Nucleotide sequence analysis and expression studies of a chloramphenicol-acetyltransferase-coding gene from Clostridium perfringens

349

Gene, 75 (1989) 349-354 Elsevier GEN 02875

Nucleotide sequence analysis and expression studies of a chloramphenicol-acetyltransferase-coding gene from Clostridium perfringens (Bacillus subtilis; Escherichia coli; promoter probing; transcription initiation)

Cornelia Steffen and Hans Matzura MolekulareGenetik der Universitiit Heidelberg, O-6900 Heidelberg (F.R.G.) Received by T.A. Bickle: 16 September 1988 Revised: 7 October 1988 Accepted: 10 October 1988

SUMMARY

The nucleotide sequence of a CmR determinant, located on the Clostridium perfrinseensplasmid pIP401, was determined and its gene product was identified as chloramphenicol acetyltransferase (CAT). The cat structural gene is preceded by transcription-initiation signals characteristic for Escherichia coli 0” or Bacillus subtilis aA promoters. By promoter probing in the heterologous hosts the direction of transcription of the clostridial cat gene was analysed and the cat mRNA start point was dete~ned in vitro using the RNA polymerases of E. coli and 3. subtilis. Comparison of the amino acid sequences of C. perfringeens CAT and other CAT proteins of Gram-positive and Gram-negative origin shows a remarkable degree of homology between the various enzymes.

INTRODUCTION

Among the various mechanisms which confer Cm resistance to bacteria, the inactivation of the antibiotic by acetylation is best understood. This acetylation Correspondence fo: Dr. H. Matzura, Molekulare Genetik, Im Neuenheimer Feld 230, D-6900 Heidelberg (F.R.G.) Tel. (06221)562676. Abbreviations: aa, amino acid(s); Ap, ampicillin; bp, base pair(s); CAT, Cm acetyltransferase; cat, gene coding for CAT; Cm, chloramphenicol; kb, 1000 bp; Km, kanamycin; nt, nucleotide(s); ORF, open reading frame; R, resistance, resistant; SD, Shine-Dalgarno; Tn, transposon.

is performed by the enzyme CAT, which acts as a trimer of identical subunits (Harding et al., 1987). The cut gene has been located on various bacterial chromosomes and antibiotic-resistant plasmids and is sometimes contained within a transposable element. The expression of the cat gene can be constitutive, especially if it is of Gram-negative origin, or inducible by subinhibitory concentrations of the drug, mainly in Gram-positive bacteria (Shaw, 1983). Cm resistance has also been described for the obligate anaerobic, sporulating and pathogenic Gram-positive C. perj%ge?zs. The CmR determinant has been located on the transferable antibiotic-

0378-l 119/89/$03.50 0 1989 Elsevier Science Publishers B.V. (Biomedical Division)

350

resistant plasmid pIP401 (Sebald and Brefort, 1975) within the 6.2-kb transposable element Tn4451 (Abraham and Rood, 1987). Recently, Clostridia have become of considerable interest for biotechnological exploitation, and chimaeric vectors have been constructed for use in and transfer between Clostridia and E. coli or B. subtilis (Squires et al., 1984; Collins et al., 1985). Because of the potential use of the clostridial CmR determinant as a genetic marker in such vectors, we have determined its nucleotide sequence. The gene product was identified as CAT both by enzyme assays and amino acid sequence comparison with other known CAT enzymes of Gram-negative and Gram-positive origin. Deviating from the general rule for the regulation of cat genes of Gram-positive origin, the clostridial CAT conferred high-level constitutive Cm resistance to E. coli, but it could not be expressed in a common B. subtilis strain.

EXPERIMENTAL

AND

DISCUSSION

(a) Nucleotide sequence and transcriptional analysis The CmR determinant of PIP401 had been located by BAL 31 deletion analysis and cloned on an approx. 1.5-kb SmaI-Hind111 fragment in the E. coli vector pUC18 to yield the 4.2-kb plasmid pJIR62, which conferred Cm resistance to E. coli (Abraham et al., 1985). From this plasmid an EcoRI-Hind111 fragment was isolated and its nucleotide sequence determined. Fig. 1 shows the sequence of the 1398 bp fragment not including the linker regions and the restriction sites for EcoRI at the 5’ and for Hind111 at the 3’ ends. It exhibits a relatively high A + T content of 64%, as is common for Clostridia, and contains an ORF for a polypeptide of 212 aa with homology to other CAT monomers (cf. Fig. 3). That the sequence codes for CAT was confirmed by the spectrophotometric CAT assay (Shaw, 1975). The A4, of the monomer was calculated to be 24 807. The ORF is preceded by a SD sequence GAGAGG and a promoter region with a TATAAT -10 and a TTGAAA -35 box, the distance between them being 17 bp. These parameters are characteristic for E. coli cr70and B. subtilis d3 promoters (Moran et al., 1982) and show some homology to other known clostridial promoters (Young et al., 1986).

To verify the function of these transcriptional start signals and to determine the exact mRNA start point, two types of experiments were carried out. First, the EcoRI-EcoRV fragment from nt position l-250 was cloned by means of EcoRI linkers in the polylinker region of the promoter probe vector pRB394. Plasmid pRB394 has been constructed from parts of the E. coli vector pBR322, of the Staphylococcus aureus plasmid pUBll0 and the structural cat gene from another S. aureus plasmid, PUB 112 (Bruckner et al., 1984); in front of this cat gene a polylinker region has been inserted (R. Bruckner, in preparation). pRB394 replicates both in E. coli and B. subtilis and confers Ap resistance to the first and Km resistance to the second host. Cm resistance is expressed only when a promoter sequence is cloned in the correct orientation in front of the PUB 112 cat gene in both hosts. After transformation of E. coli strain JMlOl with pRB394 carrying the EcoRI-EcoRV promoter fragment, some CmR colonies were obtained. Plasmids isolated from such transformants were shown by nucleotide sequencing to contain the promoter region in the 5’ to 3’ direction, as drawn in Fig. 1. Thus, the transcription initiation signals of the CmR gene of the clostridial plasmid pIP401 are active in E. coli. To confirm the direction of transcription and to find out the cat mRNA start point, in vitro transcription was performed using E. coli a7’ and B. subtilis d3 RNA polymerases and, as templates, DNA fragments including the promoter region: the EcoRIEcoRV fragment, the EcoRI-DraI and the EcoRIHinfl fragment (Fig. 1). Run-off transcripts of approximately 150, 360 and 530 nt, respectively, were obtained with both polymerases, pointing to mRNA start point between nt positions 100 and 110 (not shown). The exact start point was then determined by priming the transcription reaction with dinucleotides according to Downey et al. (1971). As shown in Fig. 2, the expected run-off transcripts were only found using GpU and UpG as primers indicating that the mRNA starts with a G or U. Finally, the synthesis of the 147-nt long run-off RNA at nt concentrations (5 PM) below that required for initiation of RNA synthesis could only be stimulated by the addition of high concentrations (250 PM) of GTP (Fig. 2). Thus, the mRNA of the clostridial cat gene is initiated in vitro with the G at nt position 104, 27 nt upstream from the SD sequence (Fig. 1).

351

266

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1130 sau 3AIHpaII PC-T 1202 ~~~~~~ 1274 Hpa II mvm:awm:p -1346 'I.'--

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Fig. I. Nucleotide sequence of the DNA fragment carrying the cat gene of the C. perfringerrr plasmid pIP401 determined from the cloned ~coRI-~~~dIII fragment {see section a). Overlapping sequencing of both strands was performed by the dideoxy chain-te~ination method (Sanger et al., 1977) after cloning the foliowing fragments in pUClg plasmid or M13mp9/18 phage DNA (Messing, 1983): EcoRI-Hind111 (total fragment), EcoRV-HindIII, DruI-HindIII, EcoRI-Sau3AI and three EcoRI-?&I fragments. Sequencing reactions were analysed by electrophoresis on 6% or 8% polyacrylamide/8 M urea gels. Initiation signals (-35, -10, SD), car mRNA start point (tail of the arrow), and restriction sites are indicated.

(b) Amino acid sequence

The comparison of the amino acid sequence of the C. peftingens CAT monomer with other known CAT sequences of Gram-positive and Gram-negative origin demonstrates a remarkable degree of homogeneity (Fig. 3). Certain positions are conserved in all CAT monomers, especially the sequence QXHHXV~DGXH close to the C terminus which is part of the active center of the enzyme (Shaw, 1983; Leslie et al., 1988). When we consider the overall homology between the clostridial and the other CAT amino acid sequences on the basis of identical aa, then it varies from 41.93 % for the Proteus mirabilis CAT to 44.65% for the pC223 CAT. Considering amino acids of the same chemical character, the best homology is exhibited with the CAT sequence of the S. aureus plasmid pC194 (71.3 %). An analogous result has been obtained by a computer program in which 10 aa of the clostridial CAT have been compared at a time with 10 aa of one of the others (DOT program, German Cancer Research Center, Heidelberg, 1985). Thus, the CAT sequence of the C. perfrigens plasmid pIP401 exhibits a higher degree of homolo~ to those of the S. au;eus CmR plasmids than to CAT monomers of either B. pumilus, P. mirabilis or E. coli. (c) The cut expression in Eschericfriu coli and Bucillus s~b?ilis

ster 10, tils rp, to CC-

TP

on :G

As mentioned before, CAT enzyme assays showed that the clostridial cat gene can be expressed in E. coli. However, all attempts to express it in B. subtifis failed. When the total gene on the Ec~RI-~i~dII1 fragment was cloned in the shuttle vector pRB373, a derivative of the bifunctional plasmid pRB273 (Bruckner et al., 1984; R. Bruckner, in preparation), E. co/i strain JMlOl could be transformed by the new plasmid to high-level Cm resistance, but no tr~sfo~~ts of B. s#~tiZisBD170 could be obtamed. This failure is not due to a lack of function of the clostridial cat promoter region in the Grampositive host because promoter activity could be detected in B. subtilis on the EcoRI-EcoRV fragment in the promoter probe vector pRB394. Unlike the usual inducibility of cat genes from Gram-positive organisms by subinhibitory concentrations of the antibiotic, CAT was always produced

353 E.coli Tn9 P_.mirabilis C_.perfrinoens B _.pumilus m S.aureus PC194 s_.aureus PC221 S.aureus pUB112 S.aureus pC223

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E.coli Tn9_ P_.mirabilis C_.perfrinaens B_.pumilus cg&& S.+ureus PC194 S.aureus PC221 S_.aureuq pUBl12 S.aureus pC223

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Fig. 3. Comparison references

of amino acid sequences

were used: E. coli Tn9 (Marcoli

from various

(Harwood

et al., 1983)

(Bruckner

et al., 1985), S. aureus pC223 (Zyprian,

of the eight sequences;

S. aureus pC194 (Horinouchi + ,identical

CAT monomers

of Gram-positive

et al., 1980), P. mirubilis (Charles and Weisblum,

1987). Designations

aa in all CAT monomers

and Gram-negative

(219) (213) (212) (216) (215) (215) (215)

origin. The following

et al., 1985), C. perjZngens (see Fig. 1), B. pumiluF cat86

1982), S. aureus pC221 (Shaw (see bottom

of Gram-positive

et al., 1985), S. aureus pUB112

lines): i, identical

origin. Amino

aa; o, identical

acid sequences

aa in seven out

were aligned

according

to best fit

constitutively in E. coli from the clostridial cat gene on pRB373 or pUC18. The expression of inducible Gram-positive cut genes depends on the translation of a short leader sequence coding for a peptide of 9 aa preceding the structural gene (Bruckner et al., 1987; Ambulos et al., 1986). Such a sequence is missing in the C. perfringeens cat gene, and no other regulatory mechanism seems to function in the foreign host.

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