The production of pediocin PA-1, a small heat-stable bacteriocin, is associated with the presence of the 9.4-kbp plasmid pSRQii in Pediococcus acidilactici PAC1.0. It was shown by subcloning of pSRQll in Escherichia coli cloning vectors that pediocin PA-1 is produced and, most probably, secreted by E. coli cells. Deletion analysis showed that a 5.6-kbp SalI-EcoRI fragment derived from pSRQ11 is required for pediocin PA-1 production. Nucleotide sequence analysis of this 5.6-kbp fragment indicated the presence of four clustered open reading frames (pedA, pedB, pedC, and pedD). The pedA4 gene encodes a 62-amino-acid precursor of pediocin PA-1, as the predicted amino acid residues 19 to 62 correspond entirely to the amino acid sequence of the purified pediocin PA-1. Introduction of a mutation in pedAl resulted in a complete loss of pediocin production. The pedB and pedC genes, encoding proteins of 112 and 174 amino acid residues, respectively, are located directly downstream of the pediocin structural gene. Functions could not be assigned to their gene products; mutation analysis showed that the PedB protein is not involved in pediocin PA-1 production. The mutation analysis further revealed that the fourth gene, pedD, specifying a relatively large protein of 724 amino acids, is required for pediocin PA-1 production in E. coli. The predicted PedD protein shows strong similarities to several ATP-dependent transport proteins, including the E. coli hemolysin secretion protein HlyB and the ComA protein, which is required for competence induction for genetic transformation in Streptococcus pneumoniae. These similarities suggest strongly that the PedD product is involved in the translocation of pediocin PA-1.

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Pediocin PA-1 is a bacteriocin which is produced by Pediococcus acidilactici PAC1.0. We demonstrate that pediocin PA-1 kills sensitive Pediococcus cells and acts on the cytoplasmic membrane. In contrast to its lack of impact on immune cells, pediocin PA-1 dissipates the transmembrane electrical potential and inhibits amino acid transport in sensitive cells. Pediocin interferes with the uptake of amino acids by cytoplasmic membrane vesicles derived from sensitive cells, while it is less effective with membranes derived from immune cells. In liposomes fused with membrane vesicles derived from both sensitive and immune cells, pediocin PA-1 elicits an efflux of small ions and, at higher concentrations, an efflux of molecules having molecular weights of up to 9,400. Our data suggest that pediocin PA-1 functions in a voltage-independent manner but requires a specific protein in the target membrane.

The bacteriocin pediocin PA-1 operon of Pediococcus acidilactici PAC1.0 encompasses four genes: pedA, pedB, pedC and pedD. Transcription of the operon results in the formation of two overlapping transcripts, probably originating from a single promoter upstream of pedA. The major transcript comprises pedA, pedB, and pedC, while a minor transcript encompasses all of these genes and pedD. By deletion analysis and overexpression of pedB in Pediococcus pentosaceus we demonstrate that this gene encodes the pediocin PA-1 immunity protein. Prepediocin is active in Escherichia coli and when pedA was expressed concomitantly with pedD both the precursor and the mature form of pediocin were observed intracellularly. Extracellular pediocin was only detected if both pedC and pedD were present. The N-terminal domains of PedD and a subgroup of bacteriocin ABC-transporters are conserved. Expression of only this domain of PedD in cells producing prepediocin was sufficient for prepediocin processing. From these results we conclude that both PedC and PedD are essential for pediocin transport, and that PedD is capable of processing prepediocin.

A 6.5-kilobase HindlIl fragment that specifies the proteolytic activity of Streptococcus cremoris Wg2 was sequenced entirely. The nucleotide sequence revealed two open reading frames (ORFs), a small ORF1 with 295 codons and a large ORF2 containing 1,772 codons. For both ORFs, there was no stop codon on the HindlIl fragment. A partially overlapping PstI fragment was used to locate the translation stop of the large ORF2. The entire ORF2 contained 1,902 coding triplets, followed by an apparently rho-independent terminator sequence. The inferred amino acid sequence would result in a protein of 200 kilodaltons. Both ORFs have their putative transcription and translation signals in a 345-base-pair ClaI fragment. ORF2 is preceded by a promoter region containing a 15-base-pair complementary direct repeat. Both the truncated 33and the 200-kilodalton proteins have a signal peptide-like N-terminal amino acid sequence. The protein specified by ORF2 contained regions of extensive homology with serine proteases of the subtilisin family. Specifically, amino acid sequences involved in the formation of the active site (viz., Asp-32, His-64, and Ser-221 of the subtilisins) are well conserved in the S. cremoris Wg2 proteinase. The homologous sequences are separated by nonhomologous regions which contain several inserts, most notably a sequence of approximately 200 amino acids between the His and Ser residues of the active site. * Corresponding author. specified two proteins, A and B, of the proteolytic systemn of S. cremoris Wg2 in S. lactis as well as in B. subtilis. Here we report on the nucleotide sequence of the gene that specifies the cell wall-bound proteinase of S. cremoris Wg2 and on some characteristics of this sequencs and of the enzyme as deduced from the predicted amino acid sequence. MATERIALS AND METHODS Bacterial strains, plasmids, and media. Growth and maintenance of bacteria and selective conditions have been previously described (14). Plasmid pGKV500 (14) and its derivatives were constructed and maintained in B. subtilis PSL1 (21). Escherichia coli JM101 (44) was used as the host for M13 and its derivatives. Molecular cloning techniques. Plasmid DNA was isolated as described previously (14). Restriction nuclease enzymes, T4 DNA ligase, and the Klenow fragment of E. coli DNA polymerase I were purchased from Boehringer Mannheim Biochemicals and used as recommended by the manufacturer. General procedures for cloning and DNA mnanipulations were essentially as described by Maniatis et al. (18). Competent cells of E. coli were transformed as described by Mandel and Higa (17). Protoplasts of B. subtilis were transformed as described by Chang and Cohen (2). DNA sequence analysis. Subfragments of the 6.5-kb Hindlll fragment of pGKV500 were cloned in both orientations in phage M13 by using M13 mplO, mpll, mpl8, and mpl9 (44). The dideoxynucleotide sequencing method of Sanger et al. (25) was used with buffer gradient gels and [a-35S]dATP (1). Synthetic 17-mer primers were prepared on a model 380A DNA synthesizer (Applied Bio...

Directly upstream of the Lactococcus lachs subsp. cremoris Wg2 proteinase gene is an oppositely directed open reading frame (ORF1). The complete nucleotide sequence of ORF1, encoding a 33-kilodalton protein, was determined. A protein of approximately 32 kilodaltons was synthesized when ORFI was expressed in Escherichia coli by using a T7 RNA polymerase-specific promoter. L. lactis subsp. lactis MG1363 transformants carrying the proteinase gene but lacking, ORFl were phenotypicaHly proteinase deficient, unlike transformants carrying both the proteinase gene and ORF1. Synthesis and secretion of proteinase antigen by L. lactis could be detected with proteinase-directed monoclonal antibodies regardless of whether ORF1 was present. The requirement of ORF1 for proteinase activation was reflected in a reduction in the molecular weight of the secreted proteinase. Furthermore, deletion of the 130 C-terminal amino acids of the Wg2 proteinase prevented attachment of the enzyme to lactococcal cells.

The structural gene and the regulatory DNA sequence of the yeast Hansenula polymorpha methanol oxidase have been isolated. According to the nucleotide sequence data obtained, the structural gene encodes a 664 amino acids long protein, contains no intervening sequences, and the 5'- and 3'-non-coding region contains several sequences implicated in transcription initiation and termination in the yeast Saccharomyces cerevisiae. Although the methanol oxidase is translocated to the peroxisomes, no cleavable signal sequence was found at the N-terminus of the protein.

Bacteriophage p2 belongs to the most prevalent lactococcal phage group (936) responsible for considerable losses in industrial production of cheese. Immunization of a llama with bacteriophage p2 led to higher titers of neutralizing heavy-chain antibodies (i.e., devoid of light chains) than of the classical type of immunoglobulins. A panel of p2-specific single-domain antibody fragments was obtained using phage display technology, from which a group of potent neutralizing antibodies were identified. The antigen bound by these antibodies was identified as a protein with a molecular mass of 30 kDa, homologous to open reading frame 18 (ORF18) of phage sk1, another 936-like phage for which the complete genomic sequence is available. By the use of immunoelectron microscopy, the protein is located at the tip of the tail of the phage particle. The addition of purified ORF18 protein to a bacterial culture suppressed phage infection. This result and the inhibition of cell lysis by anti-ORF18 protein antibodies support the conclusion that the ORF18 protein plays a crucial role in the interaction of bacteriophage p2 with the surface receptors of Lactococcus lactis.Lactococcus lactis is a gram-positive lactic acid bacterium used for the manufacture of fermented dairy products (2). The milk fermentation process is susceptible to infection by bacteriophages found in raw milk (3,19,(32)(33)(34) or by induction of prophages from lysogenic starter strains (19). The phage infection results in lysis of the bacteria, leading to production delays, variations in the taste and texture of the products, or even complete failure of fermentation. To minimize economic losses by phage infections, a variety of precautions are used (35,36). Lactococcal phages fall into three prevalent groups of DNA homology, 936-, c2-and P335-like phages (32-34). Characteristics of these phages include a double-stranded DNA genome and a long noncontractile tail. The 936 and P335 groups have a small isometric head, while members of the c2 group have a prolate head.We describe here the generation of phage-neutralizing monoclonal single-domain antibody fragments (V H H) derived from cameloid heavy-chain antibodies. In the blood of Camelidae, a high proportion of the immunoglobulins consists of homodimers of only heavy chains, devoid of light chains (17). As described in this and other papers, it is possible to elicit good immune response in camelids against complex protein mixtures, phages, or even whole organisms (26). Genes encoding V H H fragments that bind to these complex protein mixtures can be selected easily. In such libraries of binders, there is a high probability of finding V H Hs that block essential biological processes, mainly because of the long CDR3, which can block active centers (27).It was demonstrated that after immunization of a llama with lactococcal bacteriophage p2 (936 group) the fraction of heavy-chain antibodies contained about 10-fold higher neutralizing activity than conventional antibodies. We generated a phage display library (31, 3...