Bifunctional Gene Cluster lnqBCDEF Mediates Bacteriocin Production and Immunity with Differential Genetic Requirements

Abstract

A comprehensive gene disruption of lacticin Q biosynthetic cluster lnqQBCDEF was carried out. The results demonstrated the necessity of the complete set of lnqQBCDEF for lacticin Q production, whereas immunity was flexible, with LnqEF (ABC transporter) being essential for and LnqBCD partially contributing to immunity.

TEXT

Bacteriocins are a diverse group of antimicrobial peptides that are ribosomally synthesized and extracellularly released by their producers. The genes necessary for bacteriocin production and immunity are located near the structural gene and are generally arranged as an operon (1, 2). Lacticin Q (LnqQ), produced by Lactococcus lactis QU 5 (3), is an unmodified leaderless bacteriocin that has a unique gene cluster (lnqBCDEF) following the structural gene (lnqQ) (4). As predicted in silico, lnqEF encodes an ATP-binding cassette (ABC) transporter with LnqE as the membrane-spanning domain and LnqF as the cytoplasmic ATPase domain. These two open reading frames (ORFs) share some sequence similarities with ORF3 and Abc2 in the bht-b locus of Streptococcus rattus BHT (5) whose bacteriocin, BHT-B, shows some homology to LnqQ (47% identity). LnqBCD has no sequence similarity to any other bacteriocin biosynthetic proteins. However, according to a BLAST search, LnqB (79 amino acids [aa]) shows partial homology to the ABC transporter permease (407 aa) of Listeria welshimeri (36% identity) or the Na⁺/H⁺ antiporter (1,143 aa) of Puccinellia tenuiflora (30% identity), whereas LnqC (159 aa) shows homology to the membrane permease (181 aa) of Renibacterium salmoninarum (27% identity) or the membrane-flanked domain protein (165 aa) of Xylanimonas cellulosilytica (25% identity). LnqD, the largest ORF (432 aa) in the lnqQ locus, has some homology to the membrane protein (486 aa) of Lactobacillus buchneri (25% identity) and the YdbT homolog protein (484 aa) of Leuconostoc pseudomesenteroides (23% identity). Previous work has revealed that a set of lnqBCDEF confers the secretion and self-immunity of LnqQ; however, the role of each gene product has not been elucidated (4). Thus, in this study, comprehensive gene disruption was carried out to determine the necessity of each gene and to deduce its role in LnqQ biosynthesis.

The bacterial strains and plasmids used in this study are listed in Table 1. Culture conditions for the strains listed, DNA manipulation, and other molecular cloning techniques were as described previously (4, 7–9). An inverse PCR technique (11) was used to create an in-frame deletion of the gene(s) of interest from the template plasmid, pLNQ, which contains lnqQBCDEF under the control of lactococcal promoter P₃₂ (4). In one case, primers invF1 and invR1 (Table 2) were used to amplify the entire plasmid (pLNQ) without the lnqB region. The PCR products were then phosphorylated with T4 polynucleotide kinase (Toyobo, Osaka, Japan) and self-ligated with Ligation-High (Toyobo), resulting in the generation of a plasmid with lnqB disrupted (pLNQΔB). Similarly, plasmids with each gene or several genes disrupted were generated by using the primers listed in Table 2 and were introduced into Escherichia coli DH5α for plasmid replication. After the sequence was confirmed, each recombinant plasmid was introduced into L. lactis subsp. cremoris NZ9000 (L. lactis NZ9000) by the electroporation method (12). The LnqQ production (secretion) and self-immunity levels of recombinants were evaluated by the spot-on-lawn method (13). The activity titer, described in arbitrary units (AU) per milliliter, was defined as the reciprocal of the highest dilution causing a growth inhibition zone on the indicator lawn. The MIC in this study was defined as the lowest concentration of purified bacteriocin that produced a measurable inhibition zone on the indicator lawn.

Table 1.

Bacterial strains and plasmids used in this study

Bacterial strain or plasmid	Relevant characteristic(s)	Reference or source
Strains
L. lactis NZ9000	Plasmid-free derivative of L. lactis subsp. cremoris MG1363, pepN::nisRK	6
Bacillus coagulans JCM 2257T	Bacteriocin indicator	JCMb
Escherichia coli DH5α	supE44 ΔlacU169 (ϕ80lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1	TaKaRa
L. lactis QU 5	Lacticin Q producer	Lab collection (3)
L. lactis QU 14	Lacticin Z producer	Lab collection (7)
L. lactis QU 4	Lactococcin Q producer	Lab collection (8)
Lactococcus sp. strain QU 12	Lactocyclicin Q producer	Lab collection (9)
L. lactis NCDO 497	Nisin A producer	NCDOc
Enterococcus faecium T13	Enterocin L50 producer	Lab collection
Plasmids
pMG36c	pWV01-based expression vector carrying the strong lactococcal promoter P32; Cmra	10
pMGlnqQ	pMG36c derivative with lnqQ under the control of P32; Cmr	4
pLNQ	pMGlnqQ derivative with lnqBCDEF downstream of lnqQ; Cmr	4
pLNQΔQ	pLNQ derivative lacking lnqQ; Cmr	4
pLNQΔQF	pLNQ derivative lacking lnqQ, lnqF; Cmr	This study
pLNQΔB	pLNQ derivative lacking lnqB; Cmr	This study
pLNQΔC	pLNQ derivative lacking lnqC; Cmr	This study
pLNQΔD	pLNQ derivative lacking lnqD; Cmr	This study
pLNQΔE	pLNQ derivative lacking lnqE; Cmr	This study
pLNQΔF	pLNQ derivative lacking lnqF; Cmr	This study
pLNQΔBC	pLNQ derivative lacking lnqBC; Cmr	This study
pLNQΔBCD	pLNQ derivative lacking lnqBCD; Cmr	This study
pLNQΔBCDE	pLNQ derivative lacking lnqBCDE; Cmr	This study

^aCm^r, chloramphenicol resistance.

^bJCM, Japan Collection of Microorganism, Saitama, Japan.

^cNCDO, National Collection of Dairy Organisms, Reading, United Kingdom.

Table 2.

Primers used in this study

Primer	Nucleotide sequence	Application
invF1	5′-GCATTGGTAGGGAGAGCG-3′	pLNQΔB
invR1	5′-CATCTAGATGATCAAAAAATTACTTAATACC-3′	pLNQΔB/pLNQΔBC/pLNQΔBCD/pLNQΔBCDE
invF2	5′-GAGTTAATGATAAATGCTTTGGAAAGTC-3′	pLNQΔC/pLNQΔBC
invR2	5′-CGATTTGAAACCTTGAAATATAAACTACT-3′	pLNQΔC
invF3	5′-GTATTCAGGTGTTTAGAAATGAGG-3′	pLNQΔD/pLNQΔBCD
invR3	5′-GATAGATCTCATTTATTAAAGTTAGTCTCG-3′	pLNQΔD/
invF4	5′-CAATCTAGTTCTAAAAAATGTCAATATGG-3′	pLNQΔE/pLNQΔBCDE
invR4	5′-CTTGCATTATTTCCTCATTTCTAAACACC-3′	pLNQΔE
invF5	5′-AAGAGCAAAGAAAGTTGAAAGATGTG-3′	pLNQΔF/pLNQΔQF
invR5	5′-CCATATTGACATTTTTTAGAACTAGATTG-3′	pLNQΔF/pLNQΔQF

An overview of the LnqQ secretion/immunity levels of recombinants is shown in Fig. 1. With regard to LnqQ secretion, antimicrobial activity in the culture supernatant of L. lactis NZ9000(pLNQ) against Bacillus coagulans JCM 2257^T, which is a highly LnqQ-sensitive indicator strain, was estimated at 25,600 AU ml⁻¹. This activity was, however, totally lost when the lnqC, lnqD, lnqE, or lnqF gene was disrupted, whereas slight activity (800 AU ml⁻¹) was detected in an lnqB-deficient recombinant. Meanwhile, antimicrobial activity was detected in the cell extracts of all of the recombinants (data not shown), indicating that LnqQ was expressed but not extracellularly released by these recombinants. On the one hand, disruption of a single gene did not have a severe impact on LnqQ immunity, except for the disruption of lnqF, which led to a drastic loss of immunity. Even in the absence of lnqB, lnqC, or lnqD, the recombinants showed >64-fold higher immunity levels than the control strain, L. lactis NZ9000(pMG36c), while the lack of lnqE slightly reduced the immunity level. Considering the abundant immunity achieved by lnqBC- or lnqBCD-deficient recombinants, LnqEF is clearly the main contributor to LnqQ immunity. In many other cases, an ABC-type transporter mediates bacteriocin immunity by extruding the peptide and thus preventing direct access to the target molecule (14–17). Although LnqE is supposedly an ABC transporter constituent, considerable immunity was observed in an lnqE-deficient recombinant, L. lactis NZ9000(pLNQΔE). This observation suggests the possibility that another factor(s) can complement the role of LnqE; furthermore, this complementation should be accomplished by any of the LnqBCD proteins but not by host-derived proteins when considering the deficient immunity observed in an lnqBCDE-deficient recombinant. In addition, all of those membrane proteins (LnqBCDE) provided the host cells with partial immunity, even when the ABC transporter was inactive (pLNQΔF/pLNQΔQF), which was, however, far inferior to full immunity.

Fig 1.

Overview of the lacticin Q production and immunity of L. lactis NZ9000 recombinants. Gene organizations corresponding to the recombinant plasmids are schematically depicted. Arrows with patterns indicate cloned genes, bent arrows represent lactococcal promoter P₃₂, and lollipops represent the putative terminator region found downstream of lnqF. LnqQ secretion and immunity levels were evaluated as described in the text and are represented as AU of activity against B. coagulans JCM 2257^T per milliliter or MICs of purified LnqQ, respectively.

The other leaderless bacteriocins genetically characterized to date have only one ABC-type transporter encoded in their gene loci (5, 18–23). This may indicate that leaderless bacteriocins have in common the feature of having one dedicated ABC transporter mediate both secretion and immunity. LmrB, an ABC-type multidrug efflux pump, facilitates the secretion and the immunity of a leaderless bacteriocin, LsbB (20). A remarkable aspect of LmrB is its broad substrate specificity, by which it works for another bacteriocin, LsbA, from the same producer. Interestingly, LmrB shows high degrees of similarity to other leaderless bacteriocin transporters, EntQB (19) and AurT (22), despite the absence of observed sequence similarity between the structures of these bacteriocins. This fact gave rise to a simple question regarding the substrate specificity of LnqQ immunity. In this regard, several types of bacteriocins, i.e., nisin A (class I), lactococcin Q (class IIb), lactocyclicin Q (class IIc), enterocin L50 (class IId, leaderless), and lacticin Z (class IId, LnqQ homologue), were used to evaluate the specificity of LnqQ immunity. Both full immunity (lnqBCDEF) and partial immunity (lnqBCDE) to only lacticin Z and not to the other types of bacteriocins were seen (Fig. 2).

Fig 2.

Lacticin Q/Z-specific immunity facilitated by LnqBCDEF. Twofold serially diluted bacteriocin-containing culture supernatants were spotted onto a lawn containing the strains tested, i.e., L. lactis NZ9000(pMG36c) (A), NZ9000(pLNQΔQF) (B), and NZ9000(pLNQΔQ) (C).

In summary, the secretion of LnqQ is strictly controlled by the presence of LnqBCDEF, whereas immunity is flexible in that LnqEF (ABC transporter) is the minimal unit required for sufficient immunity. Here, LnqBCD could be considered accessory proteins that support the activity of LnqEF, although such proteins potentially associated with other bacteriocin transporters are usually single gene products (24–27). A distinct feature of leaderless bacteriocins is their synthesis without a leader peptide. Because this N-terminal extension in the bacteriocin prepeptide acts as a recognition signal for its dedicated transporter (2), there should be a unique bacteriocin transfer or recognition system in leaderless bacteriocin producers. The present results might indicate the involvement of LnqBCD in this unknown mechanism. The detailed function of LnqBCD, dependent on or independent of LnqEF, is now under investigation.