Allometric comparison of Georgia dairy heifers on farms and at youth shows.

Studies were conducted to determine the relationship between allometric measures of growth of Holstein dairy heifers and placing in the show ring, and to compare differences in growth between Holstein heifers that are shown and not shown. In the first study, 494 Holstein show heifers were evaluated at the 2012 and 2013 Georgia Junior National Livestock Shows. Measurements were obtained for weight, head length, withers height, hip height, thurl width, and tail length. Heifer mass index (HMI), average daily gain (ADG), and age were calculated. In total, 72.5% of Holstein show heifers were underweight. Average ADG was 0.63 kg/d, which is below the industry recommendation of 0.7 to 0.8 kg/d. Variables were ranked and converted to percentages to account for differences in class size. Withers height, head length, and HMI were most indicative of show placing. In the second study, we compared differences between growth patterns of show heifers and non-show heifers. An additional 293 non-show Holstein heifers were evaluated on 3 Georgia dairy farms during the same period as the show. In total, 43.3% of non-show heifers were underweight. Average ADG for non-show heifers was 0.71 kg/d, which is within the industry recommendation of 0.7 to 0.8 kg/d. Show heifers weighed less for their age than non-show heifers and tended to be taller at the withers than non-show heifers. The HMI scores were similar for younger show and non-show heifers, but older show heifers had lower HMI scores than non-show heifers of the same age. Show heifers had HMI scores that were lower than values calculated from standard growth data. As show heifers matured, ADG decreased, whereas as non-show heifers matured, ADG increased. Youth, leaders, and parents need to be aware of the importance of growing replacement heifers correctly so that heifers calve at 22 to 24 mo of age at an acceptable size and scale and become profitable members of the milking herd.

Sequence analysis and molecular characterization of the Lactococcus lactis temperate bacteriophage BK5-T.

The Lactococcus lactis temperate bacteriophage BK5-T is one of twelve type phages that define L. lactis phage species. This paper describes the nucleotide sequence and analysis of a 21-kbp region of the BK5-T genome and completes the nucleotide sequence of the genome of this phage. The 40,003-nucleotide linear genome encodes 63 open reading frames. Sequence runoff experiments showed that the cohesive ends of the BK5-T genome contained a 12-bp 3' single-stranded overhang with the sequence 5'-CACACACATAGG-3'. Two major BK5-T structural proteins, of approximately 30 and 20 kDa, were identified, and N-terminal sequence analysis determined that they were encoded by orf7 and orf12, respectively. A 169-bp fragment containing a 37-bp direct repeat and several smaller repeat sequences conferred resistance to BK5-T infection when introduced in trans to the host cell and is likely a part of the BK5-T origin of replication (ori).

Comparative genomics of lactococcal phages: insight from the complete genome sequence of Lactococcus lactis phage BK5-T.

Lactococcus lactis phage BK5-T and Streptococcus thermophilus phage Sfi21, two cos-site temperate Siphoviridae with 40-kb genomes, share an identical genome organization, sequence similarity at the amino acid level over about half of their genomes, and nucleotide sequence identity of 60% over the DNA packaging and head morphogenesis modules. Siphoviridae with similarly organized genomes and substantial protein sequence similarity were identified in several genera of low-GC-content Gram-positive bacteria. These phages demonstrated a gradient of relatedness ranging from nucleotide sequence similarity to protein sequence similarity to gene map similarity over the DNA packaging and head morphogenesis modules. Interestingly, the degree of relatedness was correlated with the evolutionary distance separating their bacterial hosts. These observations suggest elements of vertical evolution in phages. The structural genes from BK5-T shared no sequence relationships with corresponding genes/proteins from lactococcal phages belonging to distinct lactococcal phage species, including phage sk1 (phage species 936) that showed a closely related gene map. Despite a clearly distinct genome organization, lactococcal phages sk1 and c2 showed nine sequence-related proteins. Over the early gene cluster phage BK5-T shared nine regions of high nucleotide sequence similarity, covering at most two adjacent genes, with lactococcal phage r1t (phage species P335). Over the structural genes, the closest relatives of phage r1t were not lactococcal phages belonging to other phage species, but Siphoviridae from Mycobacteria (high-GC-content Gram-positive bacteria). Evidence for recent horizontal gene transfer between distinct phage species was obtained for dairy phages, but these transfers were limited to phages infecting the same bacterial host species.

Distances between DNA and ATP binding sites in the TyrR-DNA complex.

The Escherichia coli regulatory protein TyrR controls the expression of eight transcription units that encode proteins involved in the biosynthesis and transport of aromatic amino acids. It binds to DNA as a homodimer with a subunit molecular mass of 57 640 Da, each of which has a single site for the binding of ATP within a central structural domain. This paper reports distances between four sites on the DNA and the ATP binding site as determined by fluorescence resonance energy transfer. The DNA was a 30mer containing a centrally located binding site for TyrR. Replacement of a thymidine residue with an aminouridine residue at positions -9, -7, -3, and 2 of the palindromic oligonucleotide sequence enabled the placement of a single fluorescein group along the major groove of the DNA. The energy transfer acceptor was ATP labeled with a rhodamine group through positions 2' and 3' of the ribose, positions that are known to cause minimal interference with the binding of ATP to protein. The dissociation constant for the binding of rhodamine-ATP to TyrR was 300 nM as determined by steady-state fluorescence anisotropy titrations. The energy transfer efficiencies were determined by measuring the level of quenching of donor fluorescence on binding rhodamine-ATP to the TyrR-DNA complex. The experimental transfer efficiencies were compared to theoretical values calculated for a model of the DNA-TyrR complex in which the position of the ATP binding site was allowed to vary over the surface of the monomer unit. Theory was written to account for the transfer from one donor to two acceptors, one on each monomer unit of the TyrR dimer. The results indicate that the ATP binding site is about 40-45 A from the nearest point on the DNA and distant from the DNA helix-turn-helix binding domain. The effects of ATP binding of (i) increasing the TyrR binding affinity by a factor of 4-5 and (ii) permitting the binding of the tyrosine corepressor must therefore occur because of a significant allosteric change in the conformation of the protein.

Identification and characterization of a cystathionine beta/gamma-lyase from Lactococcus lactis ssp. cremoris MG1363.

This paper describes the nucleotide sequence of a gene encoding cystathionine beta/gamma-lyase from Lactococcus lactis ssp. cremoris MG1363, its overexpression in Escherichia coli and some functional characteristics of the purified recombinant protein.

Crystallization of the N-terminal domain of the Escherichia coli regulatory protein TyrR.

The N-terminal domain of the regulatory protein TyrR from Escherichia coli forms a dimer in solution and has been purified and crystallized. The crystals belong to space group C2 with unit-cell parameters a = 134.5, b = 72.1, c = 96.7 A, beta = 98.5 degrees. The crystals diffract to 2.8 A. Assuming a molecular weight of 23219 Da, a V(m) of 2.5 A(3) Da(-1) is obtained for two dimers in the asymmetric unit.

Thermodynamics of the interaction of the Escherichia coli regulatory protein TyrR with DNA studied by fluorescence spectroscopy.

Fluorescence quenching was used to study the site-specific binding of the Escherichia coli regulatory protein TyrR to a fluoresceinated oligonucleotide (9F30A/30B) containing a TyrR binding site. The equilibrium constant for the interaction (KL) was measured as a function of temperature and salt concentration in the presence and absence of ATPgammaS, a specific ligand for TyrR. Fluorescence titrations yielded a KL value of 1.20 x 10(7) M-1 at 20 degrees C, which was independent of the acceptor (9F30A/30B) concentration in the range 5-500 nM, indicating that the system exhibits true equilibrium binding. Clarke and Glew analysis of the temperature dependence of binding revealed a linear dependence of R ln KL on temperature in the absence of ATPgammaS. The thermodynamic parameters obtained at 20 degrees C (theta) were = -35.73 kJ mol-1, = 57.41 kJ mol-1, and = 93.14 kJ mol-1. Saturating levels of ATPgammaS (200 microM) strengthened binding at all temperatures and resulted in a nonlinear dependence of Rln KL on temperature. The thermodynamic parameters characterizing binding under these conditions were = -39.32 kJ mol-1, = 37.16 kJ mol-1, = 76.40 kJ mol-1, and = -1.03 kJ mol-1 K-1. Several conclusions were drawn from these data. First, binding is entropically driven at 20 degrees C in both the presence and absence of ATPgammaS. This can partly be accounted for by counterions released from the DNA upon TyrR binding; in the absence of ATPgammaS and divalent cations, the TyrR-9F30A/30B interaction results in the release of two to three potassium ions. Second, the more favorable value, and hence tighter binding observed in the presence of ATPgammaS, is primarily due to a decrease in (-20.3 kJ mol-1), which overcomes an unfavorable decrease in (-16.7 kJ mol-1). Third, the negative value obtained in the presence of ATPgammaS indicates that the binding of ATPgammaS favors a conformational change in TyrR upon binding to 9F30A/30B, yielding a more stable complex.

Effects of molecular crowding on the interaction between DNA and the Escherichia coli regulatory protein TyrR.

Fluorescence quenching has been used to measure quantitatively the effects of sucrose and triethylene glycol on the interaction between the Escherichia coli regulatory protein TyrR and a 30-basepair oligonucleotide containing the strong TyrR box of the TyrR operon. It was observed that the apparent binding constant increased in the presence of co-solutes, the dependence of the logarithm of the apparent binding constant on molar concentration being indistinguishable and essentially linear for both co-solutes. This activation of the TyrR-oligonucleotide interaction is attributed to thermodynamic nonideality arising from molecular crowding, an interpretation which is supported by the reasonable agreement observed between the experimental extent of reaction enhancement and that predicted on the statistical-mechanical basis of excluded volume.

Detection of bacteriocins of lactic acid bacteria isolated from foods and comparison with pediocin and nisin.

A total of 663,533 colonies from 72 dairy and meat sources showed a detection rate of 0.2% for bacteriocin producers using direct plating techniques. A further 83,000 colonies from 40 fish and vegetable sources showed a detection rate of 3.4% for bacteriocin producers using selective enrichment procedures. A collection of seven purified isolates showing a different host spectrum of bacteriocin activity and with the ability to produce bacteriocins in broth culture were compared with nisin and pediocin (with respect to their inhibitory activity, determined by the critical dilution method), against various indicator bacteria in agar and broth. The sensitivity of Listeria species to various bacteriocins was influenced by the agar and broth test systems used. A Lactobacillus curvatus strain was found to be the most suitable indicator for quantitating antimicrobial effects of all the bacteriocins investigated in both agar and broth test systems. The bacteriocin-producing isolates were characterized by biochemical reactions and DNA restriction enzyme profiles and taxonomic identification revealed species of Lactobacillus, Carnobacterium and Lactococcus assigned on the basis of 16S rDNA sequences.

Inhibition of Listeria monocytogenes by piscicolin 126 in milk and Camembert cheese manufactured with a thermophilic starter.

The effect of bacteriocin, piscicolin 126, on the growth of Listeria monocytogenes and cheese starter bacteria was investigated in milk and in Camembert cheese manufactured from milk challenged with 10(2) cfu ml(-1) L. monocytogenes. In milk incubated at 30 degrees C, piscicolin 126 added in the range of 512-2,048 AU ml(-1) effectively inhibited growth of L. monocytogenes for more than 20 d when challenged with approximately 10(2) cfu ml(-1) L. monocytogenes. At higher challenge levels (10(4) and 10(6) cfu ml(-1)), piscicolin 126 reduced the viable count of L. monocytogenes by 4-5 log units immediately after addition of the bacteriocin; however, growth of Listeria occurred within 24 h. The minimum inhibitory concentration (MIC) of piscicolin 126 against lactic acid cheese starter bacteria was generally greater than 204,800 AU ml(-1) , and the viable count and acid production of these starter cultures in milk were not affected by the addition of 2,048 AU ml(-1) piscicolin 126. Camembert cheeses made from milk challenged with L. monocytogenes and with added piscicolin 126 showed a viable count of L. monocytogenes 3-4 log units lower than those without piscicolin 126. Inactivation of piscicolin 126 by proteolytic enzymes from cheese starter bacteria and mould together with the emergence of piscicolin 126-resistant isolates was responsible for the recovery of L. monocytogenes in the cheeses during ripening.

Analysis of the DNA sequence, gene expression, origin of replication and modular structure of the Lactococcus lactis lytic bacteriophage sk1.

Bacteriophage sk1 is a small isometric-headed lytic phage belonging to the 936 species. It infects Lactococcus lactis, a commonly used dairy starter organism. Nucleotide sequence data analysis indicated that the sk1 genome is 28,451 nucleotides long and contains 54 open reading frames (ORFs) of 30 or more codons, interspersed with three large intergenic regions. The nucleotide sequence of several of the sk1 ORFs demonstrated significant levels of identity to genes (many encoding proteins of unknown function) in other lactococcal phages of both small isometric-headed and prolate-headed morphotype. Based on this identity and predicted peptide structures, sk1 genes for the terminase, major structural protein and DNA polymerase have been putatively identified. Genes encoding holin and lysin were also identified, subcloned into an Escherichia coli expression vector, and their function demonstrated in vivo. The sk1 origin of replication was located by identifying sk1 DNA fragments able to support the maintenance in L. lactis of a plasmid lacking a functional Gram-positive ori. The minimal fragment conferring replication origin function contained a number of direct repeats and 179 codons of ORF47. Although no similarity between phage sk1 and coliphage lambda at the nucleotide or amino acid sequence level was observed, an alignment of the sk1 late region ORFs with the lambda structural and packaging genes revealed a striking correspondence in both ORF length and isoelectric point of the ORF product. It is proposed that this correspondence is indicative of a strong conservation in gene order within these otherwise unrelated isometric-headed phages that can be used to predict the functions of the sk1 gene products.

Effector-induced self-association and conformational changes in the enhancer-binding protein NTRC.

The Klebsiella pneumoniae nitrogen regulatory protein NTRC is a response regulator which activates transcription in response to nitrogen limitation, and is a member of the family of sigma N-dependent enhancer-binding proteins. Using limited trypsin digestion, two domains of NTRC were detected and conformational changes within the protein in response to the binding of ligands were also observed. In the absence of ligands, the major digestion products were 42, 36 and 12.5 kDa bands corresponding to the central plus C-terminal domain, the central domain, and the N-terminal domains, respectively. Upon binding of purine but not pyrimidine nucleotides, the 36 kDa band was insensitive to further proteolysis, indicative of a conformational change in the central domain. Analysis of the dependence of this insensitivity on ATP gamma S concentration suggested an apparent dissociation constant (Kd) for ATP gamma S of 150 microM. In the presence of DNA, both the central and C-terminal domains of NTRC were insensitive to proteolytic cleavage, indicative of a further conformational change. NTRC S160F, a mutant form of NTRC that is active in the absence of phosphorylation, was more stable to proteolysis than the wild-type protein. This mutant protein is apparently locked in a conformation resembling the DNA-bound form of wild-type NTRC. The involvement of ligands in self-association was studied using sedimentation equilibrium analysis. In the absence of ligand, wild-type NTRC displayed a monomer-dimer equilibrium with a Kd of 6 microM. In the presence of ATP gamma S the equilibrium was shifted towards the dimer form (Kd = 0.8 microM). A similar dissociation constant for the monomer-dimer interaction was observed with NTRC S160F in the absence of ATP gamma S (Kd = 0.5 microM). The addition of ATP gamma S induced a significant association of NTRC S160F to higher-order states with a dimer-octamer model producing a slightly, but not significantly better fit to the data than a dimer-hexamer model. We propose that ligand-mediated self-association provides a common mechanism for activation of this class of transcriptional regulatory proteins.

The effect of self-association on the interaction of the Escherichia coli regulatory protein TyrR with DNA.

The interaction of the Escherichia coli regulatory protein TyrR, with a 42 bp oligonucleotide (42A/42B) containing a centrally located recognition sequence (TyrR box), was examined by analytical ultracentrifugation. The stoichiometry of the binding of oligonucleotide to dimeric TyrR was determined by equilibrium centrifugation of a mixture of fluorescein-5-isothiocyanate-labelled 42A/42B (F-42A/42B) in the presence of an eightfold molar excess of TyrR. The molecular mass (M) of the labelled oligonucleotide was estimated as 148,000, indicating a 1:1 complex composed of oligonucleotide (M = 27,000) and TyrR dimer (M = 113,000). The association constant (Ko,d = 2.8(+/- 0.1) x 10(6) M-1) was determined by a global analysis of sedimentation data, collected at multiple wavelengths between 230 and 285 nm. The presence of 30 microM ATP gamma S enhanced the affinity of TyrR for DNA approximately 3.5-fold, (Ko,d = 9.9(+/- 0.3) x 10(6) M-1). The effect of dimer to hexamer self-association of TyrR on the binding of 42A/42B was also examined. Multiple wavelength sedimentation data fitted a model in which the oligonucleotide could bind to one site on the dimer (Ko,d = 9.9 x 10(6) M-1), and to either one or three sites on the hexamer (Ko,h) = 2.0(+/- 0.1) x 10(6) M-1 and 3.8(+/- 0.1) x 10(6) M-1, respectively). Competitive sedimentation equilibrium and fluorescence anisotropy titrations were performed under stoichiometric conditions to resolve the number of oligonucleotide binding sites per hexamer. In these experiments, 42A/42B was used as a competitor to displace F-42A/42B from the hexamer, which was found to bind the 42mer with a 1:1 stoichiometry. Our data support a model in which ATP increases the affinity of TyrR for the DNA recognition sequence, and tyrosine induced self-association of TyrR generates a hexameric species with a single binding site for the 42A/42B oligonucleotide.

Genomic organization of lactic acid bacteria.

Current knowledge of the genomes of the lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus, and members of the genera Lactobacillus, Leuconostoc, Pediococcus and Carnobacterium, is reviewed. The genomes contain a chromosome within the size range of 1.8 to 3.4 Mbp. Plasmids are common in Lactococcus lactis (most strains carry 4-7 different plasmids), some of the lactobacilli and pediococci, but they are not frequently present in S. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus or the intestinal lactobacilli. Five IS elements have been found in L. lactis and most strains carry multiple copies of at least two of them; some strains also carry a 68-kbp conjugative transposon. IS elements have been found in the genera Lactobacillus and Leuconostoc, but not in S. thermophilus. Prophages are also a normal component of the L. lactis genome and lysogeny is common in the lactobacilli, however it appears to be rare in S. thermophilus. Physical and genetic maps for two L. lactis subsp. lactis strains, two L. lactis subsp. cremoris strains and S. thermophilus A054 have been constructed and each reveals the presence of six rrn operons clustered in less than 40% of the chromosome. The L. lactis subsp. cremoris MG1363 map contains 115 genetic loci and the S. thermophilus map has 35. The maps indicate significant plasticity in the L. lactis subsp. cremoris chromosome in the form of a number of inversions and translocations. The cause(s) of these rearrangements is (are) not known. A number of potentially powerful genetic tools designed to analyse the L. lactis genome have been constructed in recent years. These tools enable gene inactivation, gene replacement and gene recovery experiments to be readily carried out with this organism, and potentially with other lactic acid bacteria and Gram-positive bacteria. Integration vectors based on temperate phage attB sites and the random insertion of IS elements have also been developed for L. lactis and the intestinal lactobacilli. In addition, a L. lactis sex factor that mobilizes the chromosome in a manner reminiscent to that seen with Escherichia coli Hfr strains has been discovered and characterized. With the availability of this new technology, research into the genome of the lactic acid bacteria is poised to undertake a period of extremely rapid information accrual.

Characterization of the chemical and antimicrobial properties of piscicolin 126, a bacteriocin produced by Carnobacterium piscicola JG126.

A novel peptide bacteriocin produced by the lactic acid bacterium Carnobacterium piscicola JG126 isolated from spoiled ham was purified and characterized. This bacteriocin, designated piscicolin 126, inhibited the growth of several gram-positive bacteria, especially the food-borne pathogen Listeria monocytogenes, but had no effect on the growth of a number of yeasts and gram-negative bacteria. Bactericidal activity was not destroyed by exposure to elevated temperatures at low pH values; however, bactericidal activity was lost at high pH values, especially when high pH values were combined with an elevated temperature. Piscicolin 126 activity was not affected by catalase, lipase, or lysozyme but was destroyed by exposure to a range of proteolytic enzymes. Piscicolin 126 was purified to homogeneity and was found to be a peptide having a molecular weight of 4,416.6 +/- 1.9. A sequence analysis revealed that this compound is a cystibiotic (class IIa) bacteriocin containing 44 amino acid residues and one intrapeptide disulfide ring. Piscicolin 126 has regions of homology with some other bacteriocins obtained from lactic acid bacteria and is most closely related to sakacin P and pediocin PA-1 (levels of identity, 75 and 55%, respectively). Addition of piscicolin 126 to a devilled ham paste test food system inhibited the growth of L. monocytogenes for at least 14 days. Piscicolin 126 was more effective than two commercially available bacteriocin preparations tested in the same system.

Interaction between the Escherichia coli Regulatory protein TyrR and DNA: a fluorescence footprinting study.

The Escherichia coli regulatory protein TyrR controls the expression of eight transcription units that encode proteins involved in the biosynthesis and transport of aromatic amino acids. It is a homodimer of 57 600 subunit molecular weight and has a binding site for ATP and weak ATPase activity. In the presence of ATP, TyrR binds tyrosine, which induces self-association of TyrR from a dimer to a hexamer. This report examines the interaction of TyrR with a 42 bp DNA oligonucleotide containing a centrally located binding site for TyrR (TyrR box). Replacement of a thymidine residue with an aminouridine residue at positions 7, 9, 13, 15, 19, 22, and 26 from one end of the 42mer enables labeling with fluorescein and successive placement of the label along the major groove of the DNA. The fluorescence footprinting of the oligonucleotide was followed using steady-state and time-resolved fluorescence methods. Binding of the TyrR dimer caused significant changes in the fluorescent properties of the labels attached to positions 13, 15, and 26, suggesting the involvement of these bases in the binding of the protein. Except for the position 15 conjugate, binding of the TyrR dimer caused little change in fluorescence intensity. Therefore, fluorescence anisotropy was used to follow the binding equilibrium. The fluorescence of the position 15 conjugate increased 1.6-fold on binding TyrR, suggesting that the fluorophore was in close contact with the protein. For all conjugates, the addition of tyrosine at the end of the titration with TyrR increased the anisotropy markedly, suggesting that the hexameric form of TyrR could bind the oligonucleotide. Two rotational correlation times were found for the labeled conjugates: one reflecting the motion of the probe at its point of attachment to the DNA (220-290 ps), the other reflecting the global tumbling of the labeled oligonucleotide (14-21 ns). On binding TyrR, changes in the correlation times and their associated amplitudes and changes in the range of angular motion of the probe depended on the position of the label. Evidence is presented that the binding of the TyrR hexamer, but not the TyrR dimer, affects regions that flank the binding sequence. The results support the hypothesis that the binding of the TyrR hexamer is responsible for interaction between tandem TyrR boxes in the tyrR regulon.

Sequence analysis of the Lactococcus lactis temperate bacteriophage BK5-T and demonstration that the phage DNA has cohesive ends.

The Lactococcus lactis temperate bacteriophage BK5-T is a type phage in the lactococcal phage classification (A. W. Jarvis, G. F. Fitzgerald, M. Mata, A. Mercenier, H. Neve, I. B. Powell, C. Ronda, M. Saxelin, and M. Teuber, Intervirology 32:2-9, 1991). The nucleotide sequence of 18,935 bp of the genome of BK5-T was determined and analyzed for the presence of open reading frames and other structural features. Thirty-two open reading frames longer than 60 codons were identified, and these appeared to be grouped into at least seven transcriptional units. A search of the nucleotide sequence for restriction sites identified a small number of discrepancies with the previously published physical map of the BK5-T genome (G. Lakshmidevi, B. E. Davidson, and A. J. Hillier, Appl. Environ. Microbiol. 54:1039-1045, 1988). Subsequent analysis of restriction digests of BK5-T DNA which were heated prior to electrophoresis indicated that BK5-T DNA was not terminally redundant as previously reported but contained cohesive ends.

Identification of prophage genes expressed in lysogens of the Lactococcus lactis bacteriophage BK5-T.

Bacteriophage BK5-T is a small isometric-headed temperate phage that infects Lactococcus lactis subsp. cremoris. Northern (RNA) analysis of mRNA produced by lysogenic strains containing BK5-T prophage revealed four major BK5-T transcripts that are 0.8, 1.3, 1.8, and 1.8 kb in size and enabled a transcription map of the prophage genome to be prepared. The position and size of each transcript corresponded closely to the position and size of open reading frames predicted from the nucleotide sequence of BK5-T. Analysis of the transcripts suggested that one of them was derived from the gene encoding the BK5-T integrase and another was from the gene encoding the BK5-T homolog of the lambda cI repressor. Computer analysis of the nucleotide sequence upstream of the BK5-T cI homolog predicted the presence of a pair of divergent promoters and three inverted repeat sequences, features characteristic of temperature-phage immunity regions. By analogy with lambda, the three inverted repeat sequences could be binding sites for cI or Cro homologs and the two divergent promoters could initiate transcription through the BK5-T equivalents of cI and cro.

Spontaneous deletion mutants of the Lactococcus lactis temperate bacteriophage BK5-T and localization of the BK5-T attP site.

Spontaneous deletion mutants of the temperate lactococcal bacteriophage BK5-T were obtained when the phage was grown vegetatively on the indicator strain Lactococcus lactis subsp. cremoris H2. One deletion mutant was unable to form stable lysogens, and analysis of this mutant led to the identification of the BK5-T attP site and the integrase gene (int). The core sequences of the BK5-T attP and host attB regions are conserved in a number of lactococcal phages and L. lactis strains.