ABSTRACT This study reports on the identification and characterization of a novel abortive infection system, AbiU, from Lactococcus lactis. AbiU confers resistance to phages from the three main industrially relevant lactococcal phage species: c2, 936, and P335. The presence of AbiU reduced the efficiency of plaquing against specific phage from each species as follows: 3.7 × 10 −1 , 1.0 × 10 −2 , and 1.0 × 10 −1 , respectively. abiU involves two open reading frames, abiU1 (1,772 bp) and abiU2 (1,019 bp). Evidence indicates that AbiU1 is responsible for phage resistance and that AbiU2 may downregulate phage resistance against 936 and P335 type phages but not c2 type phage. AbiU appeared to delay transcription of both phage 712 and c2, with the effect being more marked on phage c2.

Dairy fermentations involving Lactococcus lactis and more recently Streptococcus thermophilus, are commonly attacked by bacteriophages. Efforts to protect these dairy starter cultures have resulted in a significant body of knowledge about the bacterio-phages, their interactions with the host, and natural phage defense mechanisms that have evolved within bacteria operating under the most dynamic and devastating phage environment faced by industrial fermentations. This paper will overview this area and discuss the novel genetic approaches that are now being investigated in an effort to provide long term phage protection to dairy starter cultures that are used extensively in the industry.

Lactacin F is a two-component class II bacteriocin produced by Lactobacillus johnsonii VPI 11088. The laf operon is composed of the bacteriocin structural genes, lafA and lafX, and a third open reading frame, ORFZ. Two strategies were employed to study the function of ORFZ. This gene was disrupted in the chromosome of NCK64, a lafA729 lafX ORFZ derivative of VPI 11088. A disruption cassette consisting of ORFZ interrupted with a cat gene was cloned into pSA3 and introduced into NCK64. Manipulation of growth temperatures and antibiotic selection resulted in homologous recombination which disrupted the chromosomal copy of ORFZ with the cat gene. This ORFZ mutation resulted in loss of immunity to lactacin F but had little effect on production of LafX, which is not bactericidal without LafA. Expression of ORFZ in this ORFZ- background rescued the immune phenotype. Expression of ORFZ in a bacteriocin-sensitive derivative of VPI 11088 also reestablished immunity. These data indicate that ORFZ, renamed lafI, encodes the immunity factor for the lactacin F system. The sensitivity of various Lactobacillus strains to lactacin F was further evaluated. Lactacin F inhibited 11 strains including several members of the A1, A2, A3, A4, B1, and B2 L. acidophilus homology groups. Expression of lafI in bacteriocin-sensitive strains L. acidophilus ATCC 4356, L. acidophilus NCFM/N2, L. fermentum NCDO1750, L. gasseri ATCC 33323, and L. johnsonii ATCC 33200 provided immunity to lactacin F. Furthermore, it was shown that lactacin F production by VPI 11088 could be used to select for L. fermentum NCDO1750 transformants containing the recombinant plasmid encoding LafI. The data demonstrate that lafI is functional in heterologous hosts, suggesting that it may be a suitable food-grade genetic marker for use in lactobacillus species.

The lactacin F complex, composed of LafA and LafX peptides, is produced by Lactobacillus johnsonii VPI 11088 and is active against five other Lactobacillus species and Enterococcus faecalis. The genetic determinants encoding the lactacin F complex are organized in a 1-kb polycistronic operon which comprises three genes, lafA, lafX, and ORFZ (encoding the putative immunity protein). The lafA and lafX genes encode the bacteriocin precursors with N-terminal extensions characterized by a Gly-Gly(-1)*Xaa(+1) cleavage site (*). The Gly-Gly motif is conserved in several other bacteriocins, including carnobacteriocins A, BM1, and B2. Carnobacterium piscicola LV17 produces carnobacteriocins which are active against Listeria monocytogenes and other lactic acid bacteria. In this study, the lactacin F operon was introduced into C. piscicola LV17. The transformants produced lactacin F concurrently with the carnobacteriocins. When the lafA and lafX genes were separated and cloned individually into LV17, production of either LafA or LafX by C. piscicoia LV17 was detected by complementation with L. johnsonii clones producing LafX or LafA, respectively. Transformants of C. piscicola LV17 which produced lactacin F, LafA, or LafX, in combination with the carnobacteriocins, were assayed for an increased and expanded inhibitory spectrum. The recombinant organisms were only active against lactacin F- and carnobacteriocin-sensitive strains. A plasmidless derivative of LV17 which does not produce the carnobacteriocins failed to produce lactacin F, LafA, or LafX when transformed with the appropriate recombinant plasmids. The ability of C. piscicola LV17 to produce lactacin F demonstrates that the machinery for the carnobacteriocins is capable of processing and exporting bacteriocins from both systems.

The lactacin F complex, composed of LafA and LafX peptides, is produced by Lactobacillus johnsonii VPI 11088 (ATCC 11506) and is active against various lactobacilli and Enterococcus faecalis. The genetic determinants encoding the lactacin F peptides, LafA and LafX, are organized in a chromosomal operon comprised of genes lafA, lafX, and ORFZ. The lactacin F operon was introduced into Leuconostoc (Lc.) gelidum UAL187-22 which produces leucocin A. Leucocin A, a plasmid-encoded bacteriocin, inhibits E. faecalis, Listeria monocytogenes, and other lactic acid bacteria. The culture supernatant of the Leuconostoc transformant containing the lactacin F operon inhibited both lactacin F-and leucocin A-sensitive indicators. Concurrent expression of both bacteriocins did not alter the production of native leucocin A. Additive inhibitory effects due to the presence of both bacteriocins were not observed. An isogenic derivative of UAL187-22, which has lost the leucocin-encoding plasmid, was unable to produce active lactacin F when transformed with the appropriate recombinant plasmid. The ability of Lc. gelidum UAL187-22 to produce lactacin F demonstrates that the export system for leucocin A is capable of producing both bacteriocins simultaneously.

Lactacin F is a membrane-active bacteriocin produced by Lactobacillus johnsonii VPI11088 (Laf+). The genetic determinants encoding lactacin F are organized in a 1-kb polycistronic operon composed of a promoter (P(laf)), three genes (lafA, lafX, and ORFZ), and a functional rho-independent transcription terminator. Two Laf- derivatives of VPI11088, designated NCK64 and NCK65, were characterized. NCK64 contained a frameshift mutation in the lafA gene causing premature termination of translation. NCK65 harbored a 10-kb chromosomal deletion covering the laf operon. When the lafA gene was cloned independently and expressed in NCK65, bacteriocin activity was limited to L. helveticus 87, only one of the six known lactacin F-sensitive (Lafs) indicators. When lafX was introduced into NCK65, no bacteriocin activity against any of the sensitive strains was detected. Genetic combination of lafA and lafX, in cis or in trans, restored bacteriocin activity against all Lafs indicators. When two NCK65 clones containing either lafA or lafX were plated slightly apart on agar plates, fully active lactacin F was present in the intervening area where the two excreted gene products, LafA and LafX, diffused together. The genetic analysis revealed that the interaction of two bacteriocinogenic peptides encoded within the laf operon is likely to participate in the formation of poration complexes in the membranes of susceptible bacteria.