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molecular analysis of the cos region of the lactobacillus casei bacteriophage a2. gene product 3, gp3, specifically binds to its downstream cos region.the terminal nucleotide sequence of the lactobacillus casei bacteriophage a2 dna revealed a single-stranded extension 13 bases in length (5'-aacggtcggcctc-3') at its 3' termini that defines the packaging initiation nicking site (cosn). the cosn sequence is bisected by an axis of hyphenated twofold rotational symmetry. directly and inverted repeated sequences located to the left (cosl) and the right (cosr) of the cosn site were observed. analysis of the 3.4 kb ecori dna sequence surrounding the c ...19979044284
isolation and characterization of promoters from the lactobacillus casei temperate bacteriophage a2.random sau3a1 dna fragments from the temperate lactobacillus bacteriophage a2 were cloned into the promoter-probe plasmid pgkv210. seven dna fragments with promoter activity were selected, after transformation of escherichia coli and lactococcus lactis, subsp, lactis, through the chloramphenicol resistance they conferred to the corresponding clones. the seven promoters were functional in lactobacillus casei. their strength was analysed by measuring the levels of chloramphenicol resistance and ch ...19979436307
identification of the repressor-encoding gene of the lactobacillus bacteriophage a2.the repressor gene of the lactobacillus phage a2 has the following properties: it (i) encodes a 224-residue polypeptide with dna binding and reca cleavage motifs, (ii) is expressed in lysogenic cultures, and (iii) confers superinfection immunity on the host. adjacent, but divergently transcribed, lies another open reading frame whose product resembles the lambda cro protein. in the 161-bp intergenic segment, putative promoters and operators have been detected.19989642205
the site-specific recombination system of the lactobacillus species bacteriophage a2 integrates in gram-positive and gram-negative bacteria.the region of the bacteriophage a2 genome involved in site-specific recombination with the dna of lactobacillus spp. has been identified. two orfs, transcribed from the same strand, have been found immediately upstream of the phage attachment site (attp). the orf adjacent to attp predicts a 385-amino-acid protein that presents significant similarity with site-specific recombinases of the integrase family. the other orf encodes a basic polypeptide of 76 amino acid residues. the junctions of the p ...19989770432
cooperative interaction of ci protein regulates lysogeny of lactobacillus casei by bacteriophage a2.the temperate bacteriophage a2 forms stable lysogens in lactobacillus casei. the a2-encoded ci product (ci), which is responsible for maintaining the a2 prophage in the lysogenic state, has been purified. the ci protein, which is a monomer of 25.3 kda in solution, specifically binds to a 153-bp dna fragment that contains two divergent promoters, pl and pr. these promoters mediate transcription from ci and a putative cro, respectively. three similar, although not identical, 20-bp inverted repeate ...199910196287
stable expression of the lactobacillus casei bacteriophage a2 repressor blocks phage propagation during milk fermentation.a general strategy was applied to implement resistance against temperate bacteriophages that infect food fermentation starters through cloning and expression of the phage repressor. lactobacillus casei atcc 393 and phage a2 were used to demonstrate its feasibility as milk fermentation is drastically inhibited when the strain is infected by this phage. the engineered strain lact. casei em40::ci, which has the a2 repressor gene (ci) integrated into the genome, was completely resistant and able to ...199910347876
comparative sequence analysis of the dna packaging, head, and tail morphogenesis modules in the temperate cos-site streptococcus thermophilus bacteriophage sfi21.the temperate streptococcus thermophilus bacteriophage sfi21 possesses 15-nucleotide-long cohesive ends with a 3' overhang that reconstitutes a cos-site with twofold hyphenated rotational symmetry. over the dna packaging, head and tail morphogenesis modules, the sfi21 sequence predicts a gene map that is strikingly similar to that of lambdoid coliphages in the absence of any sequence similarity. a nearly one to one gene correlation was found with the phage lambda genes nu1 to h, except for gene ...199910417259
a2 cro, the lysogenic cycle repressor, specifically binds to the genetic switch region of lactobacillus casei bacteriophage a2.lysogenic induction of temperate bacteriophage a2 of lactobacillus casei is controlled by the action of its ci and cro products at the phage operator region. three 20-bp inverted repeated dna segments (subsites o1, o2, and o3) and the two divergent (pl and pr) promoters were mapped within the 153-bp operator region. the a2-encoded cro product is shown to be the functional homolog of lambda cro. the binding of cro to the three operator subsites is noncooperative and yields two discrete protein-dn ...199910489355
generation of food-grade recombinant lactic acid bacterium strains by site-specific recombination.the construction of a delivery and clearing system for the generation of food-grade recombinant lactic acid bacterium strains, based on the use of an integrase (int) and a resolvo-invertase (beta-recombinase) and their respective target sites (attp-attb and six, respectively) is reported. the delivery system contains a heterologous replication origin and antibiotic resistance markers surrounded by two directly oriented six sites, a multiple cloning site where passenger dna could be inserted (e.g ...200010831443
characterization of the dna replication module of bacteriophage a2 and use of its origin of replication as a defense against infection during milk fermentation by lactobacillus casei.adjacent to the lysis/lysogeny cassette of the a2 phage genome lies a stretch of over 8 kb, which contains a series of genes probably involved in dna replication. fifteen open reading frames (orfs) were identified, 13 of which are encoded on the main coding strand and only two on the complementary strand. database searches and comparative analyses allowed the identification of an open reading frame (orf455) that shows similarity with dna helicases and contains a variant zinc-finger motif known f ...200010891412
interaction of the cro repressor with the lysis/lysogeny switch of the lactobacillus casei temperate bacteriophage a2.the transcriptional switch region of lactobacillus casei temperate bacteriophage a2 contains three similar 20 bp operator subsites, o(1), o(2) and o(3), which are interspersed between the divergent promoters p(r) and p(l). the cro protein binds initially to o(3), which overlaps the -35 region of p(l), excluding the rna polymerase (sigma(a)-rnap) from it. this results in the switching off of ci transcription and directs the incoming phage into the lytic cycle. at higher concentrations, cro also b ...200212388825
analysis of the morphogenetic cluster and genome of the temperate lactobacillus casei bacteriophage a2.the genes that encode the morphogenetic proteins of bacteriophage a2 are clustered and expressed as a single operon which originates a late transcript of more than 20 kb. this dna stretch is analyzed in the context of the whole phage genome, which presents the following peculiarities: a) the head presents two major proteins that share their nh(2) termini, i.e.: both are translated from a single gene (orf5), b) these two proteins suffer a proteolytic maturation process before being incorporated i ...200312756614
a -1 ribosomal frameshift in the transcript that encodes the major head protein of bacteriophage a2 mediates biosynthesis of a second essential component of the capsid.the two major capsid proteins of lactobacillus bacteriophage a2 share their amino termini. the smaller of these (gp5a) results from translation of orf5 and proteolytic processing after residue 123. the larger form (gp5b) originates through a -1 ribosomal frameshift at the penultimate codon of orf5 mrna, resulting in a product that is 85 amino acids longer than gp5a. frameshifting needs two cis-acting elements: a slippery region with the sequence c cca aaa (0 frame), and a stem-loop that begins 9 ...200414996802
a second case of -1 ribosomal frameshifting affecting a major virion protein of the lactobacillus bacteriophage a2.the bacteriophage a2 major tail protein gene utilizes a -1 translational frameshift to generate two structural polypeptides. frameshifting is promoted by a slippery sequence and an rna pseudoknot located 3' of the gene. the major head gene presents a similar recoding ability. a2 is the only phage described with two -1 frameshifts.200516291695
construction of recombinant lactobacillus casei strains using splicing by overlap extension.recombinant lactobacillus strains have been constructed using gene splicing by overlap extension (soe). primers were designed of which one end of an amplified product contained complementary sequences for an end of other amplified fragment. for efficient matching, we used an asymmetric pcr step that was effective at generating an excess of strands that would anneal in the final pcr. cp12, a recombinant fragment consisting of the integrase gene and attachment site of the bacteriophage a2, was con ...200819131699
LysA2, the Lactobacillus casei bacteriophage A2 lysin is an endopeptidase active on a wide spectrum of lactic acid bacteria.The lysin gene (lysA2) of the Lactobacillus casei bacteriophage A2 was cloned and expressed in Escherichia coli. LysA2 is an endopeptidase that hydrolyzes the bond between the terminal D: -alanine of the peptidoglycan tetrapeptide and the aspartic acid residue that forms the bridge with the L: -lysine of a neighboring peptidoglycan chain, characteristic of Gram-positive bacteria included into the A4 peptidoglycan subgroup. This includes most lactobacilli, Lactococcus lactis, Pediococcus acidilac ...201121952940
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