Sequence and analysis of the 60 kb conjugative, bacteriocin‐producing plasmid pMRC01 from <i>Lactococcus lactis</i> DPC3147

Ba, Dougherty; Hill, Colin; Jf, Weidman; Richardson, Des R.; Venter, J. Craig; Ross, R. Paul

doi:10.1046/j.1365-2958.1998.00988.x

Cited by 158 publications

(126 citation statements)

References 45 publications

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“…In this way, the following pORI280-ltnA1A2 derivatives were made: pOri-ltnA1S7T (primer pair [21][22], pOri-ltnA1S7G (primer pair 24-25), pOri-ltnA1S7V (primer pair 27-28), pOri-ltnA2S9T (primer pair 30-31), pOri-ltnA2S9G (primer pair 33-34), pOri-ltnA2S9V (primer pair 36-37), pOriltnA2S9,12T (primer pair 39-40), pOri-ltnA2S9,12G (primer pair 42-43), and pOri-ltnA2S9,12V (primer pair 45-46). Screening of candidate EC101 transformants was facilitated by the use of check primers 23,26,29,32,35,38,41,44, and 47, respectively, in conjunction with either primer 7 or 8, depending on the orientation of the check primer. In all cases successful mutagenesis was confirmed by DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

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Posttranslational conversion of l -serines to d -alanines is vital for optimal production and activity of the lantibiotic lacticin 3147

Cotter

O’Connor²,

Draper³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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As a general rule, ribosomally synthesized polypeptides contain amino acids only in the L-isoform in an order dictated by the coding DNA͞RNA. Two of a total of only four examples of L to D conversions in prokaryotic systems occur in posttranslationally modified antimicrobial peptides called lantibiotics. In both examples (lactocin S and lacticin 3147), ribosomally encoded L-serines are enzymatically converted to D-alanines, giving rise to an apparent mistranslation of serine codons to alanine residues. It has been suggested that this conversion results from a two-step reaction initiated by a lantibiotic synthetase converting the gene-encoded L-serine to dehydroalanine (dha). By using lacticin 3147 as a model system, we report the identification of an enzyme, LtnJ, that is responsible for the conversion of dha to D-alanine. Deletion of this enzyme results in the residues remaining as dha intermediates, leading to a dramatic reduction in the antimicrobial activity of the producing strain. The importance of the chirality of the three D-alanines present in lacticin 3147 was confirmed when these residues were systematically substituted by L-alanines. In addition, substitution with L-threonine (ultimately modified to dehydrobutyrine), glycine, or L-valine also resulted in diminished peptide production and͞or relative activity, the extent of which depended on the chirality of the newly incorporated amino acid(s).antimicrobial ͉ bacteriocin ͉ chirality T he presence of D-amino acids in ribosomally synthesized peptides was first observed in dermorphins, peptides found in the skin secretions of species of a subfamily of South American tree frogs, Phyllomedusinae. Subsequently, D-amino acids have been identified in a number of other ribosomally synthesized peptides of eukaryotic origin (1, 2). -Agatoxin IVb and bombinin H, produced by the funnel-web spider and the skin secretions of the frog Bombina variegata, respectively, represent the only instances in which the responsible enzymes, both peptide epimerases, have been identified (1, 2). For -agatoxin IVb the relevant L-serine to D-serine conversion is thought to involve a deprotonation͞reprotonation reaction (3). The presence of D-amino acids in many of these peptides is crucial, as demonstrated by the creation of synthetic analogues with Lrather than D-amino acids at the relevant locations, which are devoid of activity (4). The artificial incorporation of D-amino acids can also be beneficial. For example, a growing number of synthetic peptides͞peptide libraries containing D-amino acids have been assembled to capitalize on the ability of such residues to provide improved protease stability [enhanced pharmokinetic profile (5-7)], alter tertiary structure (new structural elements can be assembled that cannot be built solely from L-amino acids), and affect the activity of bioactive peptides [enhanced antibacterial activity with concomitant reduction in cell cytotoxicity (5, 6, 8-11)].In prokaryotic systems, there are only a few examples of L-to D-amino acid conversions. It...

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Section: Methodsmentioning

confidence: 99%

“…The genes responsible for production of and immunity to lacticin 3147 are encoded by two divergently transcribed operons (29) (Fig. 1).…”

Section: Identification Of a Dha Reductase Ltnj Responsible For The Smentioning

confidence: 99%

Posttranslational conversion of l -serines to d -alanines is vital for optimal production and activity of the lantibiotic lacticin 3147

Cotter

O’Connor²,

Draper³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

118

146

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Section: Introductionmentioning

confidence: 99%

“…For example, the majority of dairy strains have adapted to growth in milk by acquiring plasmids that encode lactose catabolism (De Vos & Gasson, 1989), casein degradation (Kok, 1990), citrate utilization (Kempler & McKay, 1979) and oligopeptide transport (Yu et al, 1996). In addition, strains isolated from plant and animal environments, which contain a wide variety of cytotoxic compounds, have developed appropriate protection mechanisms (Putman et al, 2000), such as multidrug resistance, nisin resistance and heavy metal resistance (Dougherty et al, 1998;Liu et al, 1997; Perreten et al, 1997), and the genes for these mechanisms are often carried by plasmids.Many of the properties carried by plasmids are of technological interest, and natural plasmids carrying both key industrial traits and a food-grade selectable marker have been the subject of considerable interest. In particular, heavy metal resistance is much sought after as a marker (Liu et al, 2002; O'Sullivan et al, 2001); this is because antibiotic resistance is an unsuitable marker for food micro-organisms due to the risk of promoting antibiotic resistance in the human intestinal microflora.…”

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confidence: 99%

Sequence analysis of the mobilizable lactococcal plasmid pGdh442 encoding glutamate dehydrogenase activity

2007

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A novel plasmid named pGdh442 had previously been isolated from a plant Lactococcus lactis strain. This plasmid encodes two interesting properties with applications in the dairy industry: a glutamate dehydrogenase activity that stimulates amino acid conversion to aroma compounds, and cadmium/zinc resistance that can be used as a selectable marker. Moreover, this plasmid can be transferred naturally to other strains, but appears to be incompatible with certain other lactococcal plasmids. During this study, the complete sequence of pGdh442 (68 319 bp) was determined and analysed. This plasmid contains 67 ORFs that include 20 IS elements that may have mediated transfer events between L. lactis and other genera living in the same biotope, such as Streptococcus, Pediococcus and Lactobacillus. Even though it is a low-copy-number plasmid, it is relatively stable due to a theta replication mode and the presence of two genes involved in its maintenance system. However, pGdh442 is incompatible with pSK08-derived protease/lactose plasmids because both possess the same replication and partition system. pGdh442 is not self-transmissible, but can be naturally transmitted via mobilization by conjugative elements carried by the chromosome or by other plasmids, such as the 712-type sex factor, which is widely distributed in L. lactis. In addition to several genes already found on other L. lactis plasmids, such as the oligopeptide transport and utilization genes, pGdh442 also carries several genes not yet identified in L. lactis. Finally, it does not carry genes that would trigger concern over its presence in human food. INTRODUCTIONLactococcus lactis is a lactic acid bacterium (LAB) widely used as a starter culture for the production of various fermented dairy products. Although this bacterium is commonly found in milk, it is also naturally present on plants and on parts of the bodies of cows (Nomura et al., 2006;Salama et al., 1995;Teuber, 1995). Typically, Lactococcus strains possess an abundance of plasmid DNA. Many large L. lactis plasmids are self-transmissible or mobilizable by conjugative elements carried by the chromosome or plasmids of the host strain (Gasson et al., 1995). Through their natural transfer, these plasmids increase the probability of the gene moving between bacteria, and enable strains to acquire a wide variety of new genetic traits. They thus endow their hosts with many traits which offer a selective advantage to colonize specific biotopes. For example, the majority of dairy strains have adapted to growth in milk by acquiring plasmids that encode lactose catabolism (De Vos & Gasson, 1989), casein degradation (Kok, 1990), citrate utilization (Kempler & McKay, 1979) and oligopeptide transport (Yu et al., 1996). In addition, strains isolated from plant and animal environments, which contain a wide variety of cytotoxic compounds, have developed appropriate protection mechanisms (Putman et al., 2000), such as multidrug resistance, nisin resistance and heavy metal resistance (Dougherty et al., 1998;Liu et al.,...

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“…Interestingly, TraSLVB contained high similarity to the TraJ RCR plasmid transfer proteins from pSG5 and pMEA300 and low similarity to the S. lividans linear plasmid SLP2 TraSLP2. In addition to TraSLVA and TraSLVB, pSLV.45 contains SLV.15 with homology to the Lactococcus lactis TraE protein that has been shown to be involved in the pMRC01 RCR plasmid transfer complex (Dougherty et al, 1998).…”

Section: Sequence Analysis and Deduced Functions Of Pslv45 Proteinsmentioning

confidence: 99%

Characterization of the Streptomyces lavendulae IMRU 3455 linear plasmid pSLV45

Hosted¹,

Wang²,

Horan³

2004

Microbiology

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Streptomyces lavendulae IMRU 3455 contains two large linear plasmids designated pSLV45 (45 kb) and pSLV195 (195 kb). A cosmid, pSPRX604, containing 42 kb from pSLV45 was cloned and sequenced. pSLV45 was tagged with a hygromycin-resistance marker by homologous recombination to generate the derivatives pSLV45.680 and pSLV45.681. An apramycin-resistance marker was introduced into S. lavendulae IMRU 467 using the pSPR910 integration vector to yield the recipient strain SPW910. The self-transmissible nature of pSLV45 was determined by transfer of pSLV45.680 and pSLV45.681 from the donor strains SPW680 and SPW681 into the recipient strain SPW910. Southern analysis indicated the presence of hygromycin-and pSLV45-hybridizing sequences within SPW910 exconjugants. PFGE analysis confirmed pSLV45.680 and pSLV45.681 were transferred intact and formed freely replicating linear plasmids. Sequence analysis of pSPRX604 revealed genes predicted to be involved in plasmid transfer, partitioning and regulation. The transfer of the linear plasmid pSLV45 from S. lavendulae IMRU 3455 into S. lavendulae IMRU 467 may allow the development of pSLV45 as an actinomycete-to-actinomycete conjugative shuttle vector. INTRODUCTIONLinear plasmids with 59 terminally attached proteins (TPs) and terminal inverted repeats (TIRs) have been found in bacteria, plants, filamentous fungi and yeasts, and often within actinomycetes (Griffiths, 1995;Hinnebusch & Tilly, 1993;Meinhardt et al., 1990;Rohe et al., 1992). Actinomycete linear plasmids pSLA1 (17 kb) and pSLA2 (17 kb) were first detected in the lankacidin-producer Streptomyces sp. 7434-AN4 and in Streptomyces rochei, respectively (Hayakawa et al., 1979;Hirochika & Sakaguchi, 1982;Hirochika et al., 1984). Both plasmids were shown to contain TIRs and TPs (Hirochika & Sakaguchi, 1982;Hirochika et al., 1984). With the advent of PFGE analysis, other actinomycete linear plasmids have been identified including Streptomyces lividans SLP2 (50 kb), Streptomyces clavuligerus pSCL1 (12 kb), Streptomyces avermitilis pSA1 and pSA2 (100 kb and 250 kb), Streptomyces rimosus pZG101 (387 kb), Streptomyces lasaliensis pKSL (520 kb) and Streptomyces coelicolor SCP1 (363 kb) Evans et al., 1994;Gravius et al., 1994;Keen et al., 1988;Kinashi & Shimaji, 1987;Kinashi & Shimaji-Murayama, 1991;Kinashi et al., 1994). All these plasmids were found to contain TIRs and TPs. In addition, both pSLA2 and SLP2 were shown to contain an internal origin of replication for bi-directional replication of DNA (Chang & Cohen, 1994;Chang et al., 1996;Huang et al., 2003).Unlike the chromosomes of most bacteria, which are circular in nature, the chromosomes of actinomycetes are linear (Lezhava et al., 1995;Lin et al., 1993). This was first demonstrated in S. lividans and has now been established for several other actinomycetes including S. coelicolor, Streptomyces griseus and Saccharopolyspora erythraea (formerly Streptomyces erythraeus) (Lezhava et al., 1995;Lin et al., 1993;Reeves et al., 1998). All actinomycete chromosomes characterized to da...

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Sequence and analysis of the 60 kb conjugative, bacteriocin‐producing plasmid pMRC01 from Lactococcus lactis DPC3147

Cited by 158 publications

References 45 publications

Posttranslational conversion of l -serines to d -alanines is vital for optimal production and activity of the lantibiotic lacticin 3147

Posttranslational conversion of l -serines to d -alanines is vital for optimal production and activity of the lantibiotic lacticin 3147

Sequence analysis of the mobilizable lactococcal plasmid pGdh442 encoding glutamate dehydrogenase activity

Characterization of the Streptomyces lavendulae IMRU 3455 linear plasmid pSLV45

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