ml amplclllln. Aliquots of culture were removed at Intervals during exponential growth (A 500 0.2-0.85) and assayed for both intracellular and cell-free secreted activity In, respectively, a cleared cell extract of bacterial cells lysed by lysozyme treatment and supernatant from a cell-free centrifuged culture. Lysozyme treatment entailed resuspending pelleted washed cells from 1ml. of culture in 35u.l. buffer (25mM HEPES-KOH, pHS.O, 5mM EGTA, imM DTT), freezing In liquid nitrogen, thawing at 10°C, then Incubating with 3uJ 2M KCI and 1u.g/u.l lysozyme at 0°C. Samples were refrozen and thawed, then diluted in HEPES buffer to the original culture volume. Samples were incubated for 15-30 mln. at 40°C in a 2% suspension of erythrocytes In 0.85% NaCI, 20mM CaCI2 after which hemoglobin released was measured at A54J . Activity was calculated as arbitrary hemolytlc units (1 unit causing release of 50mg of hemoglobin In 1 hr. of the standard test). (b) The extracellular presence of the previously well-characterised 110KDa hemolysln protein