Gene, 15 (1981) 343-347 Elsevier/North-HollandBiomedicalPress
The promoter-proximal region of the BaciUuslicheniformis penicillinase gene: Nucleotide sequence and predicted leader peptide sequence (B. subtilis; prepenicillinase; secretory protein;ribosome binding site, signal peptide)
James Kroyer and Shing Chang
Cetus Corporation. Berkeley, CA 94 710 (USA)
(ReceivedJune 2nd, 1981) (AcceptedJune 16th, 1981)
Penicillinase (j~.lactamase) is a major species of secreted protein produced by Bacillus licheniformis 749. From the pTB2 recombinant plasmid containing the cloned entire penicillinase (penP) gene, we have isolated and sequenced a 446.bp Hpall fragment carrying the beginning of penP. The 3°-end coding region of 216-bp on this DNA fragment codes for the t3rst 72 amino a~ids of the prepeniciUinase pzotein. The deduced structure of the leader peptide consists of a 34 amino acid signal sequence with a hydrophilic N-terminal region and a central hydrophobic core.
INTRODUCTION Bacillus licheniformis 749/C strain produces a membrane.bound prepenicillinase which is the secretory precursor of the mature penic'dlinase. Followin8 protein synthesis and subsequent translocation across the membrane, the N.terminal leader peptide of the p r e p e n i ~ is processed away by proteolyric cleavage, thus releasing the mature penicillinase into the medium (Sargent et al., 1968). The initial stable form of the secreted penicilUnase has the N-te~ sequence asSer,GIn,Pro-Ala-Glu-Lys-AsnGlu-,.. (lzui et al,, 1980; Simons et aL, 1978). A
Abbreviations: bp, base pairs; f-Met, formyl-methionine;kb, kilob&,~$.
smaller form of the penicillinase protein is also present in the culture medium; it differs from the larger form by the absence of the first eight amino acids (Ambler and Meadway, 1969). Partial information has been obtained on the s~ructure of the leader pep. tide recently; Lampen et al. (1980) have determined that the precursor molecule is a 35 000-dalton hydrophobic protein containing f-Met at the N-terminal end, and four lysine residues within the first twelve amino acids. The structural gene (penP) for the perdc'dlinase has been cloned in E. coil using both phage(Brammar et al., 1980) and plasmid (Gray and Chang, 1981) vectors. The penP gene was found to be expressed and the precursor processed in both E. coil and B. subHiishosts invivo ((;my and Chang, 1981). With subsequent manipulation of the pTB2 recombinant
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344 plasmid containing the cloned perd' gene, we have sabcloned a 1.35-kb fragment which contains the entire pen/' coding sequence as well as the adjacent noncoding regions, starting from the 5'.Hpall site to the 3'-end AsuI site as shown in Fig. 2. Since E. coli and B. subdlis cells harboring plasmids containing the 1=35 kb fragment express the penP gene (S.Y. Cb.~g and S. Chang, unpublished results), it appears the 446.bp HpalI fragment (see Fig. 2)contains promoter(s) which functions in these hosts. As revealed by sequence analyses, this HpaH fragment also contains the coding sequence for the first 72 amino acids. We report here the primary structure of the regulatory sequences for the penP gene as well as the deduced leader peptide sequence of the prepenicillinase.
MATERIALS AND METHODS
tively. Terminal transferase (for 3'-end labeling with cordycepin) was obtained from either Bethesda Research Laboratories (ribosubstitufion grade) or Retlift Biochemicals (Los Alamos, New Mexico). 3'-End labeling with cordycepin-5"triphosphate was accomplished using the procedure of Tu and Cohen (1980). AMV reverse transcriptase (for repair labeling)was kindly provided by Dr. J. Beard.
(c) DNA sequence dete~mination Determination of the DNA sequence was carded out utilizing the chemical cleavage method of Maxam and Gilbert (1980). Sequencing gels were run using 8%, 15% or 20% polyacrylamide concentrations (20 : 1 ratio of acrylamide : bisacrylamide). Autoradiograt~hy was performed at either -20°C or -80°C employing Kodak XR-5, XRP-5 or AR-5 X-ray film. The DNA sequence of both strands of the fragment from pTB2 were independently determined.
(a) Plasmids and bacterial strains Plasmid DNA was prepared from E. coli strain CS412 (Gray and Chang, 1981). Broth and afar plate media, growth conditions, preparation of plasmid DNA and transformation using plasmid DNA have been described previously (Lennox, 1955; Kupersztoch et al., 1973" Gray and Chang, 1981; Chang and Cohen, 1974; 1979). DNA sequence data were obtained from the recombinant plasmid pTB2 containing the cloned penicfllinase gene (Gray and Chang, 1981).
(b) Restriction endonuclease analyses and DNAfragment labeling All restriction endonucleases were purchased from New England Biolabs and were used according to the supplier.specified conditions. Digested fragments for I~4A sequence analyses were isolated after eiectrophoresis in 4% polyacrylamide gels by the electroelution method described by Smith (! 980). [a-a2P]Nucleotide triphosphates (dATP, dGTP, dCTP, dTTP), and [¢~-a2P]cordycepin-5'-triphosphate were purchased from New England Nuclear. Calf intestinal alkaline phosphatase and T4 polynucleotide kinase (for 5'-end labeling) were purchased from Boehringer Mannheim and Biogenics Inc., respec-
RESULTS AND DISCUSSION
(a) Se~aence of the coding region Tae nucleotide sequence of the 446.bp Hpall fragment of recombinant plasmid pTB2 is shown in Fig. 1. The deduced sequence of 72 amino acids encoded by the longest open reading frame is aLsoindicated. Amino acids 43 to 72 are identical to the first 30 residues of the smaller mature penic'dlinase as reported by Ambler and Meadway (1969). The larger penicillinase contains eight additional residues (lzui et al., 1980; Simons et al., 1978), which also agrees with our data (positions 35-42, Fig. 1). The first ATG codon in phase with this assigned reading frame is assumed to be the initiation codon. This gives a segment containing 34 residues at the N.terminal of the precursor molecule as the leader peptide. Recent work by Lampen et ai. (1980) and J.B.K. Nielsen, C.N. Chang, J.O. Lampen and G. Blobel (personal communication) indicates that the prepenicillinase protein starts with formyl-methionine and has the next methionine at position 46; furthermore, the first six lysines are located at positions 2, 9, 11, 12, 40 and 43. Their findings matca precisely with our DNA sequencing data, therefore confirming the pre-
345 TGGAAACGAG GTCATCATTT CCTTCCGAAA AAACGGTTGC ATTTAAATCT
Hinfl TACATATGTA ATACTTTCAA AGACTACATT TGTAAGATTT GATGTTTGAG
TCGGCTGAAA GATCGTACGT ACCAATTATT GTTTCGTGAT TGTTCAAGCC
Mbol ATAACACTGT AGGGATAGTG GAAAGAGTGC TTCATCTGGT TACGATCAAT
C A A A T A T T C A A A C G G A G G G A G A C G A T T T T G A T G AAA T T A TGG T T C A G T
Pstl I 5 A C T T T A AAA C T G AAA AAG G e T G C A GCA G T G T T G C T T T T C T C T T G C
10 15 20 G T C G C G C T T G C A GGA TGC G e T AAC AAT CAA ACG AAT G e e T C G C A A Val Ala Leu Ala Gl'v Cys Ala Ash Ash Gin Thr Ash A/a Ser Gin 25 30 35 C C T G C C GAG A A G A A T GAA AAG ACG GAG ATG AAA GAT G A T T T T G C A Pro
45 50 A A A C T T GAG G A A C A A T T T G A T G C A AAA C T C GGG A T C T T T G C A T T G 55 G A T A C A GGT A C A AAC CGG
,~ ~s~ 4> 44~
7O Fig. 1. DNA sequence of the Hpall fragment of the pTB2 recombinant plasmid. Nucleotides ate numbered at the end of each line. A few selected restriction enzyme sites ate shown. The positions for amino acids in the prepenicillinase are numbered below the corresponding residues. The largest mature penici/~inase protein starts at amino-acid position 35 (Ser-Gln-Pro-Ala.Glu-...), A smaller form of penicillinase starts at araino-acid position 43.
i Bglll Avail I Asul
co,, s=,, "F
, ,..... - .....
Hpall I I
Hinfl Sau3A Sau3A 1I I I
FnuDII I I
] Hpall ,!1 i
Fig. 2. Physical maps of the penP region. (a) The locations of various resUiction sites on the B. lichenifo~nis EcoRl fragment of plasmid pTB2. (b) Enlarged map of the Hpall fragment. Restriction sites were determined initially by enzyme digestions and then ¢onfi-, qed b y sequencing analysis (see Fig. 1). (c) The position of the coding region for the ,nenP gene. Open box represents the deduced leader peptide sequence. Hatched azea indicates the sequence of the mature peniciIlinase protein (see text and Fig. 1).
dicted structure for the leader peptide reported here. The leader peptide of the prepeniciltinase shares several common features with that of tl~ secretory proteins (for a recent review, see Steiner et al., 1980). Iz has a stretch of internal hydrophobic residues that can form a 0-pleated sheet. This region presumably interacts with membrane and transmembrane proteins. The N-terminal portion of the leader peptide ~s more polar and is l o o t e d within the cystosol. One unique feature is that the leng~ of this leader peptide is longer than that of other reported secretory pro. teins. Whether this has any functional significance is not clear.
Neugebauer et al. (1981) have r~cenfly reported the nucleotide sequence of the penP gene.
ACKNOWLEDGMENTS We would like to acknowledge Drs. C. Woese, H. Noller, J.B.K. Niel~on, C.N. Chang, J.O. Lampen, G. Blobel and R. Doi and his associates for communicating experimental data before publication. We thank E. Jarvis for excellent secretarial assistance.
(b) Sequence of the noncoding region Within the region immediately preceding the initiation codon for the penP gene, we can identify potential Shine-Dalgarno sequences G.G.A-G-G (positions 214-218) and CJ-G-A-G.a-c-G-A-T (positions 218-226; the lowercase letters intricate ntis. matched nucleotides). C. Woese and H. NoUer (personal communication) have determined *,he 3'-end of the 16S rRNA of B. subtilis as well as of ~everal ether Bacilli, to be 5'.G.A-U-C-A-C-C-U-C-C-UU-U-C-UoH. It differs from the corresponding E. coil sequence 5'.G-A-U-C-A-C-C-U-C-C-U-U-AoH by only the terminal residues (Shine and Dalgarno, 1974). There¢ore, one or both of these sequences on mRNA could serve as ribosome.binding site in these two" diverse bacterial species. Using restricted DNA fragments as template, 1~ Doi, T. Kudo and J. McLaughlin (persona~ ,=3mmunication) analyzed run-off RNAs made in vitro by both B. subtilis and E. coil RNA polymerases. They obtained similar sizes of transcripts using these polymerases. This suggests that the promoter sequences recognized by E. coil and B. mbtilis overlap or coincide with each other. Their preliminary data also in~cates that the promoter :3n the 446-bp HpalI fragment is located around position 100. The stretch of 50-bp sequence at positions 41.o0 is very rich in dA:dT pairs (80%). Within this region, an inverted repeat sequence can be found centered around the C at position 68, which could contain regulatory sequences for the penP gene. We are currently in~,estigating the possible function of this reL$on by generating specific deletions X, vitro.
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