Functional Analysis of the Gene Cluster Involved in Production of the Bacteriocin Circularin A by Clostridium beijerinckii ATCC 25752

Robèr Kemperman; Marnix Jonker; Arjen Nauta; Oscar P Kuipers; Jan Kok

doi:10.1128/AEM.69.10.5839-5848.2003

. 2003 Oct;69(10):5839–5848. doi: 10.1128/AEM.69.10.5839-5848.2003

Functional Analysis of the Gene Cluster Involved in Production of the Bacteriocin Circularin A by Clostridium beijerinckii ATCC 25752

Robèr Kemperman ¹, Marnix Jonker ¹, Arjen Nauta ², Oscar P Kuipers ¹, Jan Kok ^1,^*

PMCID: PMC201212 PMID: 14532033

Abstract

A region of 12 kb flanking the structural gene of the cyclic antibacterial peptide circularin A of Clostridium beijerinckii ATCC 25752 was sequenced, and the putative proteins involved in the production and secretion of circularin A were identified. The genes are tightly organized in overlapping open reading frames. Heterologous expression of circularin A in Enterococcus faecalis was achieved, and five genes were identified as minimally required for bacteriocin production and secretion. Two of the putative proteins, CirB and CirC, are predicted to contain membrane-spanning domains, while CirD contains a highly conserved ATP-binding domain. Together with CirB and CirC, this ATP-binding protein is involved in the production of circularin A. The fifth gene, cirE, confers immunity towards circularin A when expressed in either Lactococcus lactis or E. faecalis and is needed in order to allow the bacteria to produce bacteriocin. Additional resistance against circularin A is conferred by the activity of the putative transporter consisting of CirB and CirD.

Antimicrobial peptides or bacteriocins are produced by various gram-positive and gram-negative bacteria. Although many bacteriocins inhibit the growth of strains closely related to the bacteriocin producer, an ever-increasing number of bacteriocins have a broader activity range. Only a few antimicrobial peptides of clostridial origin have been characterized at the molecular level, despite their extensive use as a means of identifying and typing clostridia (24, 32). The three bacteriocins that have been partially characterized are BCN5, boticin B, and circularin A (9, 12, 25), produced by Clostridium perfringens, Clostridium botulinum, and Clostridium beijerinckii, respectively.

Circularin A, a circular bacteriocin produced by C. beijerinckii ATCC 25752, is active against a broad range of gram-positive bacteria (25). The circularization of the peptide involves a head-to-tail peptide bond formation between the fourth and last amino acid of the precursor peptide (25). Circularin A shares limited sequence homology with enterocin AS-48 (also known as Bac21), a cyclic bacteriocin from Enterococcus faecalis (34, 56), but its precursor lacks the long leader present in the enterocin AS-48 precursor. The circularin A gene cluster is chromosomally located, while the enterocin AS-48 operon is located on a plasmid.

Both circularin A and enterocin AS-48 belong to the recently defined class V bacteriocins of ribosomally synthesized, nonmodified, head-to-tail-ligated cyclic antibacterial peptides (25). Other class V bacteriocins are microcin J25 and gassericin A (4, 22). Microcin J25, peptide of 21 amino acid residues produced by Escherichia coli, is the only circular peptide known so far that is produced by a gram-negative bacterium (4). The genes involved in the production of microcin J25 are located in an operon immediately downstream of the structural gene (51). Gassericin A is produced by Lactobacillus gasseri as a 91-amino-acid precursor peptide that is circularized after removal of a leader peptide of 33 amino acids (22, 23). The coding regions of enterocin AS-48 of two strains have been sequenced and determined to be almost identical (35, 56). Enterocin AS-48 is a tightly packed peptide containing five α-helices and is structurally related to NK-lysin, a cytotoxic peptide from human natural killer or T cells (16).

Most bacteriocins require processing of a precursor peptide in order to become (fully) active. For many bacteriocins, the genes encoding processing, secretion, and immunity functions flank the structural gene. Processing can involve modification of amino acids, as is the case in lantibiotics, leader peptide removal, or, in the case of circular peptides, circularization. The mechanisms underlying these modifications are poorly understood, although the proteins involved are generally known. The secretion of most bacteriocins occurs via dedicated ABC transporters (26), while some can be secreted via the general secretion pathway (5, 21, 33).

Immunity systems for bacteriocins are poorly characterized, but it has been demonstrated that specialized immunity proteins confer immunity to bacteriocin action on cells by blocking access to a putative receptor, as is the case for the lactococcin A immunity protein LciA (58). In some cases ABC transporters have been shown to be involved, e.g., the NisFEG system in nisin resistance and McbFE in microcin B17 resistance (13, 43). Little homology exists among bacteriocin immunity proteins, even those that are involved in immunity against bacteriocins of the same class.

In this study we identified the genes required for functional heterologous expression of circularin A and showed that two independent mechanisms confer reduced circularin A sensitivity, one of which is based on the expression of cirE and the other on the combined expression of cirB and cirD. As such, this study will further the field of clostridial bacteriocins and that of class V (circular) bacteriocins in particular.

MATERIALS AND METHODS

Bacterial strains, media, and reagents.

The strains and plasmids used in this study are listed in Table 1. Clostridium beijerinckii ATCC 25752 was grown anaerobically at 30°C in AC broth (Difco, Detroit, Mich.). Anaerobicity was obtained by chemical absorption of the oxygen in closed bottles as previously described (25). Lactobacillus saké ATCC 15521 was grown in De Man Rogosa and Sharpe (MRS; Merck, Darmstadt, Germany) broth at 30°C. Twofold-diluted M17 broth (Difco) with a final concentration of 1.9% β-glycerophosphate (Merck) and 0.5% glucose (G[1/2]M17) was used for growth of Lactococcus lactis NZ9000 and Enterococcus faecalis JH2-2 at 30°C and 37°C, respectively. Escherichia coli DH5α was grown for 16 h in tryptone-yeast (TY) broth at 37°C with vigorous agitation (250 rpm). For growth on plates, medium containing 1.5% agar was used. Ampicillin (Sigma, Zwijndrecht, The Netherlands) and chloramphenicol (Sigma) were used at 100 and 10 μg/ml, respectively, for E. coli. Chloramphenicol and erythromycin (Sigma) were used at 5 μg/ml each for L. lactis NZ9000 and at 20 and 2 μg/ml, respectively, for E. faecalis JH2-2. When used together, chloramphenicol and erythromycin were employed at 2.5 μg/ml each for Lactobacillus saké ATCC 15521 or at 10 μg/ml (chloramphenicol) and 2 μg/ml (erythromycin) for E. faecalis JH2-2.

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Description^a	Reference or source^b
C. beijerinckii ATCC 25752	Circularin A producer	NIZO
E. faecalis JH2-2	Plasmid-free derivative of E. faecalis JH-2	20
L. lactis NZ9000	Plasmid-free derivative of L. lactis MG1363, pepN::nisRK	30
Lactobacillus sake ATCC 15521	Bacteriocin indicator	lab collection
E. coli Top10	F′ mcrA (mrr-hsdRMS-mcrBC) φ80lacZM15 lacX74 recA1 deoR araD139 (ara-leu)7697 galU galK rpsL (Str^r) endA1 nupG	Invitrogen
E. coli DH5α	supE44 ΔlacU169 (φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1	17
Plasmids
pMG36c	Cm^r, pWV01-based cloning vector carrying the strong lactococcal promoter P₃₂	Laboratory collection
pMG36e	Em^r, gene expression vector carrying P₃₂	57
pCR21	Amp^r Km^r	Invitrogen
pCRAE	Amp^r Km^r	This study
pIL253	Em^r, theta replicating cloning vector	49
pIL-E	Em^r, pIL253 derivative with cirE under control of P₃₂	This study
pIL-P32	Em^r, pIL253 derivative with P₃₂ promoter	This study
pMGAE1	Cm^r, pMG36c derivative with cirABCDE under control of P₃₂	This study
pCir	Cm^r, derivative of pMGAE1 overexpressing circularin A	This study
pCirΔA	Cm^r, pCir ΔcirA	This study
pCirΔB	Cm^r, pCir ΔcirB	This study
pCirΔC	Cm^r, pCir ΔcirC	This study
pCirΔD	Cm^r, pCir ΔcirD	This study
pCirΔE	Cm^r, pCir ΔcirE	This study
pCirΔAE	Cm^r, pCir ΔcirA ΔcirE	This study
pCirΔBE	Cm^r, pCir ΔcirB ΔcirE	This study
pCirΔCE	Cm^r, pCir ΔcirC ΔcirE	This study
pCirΔDE	Cm^r, pCir ΔcirD ΔcirE	This study
pCirΔACE	Cm^r, pCir ΔcirA ΔcirC ΔcirE	This study
pCirΔABCE	Cm^r, pCir ΔcirA ΔcirB ΔcirC ΔcirE	This study
pCirΔACDE	Cm^r, pCir ΔcirA ΔcirC ΔcirD ΔcirE	This study
pMG-E	Em^r, pMG36e derivative with cirE under control of P₃₂	This study

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Amp^r, ampicillin resistance; Cm^r, chloramphenicol resistance; Em^r, erythromycin resistance; Km^r, kanamycin resistance.

NIZO, NIZO Food Research Ede, The Netherlands; Invitrogen, Breda, The Netherlands.

Nucleotide sequencing.

Inverse PCR techniques with the nucleotide sequence of cirA (25) were employed to obtain the region surrounding the cirA gene. PCR products were sequenced either directly or after subcloning of restriction enzyme digestion fragments in pUC19. PCR products were purified with the High Pure PCR product purification kit of Roche (Roche Diagnostics GmbH, Mannheim, Germany). Sequencing was performed with indodicarbocyanine-labeled universal, reverse, or T7 primers (Amersham Pharmacia Biotech, Roosendaal, The Netherlands) and the ALFII system (Amersham Pharmacia Biotech) according to the protocols of the supplier with the following modifications: the power was set at 15 and 18 W for the long-read and high-resolution gels, respectively.

Bacteriocin assays.

Colony overlayer assays were performed as described previously (25). Bacteriocin activity in C. beijerinckii ATCC 25752 supernatant was quantified in triplicate by a critical dilution assay as described by Geis et al. (14), with the modification that assays were performed in microtiter plates. To 50 μl of serially diluted, bacteriocin-containing samples, 150 μl of medium containing the indicator strain Lactobacillus saké ATCC 15521(pMG36e; pMG36c), diluted 100-fold from a stationary-phase overnight culture, was added unless mentioned otherwise. Resistance to bacteriocin was determined by plating strains on plates containing 4 or 10% (vol/vol) filter-sterilized C. beijerinckii ATCC 25752 supernatant containing circularin A. Alternatively, a critical-dilution assay was performed with the strain of interest as an indicator strain. In these critical-dilution assays, 100 μl of bacteriocin-containing sample was mixed with 100 μl of freshly diluted (1,000-fold) indicator strains.

Cloning methods and materials.

Molecular cloning techniques were performed essentially as described by Sambrook et al. (45). Restriction enzymes, T4 DNA ligase, and Expand DNA polymerase were obtained from Roche (Roche Diagnostics GmbH) and used as specified by the supplier. L. lactis NZ9000 was transformed as described by Shepard and Gilmore (19) with 1% glycine (Merck). E. faecalis JH2-2 was transformed as described previously (47; http://w3.ouhsc.edu/enterococcus) with 8% glycine. After transformation, both strains were plated on G[1/2]M17 medium containing 0.5 M sucrose and the appropriate antibiotics. Plasmids from L. lactis NZ9000, Escherichia coli DH5α, and E. faecalis JH2-2 were isolated according to Birnboim (3), with the following modifications for E. faecalis: mutanolysin (1 U/ml; Sigma) was added to the suspension buffer to facilitate lysis, and plasmids isolated from 50 ml of culture were, after RNase (0.5 mg/ml; Sigma) treatment, further purified with the High Pure PCR product purification kit (Roche Diagnostics GmbH).

Cloning of the circularin A determinant.

The region encompassing cirA to cirE was amplified with primers located just upstream of cirA (B51426, 5′-ACGCGTCGACTCATGAGTTTTTCAAAAGGAGGTGATTAATT ATGTTTTTATTGCAGG-3′) and downstream of cirE (B51427, 5′-CGCGGATCCGTCGACCTCTCCCACTTTAACATTAGTTATTGCTC-3′). SalI-RcaI and BamHI-SalI sites, respectively, in the two primers are underlined. All enzymes (Roche) were used according to the manufacturer's instruction. The PCR product was cloned with the Zero-Blunt Topo PCR cloning kit (Invitrogen, Breda, The Netherlands), creating pCRAE. The plasmid pCRAE was digested with SpeI and XhoI. The fragment carrying cirA was ligated into pMG36c digested with XbaI and SalI, and the ligation mixture was used to transform E. coli DH5α. Transformants were identified by growth on TY agar with chloramphenicol. The correct plasmid, pMGAE1, was isolated as described above and introduced into E. faecalis JH2-2. Three consecutive selection steps of clean streaking and testing for a strain with a high and stable bacteriocin expression phenotype with the colony overlay assay yielded E. faecalis JH2-2 carrying a pMGAE1 derivative labeled pCir.

In-frame deletions of cirA through cirD were made by amplifying pCir by PCR with appropriate outward-facing primers, creating a PCR product of the entire plasmid but lacking the gene of interest. The primers used were 5′-AGTATGGCAAGAGCTATAGC-3′ and 5′-CACGCCTAGTGCTCCTGC-3′ for ΔcirA, 5′-TAATTATGCCTGTATCATACC-3′ and 5′-CCAAGAGTTATAGTTTGAGT CG-3′ for ΔcirB, 5′-GTGCACATAGGTAGGATTTTAAG-3′ and 5′-GAAACATTCC AACAATAATACC-3′ for ΔcirC, and 5′-GAACTTAATCTAGTTAACGGAAG-3′ and 5′-AGTTATCTCTAGCATAGGCTTC-3′ for ΔcirD.

Each PCR product was kinase treated with T4 polynucleotide kinase (Amersham Pharmacia Biotech) in T4 ligase buffer (Roche Diagnostics GmbH) and subsequently self-ligated with T4 ligase, creating the plasmids pCirΔA, pCirΔB, pCirΔC, and pCirΔD. Derivatives of pCir with a deletion of cirE and a deletion of one of the other genes cirABCD, pCirΔAE, pCirΔBE, pCirΔCE, or pCirΔDE, were made by the same method with the primers 5′-CATATATTCTACTACCTTTC-3′ and 5′-GTAATTAAAGGCTCTAATAAG-3′ for ΔcirE and the plasmids carrying the respective single deletions as templates in the PCR. A plasmid with a triple deletion, pCirΔACE, was constructed likewise by deleting cirC with pCirΔAE as a template and the primers used for the single deletion of cirC. Based on pCirΔACE, pCirΔABCE and pCirΔACDE were made with the primers employed for the single knockouts of cirB and cirD, respectively. All plasmids were isolated with E. faecalis JH2-2 as the cloning host.

The cirE gene was cloned behind the lactococcal chromosomal P₃₂ promoter by digesting the PCR product obtained with primers B51426 and B51427 and HpaI and SalI and ligating the cirE-carrying fragment into SmaI- and SalI-digested pMG36e, leading to pMG-E. Colonies obtained after transformation of L. lactis NZ9000 were replica streaked onto G[1/2]M17 plates with 4% (vol/vol) C. beijerinckii ATCC 252752 supernatant to screen for circularin A immunity. The plasmid was isolated and used to transform E. faecalis JH2-2. In order to make pCirΔE, a fragment with the immunity gene cirE behind the P₃₂ promoter was first cloned in pIL253 to avoid possible lethal effects of bacteriocin expression without immunity. This was done by digesting pMG-E with EcoRI and SalI and ligating the cirE-carrying fragment into pIL253 digested with the same enzymes. The resulting plasmid (pIL-E) was introduced into E. faecalis JH2-2. pCirΔE was subsequently made with pCir as a template and the appropriate primers. E. faecalis JH2-2(pIL-E) was used as the host for construction of pCirΔE.

Computational analyses.

Open reading frames were identified with the Glimmer 2.0 program (6). Predictions by the Glimmer 2.0 program were checked manually for validity. Homology comparisons were performed with the basic logical alignment tool (Blast) as described by Altschul et al. (1). Blast searches were performed against the NCBI nonredundant protein database and the NCBI microbial genomes database (http://www.ncbi.nlm.nih.gov/BLAST/). Homologies with conserved domains from the Pfam database (http://www.sanger.ac.uk/Software/Pfam/) (2) were also identified with Blast searches. Putative signal peptides were identified with signalP (http://www.cbs.dtu.dk/services/SignalP/) (38). Putative transmembrane helices were identified with the TMHMM2.0 program (http://www.cbs.dtu.dk/services/TMHMM-2.0/) (28). Dyad symmetries, isoelectric points, and molecular weights were determined with the program Clonemanager 4 (SEcentral; Scientific & Educational Software). Sequence alignments were performed with the ClustalW program available at http://www2.ebi.ac.uk/clustalw/ (54).

Nucleotide sequence accession number.

The cirA sequence is available under GenBank accession number AJ566621.

RESULTS

Sequence analysis of the region encompassing the structural gene of circularin A.

The structural gene of the circular bacteriocin circularin A of Clostridium beijerinckii ATCC 25752 (cirA) has previously been cloned and sequenced (25). A region of 11 kb surrounding cirA was sequenced and shown to contain 12 open reading frames (ORFs), including cirA (Fig. 1 and 2). Six putative promoters were identified, one upstream of each cfgR (cirA-flanking gene response regulator), cfg01 (cirA-flanking gene 01), cfg02, cirA, cirB, and cirG. The cirBCDE and cirGHI genes are putatively transcribed as polycistronic messengers, since no clear transcription initiation signals were detected other than the ones upstream of cirB and cirG, respectively. Translation of cirC and cirD can putatively start from alternative start codons within the same reading frame (Fig. 2). Overlap between the end of one gene (cfgR, cirC, cirD, cirH, and cirI) and the predicted start of the downstream gene, which is suggestive of regulation of expression by translational coupling, is a common feature in the entire region (Fig. 2). For cirH, translational coupling to cirG would be the only means of expression, as it lacks an obvious ribosome-binding site (Fig. 2).

FIG. 1. — (A) Physical map of the region surrounding the circularin A structural gene *cirA* of *C. beijerinckii* ATCC 25752. Solid arrows indicate genes; bent arrows show putative promoters; lollipops represent predicted regions of dyad symmetry (ΔG⁰ < −10 kcal/mol); dotted arrows show possible polycistronic messengers. Map units are base pairs. (B) Schematic representation of the *cir* DNA fragments in the indicated plasmids and locations of the deletions (indicated by the thin lines). Open arrowheads indicate a deletion in *cirE*. Promoters are shown by bent arrows. CirA⁺, circularin A production; Cir^R, circularin A resistance, denoted as ++, full protection against CirA [>24-fold increase relative to *E. faecalis* JH2-2(pMG36c)]; +, partial protection [2- to 16-fold increase relative to *E. faecalis* JH2-2(pMG36c)]; −, sensitive.

FIG. 2. — (A) Nucleotide sequences of promoter and translation initiation regions in the *cir* gene cluster. Putative −35, −10, and ribosome-binding site (RBS) sequences are underlined. Deduced amino acid sequences are indicated below the nucleotide sequences, and gene names are given below the amino acid sequences. Putative start codons are indicated in boldface and are numbered when more than one possibility exists. Termination codons are underlined and in italic.

Derived protein sequences and homologies are presented in Table 2. The gene products CirB through CirI all show some degree of homology to proteins involved in the production of enterocin AS-48, a bacteriocin produced by Enterococcus faecalis S-48 (34). Homologues of the putative proteins AS-48C1 and Bac21F are not encoded by the cir operon. CirD and CirH, like AS-48D and BacH, both contain an ATP-binding domain (Fig. 3). CirG belongs to the HlyD family of accessory proteins of ABC transporters, which includes proteins like EmrA, an accessory protein in the EmrAB multidrug transporter (31), and LcnD, an auxiliary protein involved in the secretion of the bacteriocin lactococcin A (11).

TABLE 2.

Characteristics of predicted proteins specified by the C. beijerinckii ATCC 25752 cir gene cluster

Protein	Gene size^a (bp)	Protein
Protein	Gene size^a (bp)	Size (kDa)	pl	TM^b	Homology^c	Putative function
CfgR	774	30.4	6	0	Response regulators
CfgK	1,275	49.8	6.8	7	Histidine kinases
Cfg01	597	23.2	9.8	5	AgrB regulatory protein
Cfg02	390	14.4	10.17	4	Unknown
CirA	216	7.2	10.9^d	2	Enterocin AS-48	CirA precursor
CirB	1,743	68.8	9.4	11	AS-48B (19%)	Secretion/immunity
CirC	555	20.9	10.1	4	AS-48C (21%)	Maturation
CirD	663	25.7	6.4	0	ATP-binding proteins; AS-48D (31.6%)	Secretion/immunity
CirE	147	5.7	10.6	2	AS-48D1 (30%)	Immunity
CirG	1,425	51.7	4.6	1	HlyD family of proteins/EmrA/BacG (17%)
CirH	744	27.6	6.1	0	ATP-binding proteins/BacH (40%)
CirI	1,266	45.7	9.7	4	Duf214 domain/permease of ABC transporter/Bacl (32%)

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When multiple putative starts were possible, the longest product was used.

TM, number of putative transmembrane sequences.

Homologues were identified by using Blast searches against the NCBI nonredundant protein database or by direct comparison to the proteins involved in enterocin AS-48 production. The identity of the protein to its homologue involved in enterocin AS-48 production is shown in parentheses.

The pl of CirA is based on the linear unprocessed protein.

FIG. 3. — Alignment of the putative ATP binding proteins involved in circularin A (CirD, CirH) and enterocin AS-48 (AS-48D, BacH) production with LolD, a protein involved in lipoprotein secretion. Identical residues are indicated by an asterisk. Colons and periods indicate conserved and semiconserved amino acid substitutions, respectively, according to the ClustalW grouping of amino acids. Dashes indicate gaps introduced in the sequence to maximize alignment. Walker A, Walker, and ABC transporter B motifs are indicated.

Based on the occurrence of the G-X₉-F-X₁₀-G motif, CirI can be classified in the ortholog group 3-1 of ABC transporters, as defined by Tomii and Kanehisa (55). Like the already characterized members of this group (FtsX [7] and LolC and LolE [37]), CirI contains four putative transmembrane domains, typical of group 3-1 ABC transporters (Fig. 4), and a Duf214 domain of predicted permeases, as defined in the Pfam database. CfgR and CfgK are homologous to response regulators and histidine kinases, respectively, of two-component regulatory systems. Cfg01 is homologous (24% identity) to the accessory gene regulator (AgrB) of Staphylococcus aureus, which is thought to be involved in processing and secretion of a signaling peptide (AgrD), regulating a large set of virulence factors (61). Cfg02 is homologous to two putative proteins with unknown function from C. acetobutylicum and C. perfringens (34% identity with each of these proteins) (39, 48).

FIG. 4. — Alignment of CirI with BacG (56), FtsX (6), and LolC and LolE (37). The consensus motif G-X₉-F-X₁₀-G for ortholog group 3-1 type ABC transporters is indicated. Predicted transmembrane domains are indicated in bold. The region constituting the predicted DUF214 domain is indicated by a line above the sequences. Identical residues are indicated by an asterisk, whereas colons and periods indicate conserved and semiconserved amino acid substitutions, respectively, according to the ClustalW grouping of amino acids. Gaps were introduced in the sequence to maximize alignment.

Circularin A production in a heterologous host.

The region encompassing cirABCDE was cloned in the broad-host-range vector pMG36c downstream of the constitutive lactococcal promoter P₃₂, creating pMGAE1. E. faecalis JH2-2(pMGAE1) produced a small amount of bacteriocin and was resistant to circularin A (Fig. 5), but bacteriocin production was not stable. Three consecutive cycles of selection for bacteriocin production by overlay assay and subsequent clean-streaking of producing colonies yielded a strain that stably expressed a high level of circularin A (Fig. 5). The plasmid in this strain, designated pCir, was identical to pMGAE1 by restriction enzyme analysis. The high bacteriocin expression level was maintained upon plasmid isolation and reintroduction into E. faecalis JH2-2. The copy numbers of pCir and pMGAE1 were clearly reduced compared to the copy number of the empty vector pMG36c (data not shown).

FIG. 5. — Heterologous production of circularin A by *E. faecalis* JH2-2, as visualized in a colony overlayer assay with *Lactobacillus saké* ATCC 15521 as the indicator strain. (A) *E. faecalis* JH2-2(pMGAE1); (B) *E. faecalis* JH2-2(pCir); (C) *E. faecalis* JH2-2(pMG36c).

Functional analysis of the circularin A gene cluster.

In order to determine which genes are involved in the production of circularin A, pCir was used as a template to create in-frame single deletions of cirA, cirB, cirC, or cirD. E. faecalis JH2-2 strains harboring the various plasmids all lost the CirA⁺ phenotype, as revealed by colony overlayer assays (Fig. 1). All strains remained resistant to circularin A, as they grew on plates containing filter-sterilized culture supernatant (10%, vol/vol) of C. beijerinckii ATCC 25752, while E. faecalis JH2-2(pMG36c) did not (Fig. 1). These results indicate that each of the four gene products is required for the production of active circularin A.

Removal of cirE from the cirABCDE cluster in pCir could not be achieved in several attempts with different cloning hosts [E. faecalis JH2-2 and E. faecalis JH2-2(pMGE)], while simultaneous deletion of cirA and cirE (pCirΔAE) was possible. This observation suggested that cirE is involved in bacteriocin immunity, an assumption that will be discussed further below. E. faecalis JH2-2(pCirΔAE) showed reduced sensitivity to circularin A present in filter-sterilized culture supernatant of C. beijerinckii ATCC 25752 but was clearly more resistant to circularin A than E. faecalis(pMG36c).

To determine which gene(s) is involved in this partial resistance towards circularin A, single deletions of cirB, cirC, and cirD were combined with a deletion in cirE. A mutation in either cirB or cirD in combination with deletion of cirE led to the loss of the circularin A-resistant phenotype, whereas cells carrying a pCir derivative with a deletion in cirA or cirC in combination with cirE remained partially resistant to circularin A (Fig. 1). These results indicate that both CirB and CirD are required for partial resistance in the absence of CirE. To confirm this hypothesis, three additional deletion constructs were made. E. faecalis JH2-2(pCirΔACE), specifying only CirB and CirD, still showed partial resistance to CirA. E. faecalis JH2-2 expressing only CirB (pCirΔACDE) or CirD (pCirΔABCE) was bacteriocin sensitive, confirming that both CirB and CirD are needed for the partial resistance phenotype in the absence of CirE.

Heterologous expression of circularin A in L. lactis NZ9000 was attempted, but pCir and any other vector containing cirB did not give transformants, while all control plasmids did. Apparently, an intact cirB gene is lethal to this host. Plasmids with a deletion in cirB could be stably maintained in L. lactis. L. lactis(pCirΔB) did not produce active CirA and was 2.5-fold more resistant to the bacteriocin than L. lactis(pNG8048e), a strain carrying an empty cloning vector.

cirE gene confers circularin A immunity.

To prove that cirE can confer bacteriocin resistance independent of the combination cirB and cirD, the gene was cloned downstream of the lactococcal promoter P₃₂ in pMG36e (pMG-E). Unlike L. lactis(pMG36e), L. lactis NZ9000(pMG-E) was able to grow in a medium with culture supernatant (up to 50%, vol/vol) of C. beijerinckii ATCC 25752 (CirA⁺), as determined by serial dilution assay. L. lactis NZ9000(pMG-E) also formed normal colonies on plates containing filter-sterilized C. beijerinckii ATCC 25752 culture supernatant (4%, vol/vol), whereas the control strain did not grow at all. These results indicate that cirE alone gives rise to circularin A resistance. E. faecalis JH2-2(pMG-E) was also immune to the bacteriocin present in C. beijerinckii ATCC 25752 supernatant, as determined in a plate assay.

As mentioned above, initial attempts to remove cirE from cirABCDE with either E. faecalis JH2-2 or E. faecalis JH2-2(pMG-E) as the cloning host failed. This problem was circumvented by cloning cirE downstream of the P₃₂ promoter in pIL253. The resulting strain, E. faecalis JH2-2(pIL-E), was immune to C. beijerinckii ATCC 25752 culture supernatant, as determined in a plate assay, and did not produce bacteriocin (Fig. 1), as determined by serial dilution and overlay assays. In E. faecalis JH2-2(pIL-E), we were able to introduce the cirE deletion plasmid pCirΔE (CirA⁺). E. faecalis JH2-2(pIL-E, pCirΔE) was immune to C. beijerinckii ATCC 25752 culture supernatant and produced bacteriocin (Fig. 1). Attempts to introduce pCirΔE in E. faecalis JH2-2 alone were unsuccessful, indicating that cirE is required for proper bacteriocin immunity.

The levels of resistance conferred by pMG-E and pIL-E in E. faecalis JH2-2, which is able to grow in medium containing filter-sterilized C. beijerinckii ATCC 25752 culture supernatant (at the most 3 to 6%, vol/vol), were only two- to fourfold increased compared to the control strain carrying only pMG36e, able to grow in medium containing at the most 1.5% (vol/vol) filter-sterilized C. beijerinckii ATCC 25752 culture supernatant. This level is lower than the resistance level of E. faecalis JH2-2(pCirΔB) or E. faecalis JH2-2(pCirΔD), both also expressing only cirE as a functional immunity system. Complementing pIL-E with pCirΔDE in E. faecalis JH2-2 did not restore the resistance level to that of E. faecalis JH2-2(pCirΔD), indicating that the lower resistance level is not due to the absence of auxiliary factors not present on pMG-E or pIL-E. Taken together, these results confirm that CirE is the dedicated circularin A immunity protein and that expression of cirE alone is sufficient for immunity.

DISCUSSION

The region surrounding the structural gene cirA of the circular bacteriocin circularin A of C. beijerinckii ATCC 25752 encompasses 11 genes. Upstream of cirA there are four genes, of which two could encode a two-component regulatory system. Together with the presence of an AgrB homologue (Cfg01), this presents the possibility of regulation of bacteriocin expression. Two-component systems are often involved in the regulation of bacteriocin expression, and their genes are normally located near the bacteriocin operon (27). The homology to the Agr system of Staphylococcus aureus, which consists of a two-component system, a processing protein (AgrB) and a signaling peptide (AgrD), involved in regulation of virulence factors (40, 61) suggests a similar regulatory mechanism, although we could not identify an ArgD homologue in our sequence and have not further addressed the possible involvement of CfgR and CfgK in CirA expression.

We show here that the region cirABCDEGHI is involved in bacteriocin production and secretion. The genes cirABCDE represent the minimal region required for bacteriocin processing and secretion in the heterologous host E. faecalis JH2-2, as deletion of only a single gene from this cluster causes loss of either bacteriocin production or cell viability. The genetic organization of the region cirABCDEGHI seems rather compressed, as several genes overlap. This set-up suggests that translational coupling, a gene-regulatory mechanism often used in operons in which the stoichiometry of gene expression is important (36), may occur.

The minimal requirements for extracellular circularin A activity are production, processing, circularization, and secretion of the bacteriocin, while the producer cell should be immune to the bacteriocin. All these features should in principle be encoded by cirABCDE. Here, we show that resistance to circularin A is acquired via at least two independent systems. First, expression of cirE confers a certain level of immunity to the expressing strain, which is essential for the bacteria to be able to produce and withstand CirA. CirE has a very high and contains two possible transmembrane helices, which make membrane localization of the protein very likely. Its small size, high isoelectric point, and two predicted transmembrane helices are characteristics that CirE has in common with AS-48D1, the immunity protein of the circular bacteriocin enterocin AS-48, and with the proteins PepI, EciI, LasJ, and DviA, which have all been shown or postulated to be involved in immunity to the unrelated bacteriocins Pep5, epicidin 280, lactocin S, and divergicin A, respectively (18, 41, 44, 50, 59). The immunity mechanism of these proteins is unknown, but PepI has been suggested to inhibit pore formation by Pep5 (46).

The second system conferring reduced sensitivity to CirA depends on the combined activity of CirB and CirD. Together, these proteins form a putative ABC transporter in which CirB is the transporter and CirD provides the nucleotide-binding domain. The putative transporter CirBD confers a basal level of CirA resistance, which is, however, insufficient to support bacteriocin production by the heterologous host that we used; a viable clone of E. faecalis JH2-2(pCirΔE) could not be obtained. CirBD (most likely) also function in CirA secretion, as ABC transporters are often implicated in bacteriocin secretion (10). The fact that proteins required for secretion of a bacteriocin can be involved in resistance has been shown for McbE and McbF, which are involved in microcin B17 production (13). The secretion proteins of the lantibiotic nisin were suggested to fulfill a similar role, but involvement of NisI and/or NisFEG, via a regulatory loop inducing expression of the respective genes, cannot be excluded (29, 43). Resistance is most probably obtained by the pumping out of the bacteriocin (42, 43, 46). In conclusion, CirBD confer low-level resistance by virtue of their ability to secrete CirA, while CirE shows structural homology to other bacteriocin immunity proteins, which identifies it as the dedicated CirA immunity protein.

Based on homology studies, CirGHI could constitute another transporter. CirG probably has an auxiliary function, as it is homologous to the HlyD family of proteins, many of which are accessory proteins in the export of drugs or toxic proteins, such as hemolysin (52), lactococcin A (53), and colicin V (15). CirH and CirI probably form an ABC transporter of the LolCDE type (60): CirH is homologous to LolD, while CirI is homologous to both LolC and LolE. The CirGHI homologues in the enterocin AS-48 system (BacGHI/AS-48FGH) enhance the expression of enterocin AS-48 (56) and the resistance to exogenous enterocin AS-48 (8), roles we have not investigated for CirGHI yet. The homology to the LolCDE system furthermore suggests that active transport, perhaps from the outer leaflet of the membrane, as shown for LolCDE (60), is involved in this enhancing effect by making more bacteriocin available. The NisFEG system fulfills such a function in enhancement of nisin secretion (43), but this system is not very homologous to CirGHI or LolCDE.

No experimental evidence has been obtained to identify the protein(s) involved in the processing and/or circularization of the CirA prepeptide. As CirBD together form a putative ABC transporter and cirE confers bacteriocin immunity, the essential protein CirC is a likely candidate to perform this function(s), either alone or together with CirB and/or CirD. This notion seems to be supported by the fact that the only other CirC homologue is AS-48C, encoded by the enterocin AS-48 gene cluster of E. faecalis.

In conclusion, we have identified five genes essential for circularin A production and have shown that three of these genes (cirBDE) are involved in bacteriocin resistance. Future studies will be performed to determine the mechanism of circularization and the possible role of CirC therein.

Acknowledgments

This work was supported by FCDF Corporate Research and the Dutch Ministry of Economic Affairs. Jan Kok was the recipient of a grant from the Royal Netherlands Academy of Arts and Sciences (KNAW).

We are grateful to G. Dunny (Institute of Food Research, Colney, United Kingdom) for kindly providing the E. faecalis JH2-2 strains used in this study.

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[r2] 2.Bateman, A., E. Birney, L. Cerruti, R. Durbin, L. Etwiller, S. R. Eddy, S. Griffiths-Jones, K. L. Howe, M. Marshall, and E. L. Sonnhammer. 2002. The Pfam protein families database. Nucleic Acids Res. 30:276-280. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[r4] 4.Blond, A., J. Peduzzi, C. Goulard, M. J. Chiuchiolo, M. Barthelemy, Y. Prigent, R. A. Salomon, R. N. Farias, F. Moreno, and S. Rebuffat. 1999. The cyclic structure of microcin J25, a 21-residue peptide antibiotic from Escherichia coli. Eur. J. Biochem. 259:747-755. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Functional Analysis of the Gene Cluster Involved in Production of the Bacteriocin Circularin A by Clostridium beijerinckii ATCC 25752

Robèr Kemperman

Marnix Jonker

Arjen Nauta

Oscar P Kuipers

Jan Kok

Abstract