The mechanisms of target cell recognition and producer cell selfprotection (immunity) are both important yet poorly understood issues in the biology of peptide bacteriocins. In this report, we provide genetic and biochemical evidence that lactococcin A, a permeabilizing peptide-bacteriocin from Lactococcus lactis, uses components of the mannose phosphotransferase system (man-PTS) of susceptible cells as target/receptor. We present experimental evidence that the immunity protein LciA forms a strong complex with the receptor proteins and the bacteriocin, thereby preventing cells from being killed. Importantly, the complex between LciA and the man-PTS components (IIAB, IIC, and IID) appears to involve an on-off type mechanism that allows complex formation only in the presence of bacteriocin; otherwise no complexes were observed between LciA and the receptor proteins. Deletion of the man-PTS operon combined with biochemical studies revealed that the presence of the membrane-located components IIC and IID was sufficient for sensitivity to lactococcin A as well as complex formation with LciA. The cytoplasmic component of the man-PTS, IIAB, was not required for the biological sensitivity or for complex formation. Furthermore, heterologous expression of the lactococcal man-PTS operon rendered the insensitive Lactobacillus sakei susceptible to lactococcin A. We also provide evidence that, not only lactococcin A, but other class II peptide-bacteriocins including lactococcin B and some Listeria-active pediocin-like bacteriocins also target the man-PTS components IIC and IID on susceptible cells and that their immunity proteins involve a mechanism in producer cell self-protection similar to that observed for LciA.antimicrobial peptides ͉ Mannose-PTS ͉ receptor ͉ protein complex ͉ coprecipitation

Class IIa bacteriocins target a phylogenetically defined subgroup of mannose-phosphotransferase systems (man-PTS) on sensitive cells. By the use of man-PTS genes of the sensitive Listeria monocytogenes (mpt) and the nonsensitive Lactococcus lactis (ptn) species to rationally design a series of man-PTS chimeras and site-directed mutations, we identified an extracellular loop of the membrane-located protein MptC that was responsible for specific target recognition by the class IIa bacteriocins.Bacteriocins are small, ribosomally synthesized antimicrobial peptides that normally kill bacteria closely related to the bacteriocin producers, but some also target a wider spectrum of bacteria, including a number of pathogens and food spoilage bacterial species (5, 28). Class IIa (pediocin-like) bacteriocins display a broad antimicrobial spectrum, including important pathogens such as Listeria monocytogenes and Enterococcus faecalis. These peptides consist of 37 to 48 nonmodified amino acids, contain a conserved pediocin-box sequence (Y-G-N-G-V/L) in the N-terminal region, and have defined secondary features in their structure: a cationic ␤ sheet at the conserved N terminus and a helix-containing domain at the less-conserved C terminus (16,30). Class IIa bacteriocins target sensitive cells by using the mannose phosphotransferase system (man-PTS) as a receptor (6,10,17,19,33). This sugar uptake system is the major glucose transporter for many bacteria, particularly Firmicutes and Gammaproteobacteria (39). Each man-PTS complex consists of four structural domains: IIC and IID, represented by two membrane-located proteins, and IIA and IIB, which are normally represented by a single cytoplasmic protein that can form reversible contacts with its membrane-located partners (31).It has previously been shown that coexpression of the IIC and IID genes is needed to confer sensitivity to class IIa bacteriocins as well as to the lactococcal bacteriocin lactococcin A and that the cytoplasmic IIAB partner is not involved in this process (10). However, while lactococcin A (belonging to class IIc) targets only the lactococcal man-PTS, the class IIa bacteriocins target man-PTSs of species of diverse genera (e.g., Listeria, Enterococcus, and Lactobacillus) but somehow not those of the Lactococcus genus (24). This genus specificity has been recognized for almost 2 decades (20,23,26); still, the molecular nature underlying the specificity has remained very enigmatic. In the present report we clarify this issue by demonstrating that these two types of bacteriocins exhibit different binding patterns on their receptors: class IIa bacteriocins specifically interact with a defined region of 40 amino acids in the IIC protein whereas lactococcin A has a more complex interaction involving regions from both IIC and IID. MATERIALS AND METHODSBacterial strain and growth conditions. Lactococcus lactis B488 (10), a man-PTS null mutant derived from L. lactis IL1403, was used as an expression host in this study. Lactococcal clones were grown at 30°C in M17 me...

The Abi protein family consists of putative membrane-bound metalloproteases. While they are involved in membrane anchoring of proteins in eukaryotes, little is known about their function in prokaryotes. In some known bacteriocin loci, Abi genes have been found downstream of bacteriocin structural genes (e.g., pln locus from Lactobacillus plantarum and sag locus from Streptococcus pyogenes), where they probably are involved in self-immunity. By modifying the profile hidden Markov model used to select Abi proteins in the Pfam protein family database, we show that this family is larger than presently recognized. Using bacteriocin-associated Abi genes as a means to search for novel bacteriocins in sequenced genomes, seven new bacteriocin-like loci were identified in Gram-positive bacteria. One such locus, from Lactobacillus sakei 23K, was selected for further experimental study, and it was confirmed that the bacteriocin-like genes (skkAB) exhibited antimicrobial activity when expressed in a heterologous host and that the associated Abi gene (skkI) conferred immunity against the cognate bacteriocin. Similar investigation of the Abi gene plnI and the Abi-like gene plnL from L. plantarum also confirmed their involvement in immunity to their cognate bacteriocins (PlnEF and PlnJK, respectively). Interestingly, the immunity genes from these three systems conferred a high degree of crossimmunity against each other's bacteriocins, suggesting the recognition of a common receptor. Site-directed mutagenesis demonstrated that the conserved motifs constituting the putative proteolytic active site of the Abi proteins are essential for the immunity function of SkkI, and to our knowledge, this represents a new concept in self-immunity.

The oval shape of pneumococci results from a combination of septal and lateral peptidoglycan synthesis. The septal cross-wall is synthesized by the divisome, while the elongasome drives cell elongation by inserting new peptidoglycan into the lateral cell wall. Each of these molecular machines contains penicillin-binding proteins (PBPs), which catalyze the final stages of peptidoglycan synthesis, plus a number of accessory proteins. Much effort has been made to identify these accessory proteins and determine their function. In the present paper we have used a novel approach to identify members of the pneumococcal elongasome that are functionally closely linked to PBP2b. We discovered that cells depleted in PBP2b, a key component of the elongasome, display several distinct phenotypic traits. We searched for proteins that, when depleted or deleted, display the same phenotypic changes. Four proteins, RodA, MreD, DivIVA and Spr0777, were identified by this approach. Together with PBP2b these proteins are essential for the normal function of the elongasome. Furthermore, our findings suggest that DivIVA, which was previously assigned as a divisomal protein, is required to correctly localize the elongasome at the negatively curved membrane region between the septal and lateral cell wall.

Staphylococcus aureus needs to control the position and timing of cell division and cell wall synthesis to maintain its spherical shape. We identified two membrane proteins, named CozEa and CozEb, which together are important for proper cell division in S. aureus. CozEa and CozEb are homologs of the cell elongation regulator CozE of Streptococcus pneumoniae. While cozEa and cozEb were not essential individually, the ΔcozEaΔcozEb double mutant was lethal. To study the functions of cozEa and cozEb, we constructed a CRISPR interference (CRISPRi) system for S. aureus, allowing transcriptional knockdown of essential genes. CRISPRi knockdown of cozEa in the ΔcozEb strain (and vice versa) causes cell morphological defects and aberrant nucleoid staining, showing that cozEa and cozEb have overlapping functions and are important for normal cell division. We found that CozEa and CozEb interact with and possibly influence localization of the cell division protein EzrA. Furthermore, the CozE-EzrA interaction is conserved in S. pneumoniae, and cell division is mislocalized in cozE -depleted S. pneumoniae cells. Together, our results show that CozE proteins mediate control of cell division in S. aureus and S. pneumoniae, likely via interactions with key cell division proteins such as EzrA.