Complete Genome Sequence of Lactococcus lactis subsp. cremoris 3107, Host for the Model Lactococcal P335 Bacteriophage TP901-1.
ABSTRACT: The complete genome sequence of Lactococcus lactis subsp. cremoris 3107, a dairy starter strain and a host for the model lactococcal P335 bacteriophage TP901-1, is reported here. The circular chromosome of L. lactis subsp. cremoris 3107 is among the smallest genomes of currently sequenced lactococcal strains. L. lactis subsp. cremoris 3107 harbors a complement of six plasmids, which appears to be a reflection of its adaptation to the nutrient-rich dairy environment.
Project description:Here, we report the complete genome of Lactococcus lactis subsp. cremoris UC509.9, an Irish dairy starter. The circular chromosome of L. lactis UC509.9 represents the smallest among those of the sequenced lactococcal strains, while its large complement of eight plasmids appears to be a reflection of its adaptation to the dairy environment.
Project description:The temperate phage TPW22, induced from Lactococcus lactis subsp. cremoris W22, and the evolutionarily interesting integrase of this phage were characterized. Phage TPW22 was propagated lytically on L. lactis subsp. cremoris 3107, which could also be lysogenized by site-specific integration. The attachment site (attP), 5'-TAAGGCGACGGTCG-3', of phage TPW22 was present on a 7.5-kb EcoRI fragment, a 3.4-kb EcoRI-HindIII fragment of which was sequenced. Sequence information revealed the presence of an integrase gene (int). The deduced amino acid sequence showed 42 and 28% identity with integrases of streptococcal and lactococcal phages, respectively. The identities with these integrase-encoding genes were 52 and 45%, respectively, at the nucleotide level. This could indicate horizontal gene transfer. A stable integration vector containing attP and int was constructed, and integration in L. lactis subsp. cremoris MG1363 was obtained. The existence of an exchangeable lactococcal phage integration module was suggested. The proposed module covers the phage attachment site, the integrase gene, and surrounding factor-independent terminator structures. The phages phiLC3, TP901-1, and TPW22 all have different versions of this module. Phylogenetically, the TPW22 Int links the phiLC3 lactococcal integrase with known Streptococcus thermophilus integrases.
Project description:The temperate lactococcal phage TP901-1, induced by UV light from Lactococcus lactis subsp. cremoris 901-1, was characterized. The restriction map was found to be circular, and the packaging of TP901-1 DNA was concluded to occur by a headful mechanism. The pac region was localized on the 38.4-kb phage genome. TP901-1 belongs to the class of P335 phages (V. Braun, S. Hertwig, H. Neve, A. Geis, and M. Teuber, J. Gen. Microbiol. 135:2551-2560, 1989). Evidence is presented that the phages TP936-1 (V. Braun, S. Hertwig, H. Neve, A. Geis, and M. Teuber, J. Gen. Microbiol. 135:2551-2560, 1989) and C3-T1 (A. W. Jarvis, V. R. Parker, and M. B. Bianchin, Can. J. Microbiol. 38:398-404, 1992) are very closely related to or are identical to TP901-1. The lytically propagated TP901-1 phages were able to lysogenize both indicator strains Lactococcus cremoris 3107 and Wg2. Lysogenization resulted in site-specific integration of the phage genome into the bacterial chromosome. Only one chromosomal attB site was found in 20 independent lysogens. The attP region of TP901-1 and the attL and attR regions were cloned and sequenced. The results showed a core region of only 5 bp, in which the recombination occurs, followed after a 1-bp mismatch by a 7-bp identical region, TCAAT(T/C)AAGGTAA. This result was further verified by sequencing of the attB region obtained by PCR. An integration vector was constructed with the 6.5-kb EcoRI fragment from TP901-1 containing attP. This vector also functions in the plasmid-free strains, MG1363 and LM0230 with only one specific attB site, strongly indicating a more general use of the TP901-1-based integration vector in lactococci.
Project description:Previously we showed that only one phage-expressed protein (Orf1), a 425-bp region upstream of the orf1 gene (presumably encoding a promoter), and the attP region are necessary and also sufficient for integration of the bacteriophage TP901-1 genome into the chromosome of Lactococcus lactis subsp. cremoris (B. Christiansen, L. Brondsted, F. K. Vogensen, and K. Hammer, J. Bacteriol. 178:5164-5173, 1996). In this work, a further analysis of the phage-encoded elements involved in integration was performed. Here we demonstrate that even when the orf1 gene is separated from the attP region, the Orf1 protein is able to promote site-specific integration of an attP-carrying plasmid into the attB site on the L. lactis subsp. cremoris chromosome. Furthermore, the first detailed deletion analysis of an attP region of a phage infecting lactic acid bacteria was carried out. We show that a fragment containing 56 bp of the attP region, including the core, is sufficient for the site-specific integration of a nonreplicating plasmid into the chromosome of L. lactis subsp. cremoris when the orf1 gene is donated in trans. The functional 56-bp attP region of TP901-1 is substantially smaller than minimal attP regions identified for other phages. Based on the deletion analysis, several repeats located within the attP region seem to be necessary for site-specific integration of the temperate bacteriophage TP901-1. By use of the integrative elements (attP and orf1) expressed by the temperate lactococcal bacteriophage TP901-1, a system for obtaining stable chromosomal single-copy transcriptional fusions in L. lactis was constructed. Two promoter-reporter integration vectors containing the reporter gene gusA or lacLM, encoding beta-glucuronidase or beta-galactosidase, respectively, were constructed. Immediately upstream of both genes are found translational stop codons in all three reading frames as well as multiple restriction enzyme sites suitable for cloning of the promoter of interest. By transformation of L. lactis subsp. cremoris MG1363 containing the integrase gene on a replicating plasmid, the promoter-reporter integration vectors integrated with a high frequency site specifically into the chromosomal attachment site attB used by bacteriophage TP901-1.
Project description:Phages of the P335 species infect Lactococcus lactis and have been particularly studied because of their association with strains of L. lactis subsp. cremoris used as dairy starter cultures. Unlike other lactococcal phages, those of the P335 species may have a temperate or lytic lifestyle, and are believed to originate from the starter cultures themselves. We have sequenced the genome of L. lactis subsp. cremoris KW2 isolated from fermented corn and found that it contains an integrated P335 species prophage. This 41 kb prophage (? KW2) has a mosaic structure with functional modules that are highly similar to several other phages of the P335 species associated with dairy starter cultures. Comparison of the genomes of 26 phages of the P335 species, with either a lytic or temperate lifestyle, shows that they can be divided into three groups and that the morphogenesis gene region is the most conserved. Analysis of these phage genomes in conjunction with the genomes of several L. lactis strains shows that prophage insertion is site specific and occurs at seven different chromosomal locations. Exactly how induced or lytic phages of the P335 species interact with carbohydrate cell surface receptors in the host cell envelope remains to be determined. Genes for the biosynthesis of a variable cell surface polysaccharide and for lipoteichoic acids (LTAs) are found in L. lactis and are the main candidates for phage receptors, as the genes for other cell surface carbohydrates have been lost from dairy starter strains. Overall, phages of the P335 species appear to have had only a minor role in the adaptation of L. lactis subsp. cremoris strains to the dairy environment, and instead they appear to be an integral part of the L. lactis chromosome. There remains a great deal to be discovered about their role, and their contribution to the evolution of the bacterial genome.
Project description:We report the complete genome sequence of Lactococcus lactis subsp. cremoris A76, a dairy strain isolated from a cheese production outfit. Genome analysis detected two contiguous islands fitting to the L. lactis subsp. lactis rather than to the L. lactis subsp. cremoris lineage. This indicates the existence of genetic exchange between the diverse subspecies, presumably related to the technological process.
Project description:Lactococcus lactis subsp. cremoris Ropy352 produces two distinct heteropolysaccharides, phenotypically described as ropy and mucoid, when cultured in nonfat milk. One exopolysaccharide precipitated with 50% ethanol as a series of elongated threads and was composed of glucose and galactose in a molar ratio of 3:2. The second exopolysaccharide precipitated with 75% ethanol as a fine flocculant and consisted of galactose, glucose, and mannose with a molar ratio of 67:21:12. A mutant strain, L. lactis subsp. cremoris EK240, lacking the ropy phenotype did not produce the exopolysaccharide that precipitated with 50% ethanol; however, it produced the exopolysaccharide that precipitated with 75% ethanol, indicating that the former exopolysaccharide is essential for the ropy phenotype. Cultures of L. lactis subsp. cremoris Ropy352 in 10% nonfat milk reached a viscosity of 25 Pa-s after 24 h, while those of the nonropy L. lactis subsp. cremoris EK240 mutant did not change. A mutation abolishing ropy exopolysaccharide expression mapped to a region on a plasmid containing two open reading frames, epsM and epsN, encoding novel glycosyltransferases bordered by ISS1 elements oriented in the same direction. Sequencing of this plasmid revealed two other regions involved in exopolysaccharide expression, an operon located between partial IS981 and IS982 elements, and an independent gene, epsU. Two and possibly three of these regions are involved in L. lactis subsp. cremoris Ropy352 exopolysaccharide expression and are arranged in a novel fashion different from that of typical lactococcal exopolysaccharide loci, and this provides genetic evidence for exopolysaccharide gene reorganization and evolution in Lactococcus.
Project description:Several molecular taxonomic studies have revealed that many natural (wild) Lactococcus lactis strains of dairy origin which are phenotypically representative of the L. lactis subspecies lactis cluster genotypically within subspecies cremoris and vice versa. Recently, we isolated two wild nisin-producing (Nis(+)) L. lactis strains, M78 and M104, of the lactis phenotype from Greek raw milk (J. Samelis, A. Lianou, A. Kakouri, C. Delbès, I. Rogelj, B. B. Matijašic, and M. C. Montel, J. Food Prot. 72:783-790, 2009); strain M78 possess a novel nisin A sequence (GenBank accession number HM219853). In this study, the actual subspecies identity of M78 and M104 isolates was elucidated, using 16S rRNA and acmA (encoding lactococcal N-acetylmuramidase) gene and histidine biosynthesis operon polymorphisms and 16S rRNA and ldh (encoding lactate dehydrogenase) gene phylogenies. Except the acmA gene analysis, molecular tools revealed that isolates M78 and M104 clustered with strains of the cremoris genotype, including the LMG 6897(T) strain, while they were distant from strains of the lactis genotype, including the LMG 6890(T) strain. The two wild isolates had identical repetitive sequence-based PCR (rep-PCR), randomly amplified polymorphic DNA (RAPD), plasmid, and whole-cell protein profiles and shared high 16S rRNA (99.9%) and ldh (100%) gene sequence homologies. In contrast, they exhibited identical sugar fermentation and enzymatic patterns which were similar to those of the subspecies lactis LMG 6890(T) strain. To our knowledge, this is the first complete identification report on a wild L. lactis subsp. cremoris genotype of the lactis phenotype which is capable of nisin A production and, thus, has strong potential for use as a novel dairy starter and/or protective culture.
Project description:Lactococcus lactis is of great importance for the nutrition of hundreds of millions of people worldwide. This paper describes the genome sequence of Lactococcus lactis subsp. cremoris MG1363, the lactococcal strain most intensively studied throughout the world. The 2,529,478-bp genome contains 81 pseudogenes and encodes 2,436 proteins. Of the 530 unique proteins, 47 belong to the COG (clusters of orthologous groups) functional category "carbohydrate metabolism and transport," by far the largest category of novel proteins in comparison with L. lactis subsp. lactis IL1403. Nearly one-fifth of the 71 insertion elements are concentrated in a specific 56-kb region. This integration hot-spot region carries genes that are typically associated with lactococcal plasmids and a repeat sequence specifically found on plasmids and in the "lateral gene transfer hot spot" in the genome of Streptococcus thermophilus. Although the parent of L. lactis MG1363 was used to demonstrate lysogeny in Lactococcus, L. lactis MG1363 carries four remnant/satellite phages and two apparently complete prophages. The availability of the L. lactis MG1363 genome sequence will reinforce its status as the prototype among lactic acid bacteria through facilitation of further applied and fundamental research.
Project description:Two highly autolytic Lactococcus lactis subsp. cremoris strains (CO and 2250) were selected and analyzed for their autolytic properties. Both strains showed maximum lysis when grown in M17 broth containing a limiting concentration of glucose (0.4 to 0.5%) as the carbohydrate source. Lysis did not vary greatly with pH or temperature but was reduced when strains were grown on lactose or galactose. Growth in M17 containing excess glucose (1%) prevented autolysis, although rapid lysis of L. lactis subsp. cremoris CO did occur in the presence of 1% glucose if sodium fluoride (an inhibitor of glycolysis) was added to the medium. Maximum cell lysis in a buffer system was observed early in the stationary phase, and for CO, two pH optima were observed for log-phase and stationary-phase cells (6.5 and 8.5, respectively). Autolysins were extracted from the cell wall fraction of each strain by using either 4% sodium dodecyl sulfate (SDS), 6 M guanidine hydrochloride, or 4 M lithium chloride, and their activities were analyzed by renaturing SDS-polyacrylamide gel electrophoresis on gels containing Micrococcus luteus or L. lactis subsp. cremoris CO cells as the substrate. More than one lytic band was observed on each substrate, with the major band having an apparent molecular mass of 48 kDa for CO. Each lytic band was present throughout growth and lysis. These results suggest that at least two different autolytic enzymes are present in the autolytic L. lactis subsp. cremoris strains. The presence of the lactococcal cell wall hydrolase gene, acmA (G. Buist, J. Kok, K. J. Leenhouts, M. Dabrowska, G. Venema, and A. J. Haandrikman, J. Bacteriol. 177:1554-1563, 1995), in strains 2250 and CO was confirmed by Southern hybridization. Analysis of an acmA deletion mutant of 2250 confirmed that the gene was involved in cell separation and had a role in cell lysis.