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Journal of Applied Microbiology 2000, 89, 249ÿ260

Biological and molecular characterization of a two-peptide lantibiotic produced by Lactococcus lactis IFPL105 M.C. MartõÂnez-Cuesta1, G. Buist2, J. Kok2, H.H. Hauge3, J. Nissen-Meyer3, C. PelaÂez1 and T. Requena1 1

Department of Dairy Science and Technology, Instituto del FrõÂo, Ciudad Universitaria, Madrid, Spain, 2Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen,The Netherlands, and 3 Department of Biochemistry, University of Oslo, Norway 111/12/99: received 23 December 1999, revised 20 March 2000 and accepted 22 March 2000 Â EZ AND T. M . C . M A R T ÂI N E Z - C U E S T A , G . B U I S T , J . K O K , H . H . H A U G E , J . N I S S E N - M E Y E R , C . P E L A

The lactic acid bacterium Lactococcus lactis IFPL105 secretes a broad spectrum bacteriocin produced from the 46 kb plasmid pBAC105. The bacteriocin was puri®ed to homogeneity by ionic and hydrophobic exchange and reverse-phase chromatography. Bacteriocin activity required the complementary action of two distinct peptides (a and b) with average molecular masses of 3322 and 2848 Da, respectively. The genes encoding the two peptides were cloned and sequenced and were found to be identical to the ltnAB genes from plasmid pMRC01 of L. lactis DPC3147. LtnA and LtnB contain putative leader peptide sequences similar to the known `double glycine' type. The predicted amino acid sequence of mature LtnA and LtnB differed from the amino acid content determined for the puri®ed a and b peptides in the residues serine, threonine, cysteine and alanine. Post-translational modi®cation, and the formation of lanthionine or methyllanthionine rings, could partly explain the difference. Hybridization experiments showed that the organization of the gene cluster in pBAC105 responsible for the production of the bacteriocin is similar to that in pMRC01, which involves genes encoding modifying enzymes for lantibiotic biosynthesis and dual-function transporters. In both cases, the gene clusters are ¯anked by IS946 elements, suggesting an en bloc transposition. The ®ndings from the isolation and molecular characterization of the bacteriocin provide evidence for the lantibiotic nature of the two peptides.

R E Q U E N A . 2000.

INTRODUCTION

Lactic acid bacteria (LAB) produce several types of bacteriocins. Comprehensive biochemical, structural and genetic characterization of LAB bacteriocins over the last few years has allowed the classi®cation of these substances (Klaenhammer 1993; Nes et al. 1996; Sahl and Bierbaum 1998). Major groups described differentiate between lantibiotics (Class I) and small, heat-stable non-lantibiotics (Class II). Lantibiotics are post-translationally modi®ed peptides that exert a broad spectrum of inhibitory activity against other Gram-positive bacteria (Klaenhammer 1993). Nisin is the best characterized lantibiotic produced by Correspondence to: Dr T. Requena, Department of Dairy Science and Technology, Instituto del FrõÂo (CSIC), Ciudad Universitaria, E-28040, Madrid, Spain (e-mail: [email protected]). = 2000 The Society for Applied Microbiology

LAB, and its use as a food preservative has been approved in several countries (Turtell and Delves-Broughton 1998). Most of the bacteriocins produced by LAB are included in class II, consisting of small, cationic and hydrophobic peptides. A peculiar group of these bacteriocins is characterized by the need for two peptides for full activity (Nissen-Meyer et al. 1992; Allison et al. 1994; JimeÂnezDõÂaz et al. 1995; Marciset et al. 1997; Anderssen et al. 1998). A particular two-peptide bacteriocin system has been described in Enterococcus faecalis (cytolysins CylLL and CylLS) and Staphylococcus aureus C55 (staphylococcins C55a and C55b), where both peptides have been identi®ed as lantibiotics (Booth et al. 1996; Navaratna et al. 1998). The analysis of the complete sequence of the 60 kb conjugative plasmid pMRC01 from L. lactis DPC3147 by Dougherty et al. (1998) revealed a genetic region of 20 kb, including an operon consisting of six genes responsible for the production of a two-peptide bacteriocin (lacticin 3147).

250 M . C . M A R T ÂI N E Z - C U E S T A E T A L .

Some of the putative encoded proteins were found to be homologous to enzymes involved in post-translational dehydration, lanthionine ring formation and in bacteriocin excretion, but data about amino acid sequences and possible modi®cations of the corresponding peptides are lacking. The heat-stable bacteriocin produced by L. lactis IFPL105, isolated from raw goats' milk, has been shown to have a broad spectrum of inhibitory activity against Grampositive bacteria (Casla et al. 1996). The genes responsible for the bacteriocin production were found to be located on the 46 kb plasmid. A bacteriolytic effect was observed for sensitive Lactococcus and Lactobacillus cells, resulting in the release of intracellular material (MartõÂnez-Cuesta et al. 1997). Addition of the bacteriocin-producing strain in cheese-making experiments induced cell lysis of bacteriocin-sensitive adjuncts with high peptidase activity, resulting in accelerated ripening (MartõÂnez-Cuesta et al. 1998). In the present study, the puri®cation of the bacteriocin produced by L. lactis IFPL105, and the molecular characterization of the bacteriocin gene cluster on the 46 kb plasmid (pBAC105), are described. Results provide evidence of the lantibiotic nature of the two-peptide bacteriocin produced by L. lactis IFPL105 and show that it is identical to lacticin 3147. The two plasmids, pMRC01 and pBAC105, share the same gene organization in an 18 kb region ¯anked by two IS946 elements.

MATERIALS AND METHODS Bacterial strains, plasmids and growth conditions

The strains and plasmids used in this study are listed in Table 1. Lactococcus lactis was grown at 30  C in M17 medium (Difco) or MRS (Merck) when indicated as standing cultures, or on M17 agar solidi®ed with 1
5% agar, all supplemented with 0
5% glucose. Escherichia coli was grown in TY (Difco) medium at 37  C with vigorous agitation, or on TY medium solidi®ed with 1
5% agar. Ampicillin (100 mg mlÿ1; Sigma) or 50 mg kanamicin mlÿ1 (Boehringer) was added and when required, IPTG (Sigma) and X-gal were used at concentrations of 1 mmol lÿ1 and 0
002%, respectively. Bacteriocin activity assay

The bacteriocin produced by L. lactis IFPL105 was quanti®ed during the puri®cation and complementation analyses using a microtitre plate assay as described by NissenMeyer et al. (1993). One bacteriocin unit (BU) was de®ned as the amount of bacteriocin that inhibited growth of the indicator organism by 50%. Bacteriocin production was routinely tested by an agar diffusion test as described earlier (Casla et al. 1996), and activity was expressed as activity units (AU), where one AU was de®ned as the reciprocal

Table 1 Bacterial strains and plasmids used in this study

Strain or plasmid Strains L. lactis IFPL105 IFPL359 MG1363 MG1363 (pMRC01) MG1363 (pBAC105) E. coli NM522 Plasmids PUC19 PUK21 PUKAB PUCINVA

PUCINVB

Relevant phenotype or genotype

Source or reference

Wild-type strain Wild-type strain Plasmid-free strain MG1363 containing pMRC01 MG1363 containing the 46-kb plasmid from L. lactis IFPL105 SupE thi D(lac-proAB) Dhsd5(rk± mk±) [F0 proAB lacIqZDM15]

Casla et al. (1991) Requena et al. (1991) Gasson (1983) Coakley et al. (1997) This study

Apr Kmr Kmr, 271 bp PCR fragment obtained using primers pAI and pBI in EcoRV site of pUK21 Apr, 581 bp fragment of a XhoII digest of a PCR fragment obtained using primers invA and invB in BamHI and HindII sites of pUC19 Apr, 317 bp fragment of a XhoII digest of a PCR fragment obtained using primers invA and invB in BamHI and HindII sites of pUC19

Yanish-Perron et al. (1985) Vieira and Messing (1991) This study

Stratagene

This study This study

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of the highest dilution of the ®lter-sterilized culture supernatant ¯uid that produced a clear zone of inhibition. Lactococcus lactis IFPL359 was used as the indicator organism in all cases. Purification of the bacteriocin

The bacteriocin was puri®ed from the supernatant fraction of early stationary phase (at an optical density of 2 at 600 nm) cultures of L. lactis IFPL105, essentially following the four-step puri®cation procedure described by NissenMeyer et al. (1992). The bacteriocin was precipitated by adding 400 g ammonium sulphate per litre of culture supernatant ¯uid, pelleted by centrifugation (8000 g, 20 min), and solubilized in 20 mmol lÿ1 sodium phosphate buffer, pH 6
8 (buffer A; fraction I). The bacteriocin preparation was applied to a 5 ml SP-Sepharose Fast Flow (Pharmacia Biotech, Uppsala, Sweden) cation-exchange column equilibrated with buffer A. The ¯ow-through fraction (fraction II) containing the bacteriocin activity was applied to a 5 ml Q-Sepharose Fast Flow (Pharmacia Biotech, Uppsala, Sweden) anion exchange column equilibrated with buffer A. The ¯ow-through fraction contained the bacteriocin activity and it was designated fraction III. Ammonium sulphate was added to fraction III to a ®nal concentration of 10% (w/v), after which the fraction was applied to a 1 ml Phenyl Sepharose 6 Fast Flow (HighSub; Pharmacia) column equilibrated with 10% (w/v) ammonium sulphate in buffer A. The column was subsequently washed with 10 ml buffer A, after which the bacteriocin activity was eluted with 10 ml 70% ethanol and 30% buffer A (fraction IV). Fraction IV was diluted to 50 ml with water containing 0
1% tri¯uoroacetic acid (TFA) and was applied to a 3 ml Resource RPC column (Pharmacia) equilibrated with 2-propanol/water (10:90, v/ v), containing 0
1% TFA. The bacteriocin was eluted with a linear gradient ranging from 0 to 100% 2-propanol/water containing 0
1% TFA (fraction V). The bacteriocin peptides in fraction V were diluted ®vefold with water containing 0
1% TFA and rechromatographed on a C2/C18 reverse-phase column (PepRPC HR 5/5, Pharmacia), with a linear gradient ranging from 20 to 60% 2-propanol/water containing 0
1% TFA by which the two peptides of the bacteriocin were separated. Mass spectrometry, amino acid composition and sequence analyses

Determination of the molecular mass of the peptides was performed with a Bioion mass analyser (Applied Biosystems, Foster City, CA, USA). For determination of the amino acid composition, the puri®ed bacteriocin peptides were hydrolysed in 6 mol lÿ1 HCl in a vacuum at 110  C for 24 h. The amino acids were analysed using a

251

Biotronic LC 5000 analyser connected to a SP 4100 Computing Integrator (Spectra Physics, San Jose, CA, USA). The isolated peptides were cleaved with cyanogen bromide, trypsin and chymotrypsin as described by Sletten and Husby (1974), and the cleavage products were separated by SMART (Pharmacia Biotech) reverse-phase chromatography. The amino acid sequence was determined by Edman degradation using an Applied Biosystems model 477 A pulse liquid sequator, on-line connected to a Model 120 A Applied Biosystems RP-HPLC unit for identi®cation of the step-wise released phenylthiohydantoin-amino acids (Eurosequence, Groningen, The Netherlands). General DNA techniques and transformation

Molecular cloning techniques were performed essentially as described by Sambrook et al. (1989). Restriction enzymes and T4 DNA ligase were obtained from Boehringer and were used according to the instructions of the supplier. Plasmid DNA used for sequencing was isolated from E. coli cells using the High pure plasmid isolation kit (Boehringer). The plasmid mixture of L. lactis IFPL105 and plasmid pMRC01 contained in L. lactis MG1363 were isolated by the method of Anderson and McKay (1983), and further puri®ed by caesium chloride-gradient centrifugation followed by dialysis against a 10 mmol lÿ1 Tris, 1 mmol lÿ1 EDTA (pH 7) solution. Escherichia coli and L. lactis were transformed by electroporation using a Gene Pulser (Bio-Rad) as described by Zabarovsky and Winberg (1990) and Holo and Nes (1989), with the modi®cations suggested by Leenhouts and Venema (1993), respectively. The 46 kb plasmid pBAC105 of L. lactis IFPL105, which has been shown to be responsible for the bacteriocin production (Casla et al. 1996), was separated from the other plasmids using the plasmid mixture isolated from L. lactis IFPL105 to transform L. lactis MG1363 cells. Selection of bacteriocin-producing transformants was conducted on glucose-M17 agar plates containing the bacteriocin (150 AU mlÿ1). After overnight growth, the colonies were overlayed with 3 ml soft glucose-M17 agar (0
7%), containing 150 ml of an overnight culture of L. lactis IFPL359. Polymerase chain reaction (PCR), molecular cloning and DNA sequencing

PCR was performed in a Bio-Med thermocycler 60 using the ExpandTM high ®delity PCR system (Boehringer) according to the manufacturer's instructions, and using pBAC105 as a template. PCR fragments were puri®ed using the QIA quick PCR puri®cation kit (QIAGEN GmbH, Hiden, Germany). Synthetic oligo deoxyribonucleotides were purchased from GibcoBRL (Life Technologies).

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252 M . C . M A R T ÂI N E Z - C U E S T A E T A L .

The primers pAI (50 -TACTGGGGAAATAACGG) and pBI (50 -TGGACAAGTATTGGTAC), derived from the nucleotide sequence of pMRC01 (Dougherty et al. 1998) and the internal amino acid sequence determined for the b peptide, respectively, were used to clone the DNA fragment of pBAC105 encoding parts of the a and b peptides. The puri®ed PCR fragment of 271 bp was treated with Klenow enzyme and subcloned into the EcoRV site of plasmid pUK21. The ligation mixture was used to electrotransform E. coli NM522. Primers invA (50 -CATTCAT GAGTGAGTGTACACC) and invB (50 -CCAGCAACT CCTGCAATC), located within the ltnA and ltnB genes (Dougherty et al. 1998), were used in a PCR on a re-ligated XhoII digest of pBAC105 to clone the ¯anking sequences of the PCR fragment obtained with primers pAI and pBI. The 898 bp PCR fragment was treated with Klenow enzyme and digested with XhoII. The two DNA fragments of 581 and 317 bp were subcloned into the BamHI and HindII sites of pUC19. The ligation mixture was used to electrotransform E. coli NM522. As subcloning of the 581 bp fragment, containing the upstream region of ltnA, in E. coli resulted in deletions, the PCR fragment was sequenced using primer invA. The sequence was completed by sequencing directly on pBAC105. Nucleotide sequences of double-strand plasmid templates were determined, using the dideoxy chain termination method (Sanger et al. 1977), with the T7 sequencing kit and protocol (Pharmacia), or the automated ¯uorescent DNA sequencer 725 of Vistra systems (Amersham International, UK), using Texas red-labelled forward and reverse puc primers (Amersham). DNA and protein homology searches against the Genbank were carried out using the Blast program (Altschul et al. 1997). The nucleotide sequence identi®ed in this study has been assigned Genbank accession number AF167432.

Southern transfer and DNA hybridization

After agarose gel electrophoresis, DNA was transferred to Nytran-plus membranes (Schleicher and Schuell) by the protocol of Southern, as modi®ed by Chomczynski and Quasba (1984). Probe labelling and hybridization were done with the ECL labelling and detection system according to the instructions of the manufacturer (Amersham). The probes in the hybridization experiments were a PCR fragment obtained using the primers pAI and pBI, a PCR fragment of the internal region of the gene for the insertion sequence (IS) element IS946 using the primers IS946P3 (50 -GATCGTGGAATAAATGTTTGTC) and IS946P4 (50 -ATCGTGGTTGAGGCAGTTCG), using pBAC105 as a template, or a Sau3A digest of plasmid pMRCO1.

Primer extension analysis

RNA was isolated as previously described (Van Asseldonk et al. 1993) from exponentially-growing L. lactis MG1363 (pBAC105) at an optical density of 0
5 at 600 nm. Oligonucleotide invA was used for primer extension reactions and nucleotide sequence reactions on plasmid pBAC105. A 25 ng portion of primer was added to 3
5 mg of RNA in a reaction mixture containing dATP, dGTP, dTTP and [a-P32]dCTP, and cDNA was synthesized with SUPERSCRIPT transcriptase (Boehringer). After 10 min of incubation at 42  C, an excess of cold dCTP was added and incubation was continued for another 10 min at 42  C. The products were analysed on a 6% polyacrylamide sequencing gel. RESULTS Bacteriocin purification

An investigation of the binding of the bacteriocin precipitated from the culture supernatant ¯uid of L. lactis IFPL105 to various hydrophobic ligands (Butyl-, Octyland Phenyl-Sepharose columns, Pharmacia) revealed that the bacteriocin bound most ef®ciently to a Phenyl Sepharose Fast Flow (High-Sub) column. The bacteriocin did not bind to either a cationic or an anionic exchanger at pH values of 6
8, 6
0 or 5
0. After ammonium sulphate precipitation, passage through both anion and cation exchangers, and chromatography on a phenyl hydrophobic interaction column, the bacteriocin was chromatographed on a Resource reverse-phase column. The results of this ®ve-step puri®cation procedure are summarized in Table 2. Rechromatography of fraction V (see Table 2) on a reversephase column (PepRPC 5/5) resulted in the appearance of two distinct peptides, each of which was rechromatographed on the reverse phase column (Fig. 1). These two peptides eluted at approximately 37 and 46% 2-propanol and were termed a and b, respectively. The average molecular masses of the a and b peptides were 3322 and 2848 Da, respectively, as determined by mass spectrometry. No bacteriocin activity was detected when either a or b were assayed separately. However, activity was recovered when the fraction containing the b peptide was combined with the fraction containing the a peptide, indicating that the complementary action of the two peptides was necessary to obtain bacteriocin activity (Fig. 1). Amino acid composition and sequence analysis of the bacteriocin

The N-terminal residues of both puri®ed peptides appeared likely to be blocked, since direct sequence determination by Edman degradation did not succeed.

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253

Table 2 Puri®cation of the bacteriocin produced by Lactococcus lactis IFPL105

Fraction Culture supernatant ¯uid Fractions I (Ammonium sulphate precipitation) II (Cation exchange) III (Anion exchange) IV (Phenyl sepharose) V (C2/C18 column)

Volume (ml)

Total A280*

Total activity (BU){

2000

71 800

4
44  105

6
2

250 260 270 20 2

2597 4316 3375 57
6 1
2

2
77  105 3
33  105 3
30  105 1
50  105 0
57  105

106
6 77
2 97
7 2604 21 250

Speci®c activity{

Puri®cation (fold)

Yield (%)

1

100

17 12 16 421 3438

63 75 74 34 11

*Total A280 is A280 multiplied by the volume (ml). {BU; bacteriocin units. {Speci®c activity is total activity divided by total A280.

Treatment of the a peptide with cyanogen bromide, trypsin or chymotrypsin did not result in peptide fragments of which the N-terminal sequence could be determined by Edman degradation. However, upon cleavage of the b peptide with chymotrypsin, Edman degradation succeeded and the following sequence was obtained: Ile-(Ser)-Thr-AsnThr-(Glu)-Pro, where parentheses indicate amino acids not determined with certainty. A homology comparison of the determined sequence of the b peptide revealed that its sequence was homologous to an internal sequence of the

peptide LtnB of the two-peptide bacteriocin lacticin 3147 (Dougherty et al. 1998). A degenerate primer designed on the determined sequence, and a second one homologous to a 17 nucleotide DNA sequence located in ltnA, were used in a PCR to obtain the DNA fragment of pBAC105 encoding parts of the two peptides. For this purpose, the plasmid content of L. lactis IFPL105 was used to transform L. lactis MG1363, in order to separate the 46 kb bacteriocin plasmid (pBAC105) from the other plasmids present. Bacteriocin-producing transformants only contained

Fig. 1 Reverse-phase chromatography of the (a) a and (b) b peptides of the bacteriocin speci®ed by pBAC105. Bacteriocin activity is

indicated without (open circles) and with complementation (closed circles) of one peptide with the other. Ð, A280; - - -, 2-propanol gradient

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254 M . C . M A R T ÂI N E Z - C U E S T A E T A L .

pBAC105, which was isolated and used to clone and sequence the complete bacteriocin encoding genes as described in Materials and Methods. The entire sequence determined in this work (1069 bp) was found to be identical to a region in pMRC01. The a peptide corresponds to LtnA, while the b peptide puri®ed in this work matches LtnB. Comparison of the putative leader peptides of LtnA and LtnB indicates that they are of the `double Gly' type and share homology with other leader peptides of lantibiotics that require hybrid transporter proteases (Sahl and Bierbaum 1998) (Fig. 2). These leader sequences are also characterized by a high proportion of glutamic acid and aspartic acid amino acid residues, which is also the case for LtnA and LtnB (24% and 25%, respectively). The amino acid composition deduced from putative mature LtnA and LtnB differs mainly in the number of serine, threonine and alanine residues compared with the composition of the isolated a and b peptides (Table 3). A comparison of the molecular masses determined for the puri®ed peptides, and those predicted for mature LtnA and LtnB presuming they are processed at the Gly-X site (3322 vs 3428
4 for the a peptide and 2848 vs 2985
5 for the b peptide), indicates that the experimentally-derived mass for the a peptide is 106
4 less than predicted, and that for the b peptide is 137
5 less than predicted. These results are suggestive of post-translational modi®cations in the two components of the bacteriocin. The gene cluster for bacteriocin production in pBAC105 is identical to that of pMRC01

The 271 bp PCR fragment obtained using primers pAI and pBI containing parts of ltnA and ltnB, a Sau3A digest of

plasmid pMRC01, as well as an internal DNA fragment of the IS element IS946, were used as probes in a Southern hybridization on RcaI, ScaI and XbaI digests of pBAC105 and pMRC01 (Fig. 3). The signals obtained with the probe against ltnA and ltnB show that these genes are located on similar sized DNA fragments of the plasmids pBAC105 and pMRC01. When using the IS-element as a probe, the only similar signal obtained in all three digests of the two plasmids is the 1489 bp XbaI fragment which borders the left side of the bacteriocin region in pMRC01. Digestion of pBAC105 and pMRC01 with XbaI results in two and three stronger hybridizing signals, respectively, while only one is present in the RcaI and ScaI digests. This shows that pBAC105 contains at least one, and most likely two extra IS946 elements (Fig. 3a) compared with pMRC01, which has been shown to contain two IS946 elements. The size of the ‹6300 bp XbaI fragment that hybridized with the IS probe and the digest of pMRC01 shows that an IS946 element is located shortly downstream of orf40. Analysis of the hybridization pattern of the RcaI digest of pBAC105 probed with the Sau3A digest of pMRC01 shows that the organization of the bacteriocin encoding region is similar in both plasmids, except for the ‹5300 bp RcaI fragment containing the IS946 element, ltnE and a part of ltnF. The 441 bp RcaI fragment in Fig. 3b was only present after a longer exposure of the blot, which also accounts for the other fragments smaller than 500 bp (result not shown). The organization of the region of pBAC105 for the synthesis of the bacteriocin is similar to that in pMRC01 (Fig. 3b). No hybridization with the other domains of pMRC01 was obtained, indicating that the remaining part of pBAC105 is completely different from that of pMRC01.

Fig. 2 Alignment of the putative LtnA and LtnB leader peptides with those of the `double glycine' type lantibiotics lacticin 481 (Piard

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TWO-PEPTIDE LANTIBIOTIC FROM LACTOCOCCUS LACTIS

Table 3 Amino acid composition of the two-peptide bacteriocin

produced by Lactococcus lactis IFPL105

Number of residues per molecule Amino acid

a-peptide*

LtnA{

b-peptide*

LtnB{

Ala Arg Asx Cys Glx Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Total

3±4 0 4 3 1±2 2±3 1 0 2 1 1 1 0 0 0 ND{ 1 0 20±23

2 0 4 4 1 2 1 0 2 1 1 1 0 3 4 3 1 0 30

7 1 1 3 0 0 0 3 1±2 1 0 0 3±4 0 3 ND 1 0 24±26

4 1 1 3 0 0 0 3 1 1 0 0 3 3 8 0 1 0 29

*The number of amino acid residues was calculated from the molar ratio relative to His (a) and Arg (b) after hydrolysis in 6 mol lÿ1 HCl of the puri®ed peptides. Unidenti®ed residues are not shown. {Amino acid residues predicted from the gene sequence after processing of LtnA and LtnB at the putative Gly-X cleavage site. {ND ˆ Not determined.

Determination of the transcription start site of ltnAB

ltnA and ltnB are preceded by putative ribosome-binding sites complementary to the 30 end of the lactococcal 16S rRNA (Chiaruttini and Milet 1993) and have DG values of ÿ 18
1 and ÿ 12
8 kcal molÿ1, respectively (Tinoco et al. 1973). The transcription start site upstream of ltnA was found to be located 23 nucleotides upstream of the ATG start codon. Seven bases upstream of the transcription start site, a putative ÿ10 sequence, separated by 17 bp from a preceding perfect ÿ35 sequence (TTGACA), is present. The transcription start site is located in a direct repeat that partly overlaps the ribosome binding site of ltnA (Fig. 4). A similar direct repeat, also partly overlapping the ribosome binding site, is present upstream of ltnB. In between the ÿ10 and ÿ35 sequence, a sequence of seven nucleotides (ATGGAAT) is present, which is also present twice in the sequence between the ÿ35 and the start codon of orf34. One of these sequences is located in a stem-loop. Whether

255

these sequences have any importance in regulating the bacteriocin expression will have to be investigated. DISCUSSION

Lactococcus lactis IFPL105 produces a two-peptide bacteriocin. The peptides, designated a and b were puri®ed to homogeneity and appeared not to have a net charge at neutral pH, as none of them bound to ion exchangers. Both peptides, however, showed high af®nity for the reversephase column, which indicates a considerable grade of hydrophobicity. The bacteriocin activity required the complementary action of both peptides. Activity appeared to be optimal when the amount of the b peptide was ®vefold higher than that of the a peptide. It should be pointed out that this is an estimate, since the peptides were quanti®ed on the basis of their theoretical extinction coef®cients at 280 nm. Other two-peptide bacteriocins that have been characterized with respect to optimal assay conditions require approximately equimolar amounts of the two peptides (JimeÂnez-DõÂaz et al. 1995; Moll et al. 1996; Marciset et al. 1997; Anderssen et al. 1998; Navaratna et al. 1998). Sequencing of the bacteriocin encoding genes of the 46 kb plasmid (pBAC105) and their ¯anking DNAs showed that this region is identical to the bacteriocin encoding region of the 60 kb plasmid pMRC01 of L. lactis DPC3147 (Dougherty et al. 1998). On both plasmids, the gene clusters are ¯anked by IS946 elements. From the hybridization analysis it was shown that there was no homology between pBAC105 and pMRC01 outside the bacteriocin gene cluster, although both plasmids carry other IS elements. This indicates that pBAC105 does not contain the conjugative transfer region present on pMRC01, and it would explain the failure to transfer the plasmid from L. lactis IFPL105 to other lactococcal strains by conjugation (unpublished results). Also, the lack of hybridization with the region responsible for replication of pMRC01 suggests that pBAC105 contains a different replicon. The presence of three copies of IS946 suggests that pBAC105, like pMRC01, has a modular structure. From the hybridization experiments it was also clear that the open reading frames 41, 42 and 43 present in the lacticin 3147 biosynthetic gene cluster are not present in this cluster on pBAC501, suggesting that the proteins encoded by these open reading frames are not involved in bacteriocin production. An important result of this research is the strong suggestion of the lantibiotic nature of the bacteriocin produced by L. lactis IFPL105. This conclusion is supported by several observations. The ®rst is the fact that the amino acid sequencing of both peptides failed, probably due to Nterminal blocking groups. Blockage of Edman degradation is a common feature of dehydrated amino acid residues (Sahl and Bierbaum 1998). The second support comes

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256 M . C . M A R T ÂI N E Z - C U E S T A E T A L .

Fig. 3 (a) Southern hybridization analysis of RcaI, ScaI and XbaI digests of pBAC105 (1) and pMRC01 (2). The probes used were the

PCR fragment obtained with the primer combination pAI/pBI (ltnAB), a Sau3A digest of plasmid pMRC01 (pMRC01) and an internal fragment of IS946 obtained using the primer combination IS946P3±IS946P4 (IS946). Sizes of the molecular weight marker (bacteriophage SppI DNA cut with EcoRI) are indicated on the left. Bands indicated with an asterisk consist of two hybridizing DNA fragments of the same size. The lower panel shows the organization of the RcaI, ScaI and XbaI fragments in pBAC105. (b) Map of the region of plasmid pMRC01 responsible for bacteriocin production (Dougherty et al. 1998). The region between the arrowheads indicates the region that was cloned and sequenced from plasmid pBAC105. The black boxes labelled with LtnAB and IS represent the PCR fragments used as probes in the hybridizations shown in (a). The position of the restriction enzyme sites shown is similar to that in pMRC01. Only relevant restriction enzyme sites are shown

from the presence of a putative `double-Gly' containing leader sequence, which shares homology with other lantibiotic leader peptides, in the deduced amino acid sequence of LtnA and LtnB. This is consistent with the presence in the lacticin 3147 gene cluster of ltnT, which encodes an ABC-transporter, as judged from its nucleotide sequence (Dougherty et al. 1998; Fig. 3b). Dedicated ABC-transporters process and transport across the cell membrane `double-Gly' lantibiotics (Sahl and Bierbaum 1998). The leader sequences are characterized by a high proportion of acidic residues, and they possess the characteristic cleavage site with glycine in position ÿ2 and alanine or glycine in position ÿ1 (Fig. 2). Another observation supporting the idea that the bacteriocin is a lantibiotic comes from the comparison of the amino acid composition and the determined molecular masses of the puri®ed a and b peptides with those pre-

dicted for mature LtnA and LtnB. The main differences found between the amino acid content of the peptide hydrolysates and the predicted peptides were in the amino acids serine, threonine, cysteine and alanine (Table 3). It appears as if most of the serine and threonine residues predicted from ltnA and ltnB have been transformed by posttranslational modi®cation. Likewise, one cysteine residue in the a peptide seems to have been modi®ed. Dehydration of serine and threonine residues into didehydroalanine and didehydrobutyrine, respectively, and their reaction with cysteine residues to form the thioether-containing residues lanthionine and methyllanthionine, may be catalysed by the enzymes encoded by ltnM1 and ltnM2 (Dougherty et al. 1998; Fig. 3b). The N-terminal Cys residue in the a peptide might form a lanthionine or a methyllanthionine residue with a nearby didehydroalanine or didehydrobutyrine residue, whereas the N-terminal threonine residue in the b

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Fig. 4 Determination by primer extension of the start site of

transcription of ltnAB (indicated by the ¯at arrow on the A residue). Lane P shows the primer extension product on total RNA isolated from Lactococcus lactis MG1363 (pBAC105). At the right, the sequence obtained with primer invA on plasmid pBAC105 is indicated; ÿ10 (TAAAT), ÿ35 (TTGACA) and ribosome binding sites are indicated (RBS). The two thin facing arrows indicate a putative inverted repeat. The bold arrow indicates the nucleotide sequence that is present three times between orf34 and ltnA

peptide might become dehydrated and converted to didehydrobutyrine, which in turnÐbeing an N-terminal residueÐconverts to a 2-oxobutyryl residue upon deamination (Skaugen et al. 1994; Van de Kamp et al. 1995; Heidrich et al. 1998). These reactions would block the N-terminal residues of both a and b peptides and explain why the peptides could not be sequenced directly by Edman degrada-

257

tion. Serine residues might also be converted to D-alanine by dehydration to didehydroalanine followed by the reduction of the double bond in didehydroalanine, as described for lactocin S (Skaugen et al. 1994). This could explain why the puri®ed peptides had a higher content of alanine residues than predicted from the gene sequence (Table 3). The modi®cations proposed for LtnB, deamination of N-terminal didehydrobutyrine to a 2-oxobutyryl residue after addition of a water molecule, which will result in the loss of a mass of 17 Da, four dehydrations of threonine (loss of 4  18 Da), and three serine residues converted into alanine (loss of 3  16 Da), imply that the mature b peptide would have the predicted molecular mass of 2985
5 Da minus 137, which is quite close to the detected molecular mass of 2848 Da. The modi®cations in LtnA, namely formation of one lanthione or b- methyllanthione residue, dehydration of four hydroxylated amino acids, and one serine converted in alanine, would result in the loss of a mass of 106 Da. These modi®cations agree with the differences found between the predicted molecular mass of LtnA (3428
4 Da) and the mass of the a peptide measured by mass spectrometry (3322 Da). However, this estimation would not explain the absence of an additional hydroxylated amino acid residue in the amino acid composition of the a peptide (Table 3). Nevertheless, the bacteriocin produced by L. lactis IFPL105 shows certain distinctive characteristics among lantibiotics. The a peptide has no net charge at neutral pH and the b peptide would have a ‡1 net charge if the Nterminal threonine residue were deaminated. In this sense, the bacteriocin differs from the typically cationic type-A lantibiotics (nisin group) (Sahl and Bierbaum 1998) and most antimicrobial peptides in general (Nissen-Meyer and Nes 1997). The cysteine residue on the N-terminus is another distinct feature of the mature a peptide, although it has been observed among the type-B lantibiotics (Chatterjee et al. 1992; Zimmermann et al. 1995). However, the main feature of the bacteriocin is that two distinct peptides, both subject to post-translational modi®cations, have to interact to exert the inhibitory effect. In this sense, the bacteriocin could be related to cytolysins from enterococci and staphylococcins C55a and C55b (Booth et al. 1996; Navaratna et al. 1998). Finally, a point of particular interest is that the same bacteriocin is produced by two strains isolated from distant natural sources, L. lactis DPC3147 isolated from Irish ke®r grain (Ryan et al. 1996) and L. lactis IFPL105 from Spanish raw goats' milk (Casla et al. 1996). The IS946 elements ¯anking the gene clusters on both plasmids may have mediated the transposition of the gene block and the horizontal transfer of the bacteriocin production between lactococcal strains by IS-mediated cointegration. Recently, such an en bloc transfer of a gene cluster of seven open

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258 M . C . M A R T ÂI N E Z - C U E S T A E T A L .

reading frames responsible for the production of the lantibiotic Mutacin II has been shown to occur in Streptococcus mutans (Chen et al. 1999). Also, the gene cluster involved in the production of nisin is located on a conjugative transposon (Horn et al. 1991; Rauch and De Vos 1992). These observations strengthen the idea Buchman et al. (1998) that lantibiotics probably evolved from one common ancestor which dispersed among Gram-positive bacteria by transfer of mobile elements. The bacteriocin produced by L. lactis IFPL105 has interesting technological applications based on its broad spectrum of activity (Casla et al. 1996) and the bacteriolytic effect on sensitive strains (MartõÂnez-Cuesta et al. 1998).

ACKNOWLEDGEMENTS

This work was supported by Research Projects ALI 97± 0737 (Spanish Commission for Science and Technology) and FAIR CT97±3173. The authors are grateful to S. Bayne, Novo Nordisk A/S, Denmark, for analysing the peptides by mass spectrometry, and to Dr K. Sletten, Department of Biochemistry, University of Oslo, for amino acid composition analysis. ADDENDUM IN PROOF

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