Putative Immunity Protein Research Articles

Regulation of Bacteriocin Production in Streptococcus mutans by the Quorum-Sensing System Required for Development of Genetic Competence

In Streptococcus mutans, competence for genetic transformation and biofilm formation are dependent on the two-component signal transduction system ComDE together with the inducer peptide pheromone competence-stimulating peptide (CSP) (encoded by comC). Here, it is shown that the same system is also required for expression of the nlmAB genes, which encode a two-peptide nonlantibiotic bacteriocin. Expression from a transcriptional nlmAB'-lacZ fusion was highest at high cell density and was increased up to 60-fold following addition of CSP, but it was abolished when the comDE genes were interrupted. Two more genes, encoding another putative bacteriocin and a putative bacteriocin immunity protein, were also regulated by this system. The regions upstream of these genes and of two further putative bacteriocin-encoding genes and a gene encoding a putative bacteriocin immunity protein contained a conserved 9-bp repeat element just upstream of the transcription start, which suggests that expression of these genes is also dependent on the ComCDE regulatory system. Mutations in the repeat element of the nlmAB promoter region led to a decrease in CSP-dependent expression of nlmAB'-lacZ. In agreement with these results, a comDE mutant and mutants unable to synthesize or export CSP did not produce bacteriocins. It is speculated that, at high cell density, bacteriocin production is induced to liberate DNA from competing streptococci.

Macroarray analysis of coelomocyte gene expression in response to LPS in the sea urchin. Identification of unexpected immune diversity in an invertebrate

The purple sea urchin, Strongylocentrotus purpuratus, is a member of the phylum Echinodermata, which is basal to the phylum Chordata within the deuterostome lineage of the animal kingdom. This relationship makes the analysis of the sea urchin immune system relevant to understanding the evolution of the deuterostome immune system leading to the Vertebrata. Subtractive suppression hybridization was employed to generate cDNA probes for screening high-density arrayed, conventional cDNA libraries to identify genes that were upregulated in coelomocytes responding to lipopolysaccharide. Results from 1,247 expressed sequence tags (ESTs) were used to infer that coelomocytes upregulated genes involved in RNA splicing, protein processing and targeting, secretion, endosomal activities, cell signaling, and alterations to the cytoskeletal architecture including interactions with the extracellular matrix. Of particular note was a set of transcripts represented by 60% of the ESTs analyzed, which encoded a previously uncharacterized family of closely related proteins, provisionally designated as 185/333. These transcripts exhibited a significant level of variation in their nucleotide sequence and evidence of putative alternative splicing that could yield up to 15 translatable elements. On the basis of the striking increase in gene expression in response to lipopolysaccharide and the unexpected level of diversity of the 185/333 messages, we propose that this set of transcripts encodes a family of putative immune response proteins that may represent a major component of an immunological response to bacterial challenge.

DNA analysis of the genes encoding acidocin LF221 A and acidocin LF221 B, two bacteriocins produced by Lactobacillus gasseri LF221.

Lactobacillus gasseri LF221, an isolate from the feces of a child, produces two bacteriocins. Standard procedures for molecular techniques were used to locate, clone and sequence the fragments of LF221 chromosomal DNA carrying the acidocin LF221 A and B structural genes, respectively. Sequencing analysis revealed the gene of acidocin LF221 A to be an open reading frame encoding a protein composed of 69 amino acids, including a 16-amino-acid N-terminal extension. The acidocin LF221 B gene was found to encode a 65-amino-acid bacteriocin precursor with a 17-amino-acid N-terminal leader peptide. DNA homology searches showed similarities of acidocin LF221 A to brochocin B, lactococcin N and thermophilin B, whereas acidocin LF221 B exhibited some homology to lactacin F and was virtually identical to gassericin X. The peptides encoded by orfA1 and orfB3 showed characteristics of class II bacteriocins and are suspected to be the complementary peptides of acidocin A and B, respectively. orfA3 and orfB5 are proposed to encode putative immunity proteins for the acidocins. Acidocin LF221 A and acidocin LF221 B are predicted to be members of the two-component class II bacteriocins, where acidocin LF221 A appears to be a novel bacteriocin. L. gasseri LF221 is being developed as a potential probiotic strain and a food/feed preservative. Detailed characterization of its acidocins is an important piece of background information useful in applying the strain into human or animal consumption. The genetic information on both acidocins also enables tracking of the LF221 strain in mixed populations and complex environments.

Purification and genetic characterization of plantaricin NC8, a novel coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8.

A new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated from Lactobacillus plantarum NC8 cultures which had been induced with Lactococcus lactis MG1363 or Pediococcus pentosaceus FBB63. This bacteriocin consists of two distinct peptides, named alpha and beta, which were separated by C(2)-C(18) reverse-phase chromatography and whose complementary action is necessary for full plantaricin NC8 activity. N-terminal sequencing of both purified peptides showed 28 and 34 amino acids residues for PLNC8 alpha and PLNC8 beta, respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da (alpha) and 4,000 Da (beta). The corresponding genes, designated plNC8A and plNC8B, were sequenced, and their nucleotide sequences revealed that both peptides are produced as bacteriocin precursors of 47 and 55 amino acids, respectively, which include N-terminal leader sequences of the double-glycine type. The mature alpha and beta peptides contain 29 and 34 amino acids, respectively. An open reading frame, orfC, which encodes a putative immunity protein was found downstream of plNC8B and overlapping plNC8A. Upstream of the putative -35 region of plNC8B, two direct repeats of 9 bp were identified, which agrees with the consensus sequence and structure of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism.

Characterization and heterologous expression of a class IIa bacteriocin, plantaricin 423 from Lactobacillus plantarum 423, in Saccharomyces cerevisiae

Lactobacillus plantarum 423 produces a small heat-stable antimicrobial protein designated plantaricin 423. This protein is bactericidal for many Gram-positive foodborne pathogens and spoilage bacteria, including Listeria spp., Staphylococcus spp., Pediococcus spp., Lactobacillus spp., etc. The DNA sequence of the plantaricin 423-encoding region on plasmid pPLA4 revealed a four open reading frame (ORF) operon structure similar to pediocin PA-1/AcH from Pediococcus acidilactici and coagulin from Bacillus coagulans I 4. The first ORF, plaA, encodes a 56-amino acid prepeptide consisting of a 37-amino acid mature molecule, with a 19-amino acid N-terminal leader peptide. The second ORF, plaB, encodes a putative immunity protein with protein sequence similarities to several bacteriocin immunity proteins. The plaC and plaD genes are virtually identical to pedC and pedD of the pediocin PA-1 operon, as well as coaC and coaD of the coagulin operon. Plantaricin 423 was cloned on a shuttle vector under the control of a yeast promoter and heterologously produced in Saccharomyces cerevisiae.

Self-protection against cell wall hydrolysis in Streptococcus milleri NMSCC 061 and analysis of the millericin B operon.

Streptococcus milleri NMSCC 061 produces an endopeptidase, millericin B, which hydrolyzes the peptide moiety of susceptible cell wall peptidoglycan. The nucleotide sequence of a 4.9-kb chromosomal region showed three open reading frames (ORFs) and a putative tRNA(Leu) sequence. The three ORFs encode a millericin B preprotein (MilB), a putative immunity protein (MilF), and a putative transporter protein (MilT). The milB gene encodes a 277-amino-acid preprotein with an 18-amino-acid signal peptide with a consensus IIGG cleavage motif. The predicted protein encoded by milT is homologous to ABC (ATP-binding cassette) transporters of several bacteriocin systems and to proteins implicated in the signal-sequence-independent export of Escherichia coli hemolysin A. These similarities strongly suggest that the milT gene product is involved in the translocation of millericin B. The gene milF encodes a protein of 302 amino acids that shows similarities to the FemA and FemB proteins of Staphylococcus aureus, which are involved in the addition of glycine to a pentapeptide peptidoglycan precursor. Comparisons of the cell wall mucopeptide of S. milleri NMSCC 061(resistant to lysis by millericin B) and S. milleri NMSCC 051(sensitive) showed a single amino acid difference. Serial growth of S. milleri NMSCC 051 in a cell wall minimal medium containing an increased concentration of leucine resulted in the in vivo substitution of leucine for threonine in the mucopeptide of the cell wall. A cell wall variant of S. milleri NMSCC 051 (sensitive) that contained an amino acid substitution (leucine for threonine) within its peptidoglycan cross bridge showed partial susceptibility to millericin B. The putative tRNA(Leu) sequence located upstream of milB may be a cell wall-specific tRNA and could together with the milF protein, play a potential role in the addition of leucine to the pentapeptide peptidoglycan precursor and thereby, contributing to self-protection to millericin B in the producer strain.

Intra- and interspecies signaling between Streptococcus salivarius and Streptococcus pyogenes mediated by SalA and SalA1 lantibiotic peptides.

Streptococcus salivarius 20P3 produces a 22-amino-acid residue lantibiotic, designated salivaricin A (SalA), that inhibits the growth of a range of streptococci, including all strains of Streptococcus pyogenes. Lantibiotic production is associated with the sal genetic locus comprising salA, the lantibiotic structural gene; salBCTX genes encoding peptide modification and export machinery proteins; and salYKR genes encoding a putative immunity protein and two-component sensor-regulator system. Insertional inactivation of salB in S. salivarius 20P3 resulted in abrogation of SalA peptide production, of immunity to SalA, and of salA transcription. Addition of exogenous SalA peptide to salB mutant cultures induced dose-dependent expression of salA mRNA (0.2 kb), demonstrating that SalA production was normally autoregulated. Inactivation of salR encoding the response regulator of the SalKR two-component system led to reduced production of, and immunity to, SalA. The sal genetic locus was also present in S. pyogenes SF370 (M type 1), but because of a deletion across the salBCT genes, the corresponding lantibiotic peptide, designated SalA1, was not produced. However, in S. pyogenes T11 (M type 4) the sal locus gene complement was apparently complete, and active SalA1 peptide was synthesized. Exogenously added SalA1 peptide from S. pyogenes T11 induced salA1 transcription in S. pyogenes SF370 and in an isogenic S. pyogenes T11 salB mutant and salA transcription in S. salivarius 20P3 salB. Thus, SalA and SalA1 are examples of streptococcal lantibiotics whose production is autoregulated. These peptides act as intra- and interspecies signaling molecules, modulating lantibiotic production and possibly influencing streptococcal population ecology in the oral cavity.

Isolation and characterization of enterocin BC25 and occurrence of the ent A gene among ruminal Gram-positive cocci

Enterocin BC25, a bacteriocin produced by Enterococcus faecium BC25 isolated from the rumen of cow was purified to homogeneity and sequenced. Twenty amino acids were identified in the peptide chain (TTHSGKYYGNGVYCT-KNKCT), identical to the N-terminal sequence of enterocin A. The DNA sequence of the enterocin BC25 structural gene and putative immunity protein exhibited high similarity to the entA gene. The occurrence of a 726 bp amplicon containing the enterocin A structural gene was studied among gram-positive ruminal cocci by PCR. Our results showed wide occurrence of the entA structural gene among ruminal enterococcal and streptococcal bacterial strains tested, and indicate variable ability to express bacteriocin production and resistance.

Characterization of bacteriocin N15 produced by enterococcus faecium N15 and cloning of the related genes

Enterococcus faecium N15 was isolated from nuka (Japanese rice-bran paste), which is utilized as starter in the fermenting of vegetables, and was found to produce a bacteriocin that exhibited a broad spectrum of activity, including activity against Listeria monocytogenes and Bacillus circulans JCM2504. The bacteriocin was sensitive to proteases (α-chymotrypsin, proteinase K, trypsin, and pepsin) and α-amylase, but it was resistant to lipase. The bacteriocin was resistant to heat treatment at 100°C for 2 h, but its activity was completely lost after autoclaving at 121°C for 15 min. It was active over a wide pH range from 2.0 to 10.0. The bacteriocin showed bactericidal activity against Lactobacillus sake JCM1157 at a concentration of 40 AU/ml. Its molecular weight was estimated by SDS—PAGE to be about 3–5 kDa. PCR primers were designed based on the conserved amino acid sequences of class IIa bacteriocins. A 3-kb DNA fragment was amplified and three open reading frames (ORFs) were found. The first encodes a probable immunity protein of 103 amino acid residues and shows complete homology with the putative immunity protein of E. faecium DPC1146. The second and third ORFs respectively encode a probable transposase gene and an inducing factor. The upstream region of the immunity gene, in which the bacteriocin structural gene is located, was amplified. A homology search revealed that the bacteriocin produced by E. faecium N15 exhibits complete identity to enterocin A, a bacteriocin produced by E. faecium DPC1146. PCR using the primers designed in this study is a rapid and sufficient method of screening for bacteriocin-producing strains.

Characterization of Bacteriocin N15 Produced by Enterococcus faecium N15 and Cloning of the Related Genes.

Enterococcus faecium N15 was isolated from nuka (Japanese rice-bran paste), which is utilized as starter in the fermenting of vegetables, and was found to produce a bacteriocin that exhibited a broad spectrum of activity, including activity against Listeria monocytogenes and Bacillus circulans JCM2504. The bacteriocin was sensitive to proteases (alpha-chymotrypsin, proteinase K, trypsin, and pepsin) and alpha-amylase, but it was resistant to lipase. The bacteriocin was resistant to heat treatment at 100 degrees C for 2 h, but its activity was completely lost after autoclaving at 121 degrees C for 15 min. It was active over a wide pH range from 2.0 to 10.0. The bacteriocin showed bactericidal activity against Lactobacillus sake JCM1157 at a concentration of 40 AU/ml. Its molecular weight was estimated by SDS-PAGE to be about 3-5 kDa. PCR primers were designed based on the conserved amino acid sequences of class IIa bacteriocins. A 3-kb DNA fragment was amplified and three open reading frames (ORFs) were found. The first encodes a probable immunity protein of 103 amino acid residues and shows complete homology with the putative immunity protein of E. faecium DPC1146. The second and third ORFs respectively encode a probable transposase gene and an inducing factor. The upstream region of the immunity gene, in which the bacteriocin structural gene is located, was amplified. A homology search revealed that the bacteriocin produced by E. faecium N15 exhibits complete identity to enterocin A, a bacteriocin produced by E. faecium DPC1146. PCR using the primers designed in this study is a rapid and sufficient method of screening for bacteriocin-producing strains.

Biochemical and genetic evidence that Enterococcus faecium L50 produces enterocins L50A and L50B, the sec-dependent enterocin P, and a novel bacteriocin secreted without an N-terminal extension termed enterocin Q.

Enterococcus faecium L50 grown at 16 to 32 degrees C produces enterocin L50 (EntL50), consisting of EntL50A and EntL50B, two unmodified non-pediocin-like peptides synthesized without an N-terminal leader sequence or signal peptide. However, the bacteriocin activity found in the cell-free culture supernatants following growth at higher temperatures (37 to 47 degrees C) is not due to EntL50. A purification procedure including cation-exchange, hydrophobic interaction, and reverse-phase liquid chromatography has shown that the antimicrobial activity is due to two different bacteriocins. Amino acid sequences obtained by Edman degradation and DNA sequencing analyses revealed that one is identical to the sec-dependent pediocin-like enterocin P produced by E. faecium P13 (L. M. Cintas, P. Casaus, L. S. Hâvarstein, P. E. Hernández, and I. F. Nes, Appl. Environ. Microbiol. 63:4321-4330, 1997) and the other is a novel unmodified non-pediocin-like bacteriocin termed enterocin Q (EntQ), with a molecular mass of 3,980. DNA sequencing analysis of a 963-bp region of E. faecium L50 containing the enterocin P structural gene (entP) and the putative immunity protein gene (entiP) reveals a genetic organization identical to that previously found in E. faecium P13. DNA sequencing analysis of a 1,448-bp region identified two consecutive but diverging open reading frames (ORFs) of which one, termed entQ, encodes a 34-amino-acid protein whose deduced amino acid sequence was identical to that obtained for EntQ by amino acid sequencing, showing that EntQ, similarly to EntL50A and EntL50B, is synthesized without an N-terminal leader sequence or signal peptide. The second ORF, termed orf2, was located immediately upstream of and in opposite orientation to entQ and encodes a putative immunity protein composed of 221 amino acids. Bacteriocin production by E. faecium L50 showed that EntP and EntQ are produced in the temperature range from 16 to 47 degrees C and maximally detected at 47 and 37 to 47 degrees C, respectively, while EntL50A and EntL50B are maximally synthesized at 16 to 25 degrees C and are not detected at 37 degrees C or above.

Allelic variation in a peptide-inducible two-component system of Streptococcus pneumoniae.

The peptide SpiP of Streptococcus pneumoniae regulates the induction of a complex signal transduction system spiR1spiR2spiH. Distinct alleles of spiP and the receptor histidine protein kinase gene spiH were recognized in different pneumococcal clones. The spi system in strain KNR7/87 is adjacent to a bacteriocin gene cluster encoding putative double glycine-type bacteriocins, immunity proteins, and translocator proteins. A direct repeat element upstream of the spiR1 promoter and another three potential transcription start sites within the bacteriocin cluster indicate that SpiP functions as an inducing peptide for bacteriocin synthesis in S. pneumoniae.

Allelic variation in a peptide-inducible two-component system of Streptococcus pneumoniae

The peptide SpiP of Streptococcus pneumoniae regulates the induction of a complex signal transduction system spiR1spiR2spiH. Distinct alleles of spiP and the receptor histidine protein kinase gene spiH were recognized in different pneumococcal clones. The spi system in strain KNR7/87 is adjacent to a bacteriocin gene cluster encoding putative double glycine-type bacteriocins, immunity proteins, and translocator proteins. A direct repeat element upstream of the spiR1 promoter and another three potential transcription start sites within the bacteriocin cluster indicate that SpiP functions as an inducing peptide for bacteriocin synthesis in S. pneumoniae.

Synthesis of lactococcin 972, a bacteriocin produced by Lactococcus lactis IPLA 972, depends on the expression of a plasmid-encoded bicistronic operon.

Synthesis of lactococcin 972 is plasmid-encoded. An operon composed of two genes that encode pre-bacteriocin and a putative immunity protein has been identified. The first gene encodes a 91-residue polypeptide that is exported via a sec-dependent system to give the mature 66-aa bacteriocin. The immunity protein is a 563-residue polypeptide with seven potential transmembrane domains. Two transcripts were observed from this region: one comprises the whole operon and is synthesized during the exponential phase of growth while the other, which corresponds just to the bacteriocin structural gene, presents a maximum in exponential cultures but is still present in late-stationary-phase cells.

Characterization and heterologous expression of the genes encoding enterocin a production, immunity, and regulation in Enterococcus faecium DPC1146.

Enterocin A is a small, heat-stable, antilisterial bacteriocin produced by Enterococcus faecium DPC1146. The sequence of a 10, 879-bp chromosomal region containing at least 12 open reading frames (ORFs), 7 of which are predicted to play a role in enterocin biosynthesis, is presented. The genes entA, entI, and entF encode the enterocin A prepeptide, the putative immunity protein, and the induction factor prepeptide, respectively. The deduced proteins EntK and EntR resemble the histidine kinase and response regulator proteins of two-component signal transducing systems of the AgrC-AgrA type. The predicted proteins EntT and EntD are homologous to ABC (ATP-binding cassette) transporters and accessory factors, respectively, of several other bacteriocin systems and to proteins implicated in the signal-sequence-independent export of Escherichia coli hemolysin A. Immediately downstream of the entT and entD genes are two ORFs, the product of one of which, ORF4, is very similar to the product of the yteI gene of Bacillus subtilis and to E. coli protease IV, a signal peptide peptidase known to be involved in outer membrane lipoprotein export. Another potential bacteriocin is encoded in the opposite direction to the other genes in the enterocin cluster. This putative bacteriocin-like peptide is similar to LafX, one of the components of the lactacin F complex. A deletion which included one of two direct repeats upstream of the entA gene abolished enterocin A activity, immunity, and ability to induce bacteriocin production. Transposon insertion upstream of the entF gene also had the same effect, but this mutant could be complemented by exogenously supplied induction factor. The putative EntI peptide was shown to be involved in the immunity to enterocin A. Cloning of a 10.5-kb amplicon comprising all predicted ORFs and regulatory regions resulted in heterologous production of enterocin A and induction factor in Enterococcus faecalis, while a four-gene construct (entAITD) under the control of a constitutive promoter resulted in heterologous enterocin A production in both E. faecalis and Lactococcus lactis.

Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum.

Enterocin P is a new bacteriocin produced by Enterococcus faecium P13 isolated from a Spanish dry-fermented sausage. Enterocin P inhibited most of tested spoilage and food-borne gram-positive pathogenic bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum. Enterocin P is produced during growth in MRS broth from 16 to 45 degrees C; it is heat resistant (60 min at 100 degrees C; 15 min at 121 degrees C) and can withstand exposure to pH between 2.0 and 11.0, freeze-thawing, lyophilization, and long-term storage at 4 and -20 degrees C. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation, gel filtration, cation-exchange, hydrophobic-interaction, and reverse-phase liquid chromatography. The sequence of 43 amino acids of the N terminus was obtained by Edman degradation. DNA sequencing analysis of a 755-bp region revealed the presence of two consecutive open reading frames (ORFs). The first ORF encodes a 71-amino-acid protein containing a hydrophobic N-terminal sec-dependent leader sequence of 27 amino acids followed by the amino acid sequence corresponding to the purified and sequenced enterocin P. The bacteriocin is apparently synthesized as a prepeptide that is cleaved immediately after the Val-Asp-Ala residues (positions -3 to -1), resulting in the mature bacteriocin consisting of 44 amino acids, and with a theoretical molecular weight of 4,493. A second ORF, encoding a putative immunity protein composed of 88 amino acids with a calculated molecular weight of 9,886, was found immediately downstream of the enterocin P structural gene. Enterocin P shows a strong antilisterial activity and has the consensus sequence found in the pediocin-like bacteriocins; however, enterocin P is processed and secreted by the sec-dependent pathway.

The putative immunity protein of the gram-positive bacteria Leuconostoc mesenteroides is preferentially located in the cytoplasm compartment.

Immunity proteins are though to protect bacteriocin-producing bacterial strains against the bactericidal effects of their own bacteriocin. The immunity protein which protects the lactic acid bacterium Leuconostoc mesenteroides against mesentericin Y105(37) bacteriocin was detected and localized by immunofluorescence and electron microscopy, using antibodies directed against the C-terminal end of the predicted immunity protein. The antibodies recognized the immunity proteins of various strains of Leuconostoc, including Leuconostoc mesenteroides and Leuconostoc gelidum. This study demonstrated that immunity proteins produced by Leuconostoc mesenteroides accumulated in the cytoplasmic compartment of the bacteria. This is in contrast with other known immunity proteins, such as the colicin immunity proteins, which are integral membrane proteins possessing three to four transmembrane domains.

The putative immunity protein of the Gram-positive bacteria Leuconostoc mesenteroides is preferentially located in the cytoplasm compartment

Immunity proteins are thought to protect bacteriocin-producing bacterial strains against the bactericidal effects of their own bacteriocin. The immunity protein which protects the lactic acid bacterium Leuconostoc mesenteroides against mesentericin Y105 37 bacteriocin was detected and localized by immunofluorescence and electron microscopy, using antibodies directed against the C-terminal end of the predicted immunity protein. The antibodies recognized the immunity proteins of various strains of Leuconostoc, including Leuconostoc mesenteroides and Leuconostoc gelidum. This study demonstrated that immunity proteins produced by Leuconostoc mesenteroides accumulated in the cytoplasmic compartment of the bacteria. This is in contrast with other known immunity proteins, such as the colicin immunity proteins, which are integral membrane proteins possessing three to four transmembrane domains.

Sea urchin genes expressed in activated coelomocytes are identified by expressed sequence tags. Complement homologues and other putative immune response genes suggest immune system homology within the deuterostomes.

To identify some of the genes expressed in LPS-activated coelomocytes, we sequenced randomly chosen clones from a directionally constructed cDNA library to produce a set of expressed sequence tags (ESTs). Deduced amino acid sequences from 307 ESTs were compared with known protein sequences in GenBank, and significant matches to approximately 30% of the clones were identified. Eighty-nine clones matched to 55 different proteins, including several putative immune effector proteins. In this work, we show the first identification of an invertebrate homologue of a vertebrate C component. Another EST matches to several short consensus repeats that are characteristic of a variety of proteins, including CR/regulatory proteins and clotting factors. Additional putative immune effector genes include 1) a Kazal-type protease inhibitor that may function to inactivate bacterial proteases, 2) a C-type lectin similar to echinoidin, and 3) a serine protease with similarities to thrombin, elastase, haptoglobin, and plasmin. Other EST categories include 1) cell surface proteins and receptors, 2) proteins involved in signaling systems, 3) lysosomal and secreted proteins, 4) cytoskeletal and cytoskeletal modifying proteins, 5) general cell function proteins, 6) proteins with unknown function, and 7) ESTs without significant matches, 25 with open reading frames. Many of the ESTs identified in this study represent the types of genes expected to be used in lower deuterostome immune functions.

Heterologous expression of the lactacin F peptides by Carnobacterium piscicola LV17

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. piscicola 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.