Proteinase PI and lactococcin A genes are located on the largest plasmid inLactococcus lactissubsp.lactisbv. diacetylactis S50

2005 ◽  
Vol 51 (4) ◽  
pp. 305-314 ◽  
Author(s):  
Milan Kojic ◽  
Ivana Strahinic ◽  
Ljubisa Topisirovic
Keyword(s):  
Dna Hybridization ◽  
Cell Envelope ◽  
Bacteriocin Production ◽  
Conjugative Plasmid ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Plasmid Curing ◽  
Test Frequency ◽  
Genes Encoding ◽  
Large Plasmids

Lactococcus lactis subsp. lactis bv. diacetylactis S50 produces a lactococcin A-like bacteriocin named bacteriocin S50, and cell envelope-associated PI-type proteinase activity. This strain harbours 3 small size plasmids: pS6 (6.3 kb), pS7a (7.31 kb), and pS7b (7.27 kb). Plasmid curing using a combination of novobiocin treatment (10 µg·mL–1) and sublethal temperature (40 °C) resulted in a very low yield (0.17%) of Prt–, Bac–, Bacsderivatives, which retained all 3 small size resident plasmids. Pulsed-field gel electrophoresis of DNA isolated from the strain S50 and cured derivatives in combination with restriction enzyme analysis and DNA–DNA hybridization revealed that S50 contains 2 additional large plasmids: pS140 (140 kb) and pS80 (80 kb). Conjugation experiments using strain S50 as a donor and various lactococcal recipients resulted in Prt+, Bac+, Bacrtransconjugants. Analysis of these transconjugants strongly indicated that plasmid pS140 harbours the prt and bac genes encoding proteinase and bacteriocin production, and immunity to bacteriocin, since each Prt+, Bac+, Bacrtranconjugant contained pS140. Accordingly, none of the Prt–, Bac–, Bacstransconjugants contained this plasmid. pS140 was a self-transmissible conjugative plasmid regardless of the host lactococcal recipient used in the test. Frequency of conjugation of plasmid pS140 did not depend on either the donor or recipient strain.Key words: Lactococcus, plasmids, conjugation, bacteriocin, proteinase.

scholarly journals Characterization of lactococci isolated from homemade kefir

2007 ◽  
Vol 59 (1) ◽  
pp. 13-22 ◽  
Author(s):  
M. Kojic ◽  
Jelena Lozo ◽  
Jelena Begovic ◽  
B. Jovcic ◽  
Lj. Topisirovic
Keyword(s):  
Dna Hybridization ◽  
Antimicrobial Compounds ◽  
Plasmid Curing ◽  
Natural Isolate ◽  
Bacteriocin Activity ◽  
Proteinase Production ◽  
Genes Encoding ◽  
Traditionally Prepared ◽  
Cross Inhibition

Five bacteriocin-producing lactococci isolates from traditionally prepared kefir were determined as Lactococcus lactis subsp. lactis. The analyzed isolates showed different plasmid profiles and no cross inhibition between them was detected. Moreover, natural isolate BGKF26 was resistant to the antimicrobial activity of nisin producing strain NP45. Plasmid curing experiments revealed that the genes encoding bacteriocin and proteinase production are located on separate genetic elements, except in BGKF26. Production of the tested bacteriocins depends on the concentration of casitone or triptone in the medium. Higher concentrations of casitone or triptone induce bacteriocin activity. Our DNA-DNA hybridization analyses suggest that the analyzed antimicrobial compounds probably are lactococcin-like bacteriocins.

Restriction enzyme and DNA hybridization analysis of cellulolytic Streptomyces isolates of different origin

1997 ◽  
Vol 43 (4) ◽  
pp. 395-399 ◽  
Author(s):  
Laura Marri ◽  
Emanuela Barboni ◽  
Tiziana Irdani ◽  
Brunella Perito ◽  
Giorgio Mastromei
Keyword(s):  
Restriction Enzyme ◽  
Dna Hybridization ◽  
Genomic Dna ◽  
Restriction Pattern ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Restriction Maps ◽  
E Coli ◽  
The One ◽  
Different Sources

Streptomyces rochei A2 endoglucanase (eglS) and β-glucosidase (bgs1) genes were used as probes to survey their distribution among 16 Streptomyces strains isolated from different sources and characterized for their cellulolytic activities. The eglS probe hybridized to the genomic DNA of 12 strains with a restriction pattern different from that of S. rochei A2. The DNA from all strains, except one, hybridized with the bgsl probe and one strain showed the same restriction pattern as seen in S. rochei A2. The sequence localized by the eglS probe in S. thermoviolaceus and the one localized by the bgs1 probe in strain EC1 were cloned and expressed in E. coli in plasmids pTAE and pCSF203, respectively. The restriction maps showed that the cloned genes were identical to eglS and bgs1. The restriction enzyme analysis of genomic DNA from all the strains identified nine different groups, each characterized by a distinctive pattern and in agreement with the results of the hybridization experiments.Key words: Streptomyces, cellulase genes, hybridization, restriction enzyme analysis.

Isolation and characterization of two temperate phages from Lactococcus lactis ssp. cremoris C3

1992 ◽  
Vol 38 (5) ◽  
pp. 398-404 ◽  
Author(s):  
Audrey W. Jarvis ◽  
Vaughan R. Parker ◽  
Moana B. Bianchin
Keyword(s):  
Lactococcus Lactis ◽  
Dna Hybridization ◽  
Base Plate ◽  
Restriction Endonuclease Analysis ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Isolation And Characterization ◽  
Dna Fragment ◽  
Endonuclease Analysis

Lactococcus lactis ssp. cremoris C3 was shown to contain two prophages, C3-T1 and C3-T2, both inducible bymitomycin C. The phages were morphologically indistinguishable, having an isometric head (55 nm), a noncontractile tail (142 nm), and a distinctive base plate. The phages C3-T1 and C3-T2 were differentiated from one another by restriction endonuclease analysis of their DNA, and genome sizes of 37.8 (C3-T1) and 32.5 kb (C3-T2). Southern blot DNA hybridization suggested a maximum homology between the phage genomes of 50%. Phage C3-T1 was propagated and the phage obtained was designated C3-TIℓ. Phage C3-T2 was not propagated despite tests with a large number of possible indicator strains. Restriction enzyme analysis of phage C3-T1ℓ genome yielded a 37.8-kb circular restriction map. The packaging site (pac) and site of integration into the bacterial chromosome (att) were located and an EcoRI DNA fragment containing the att site was cloned. Only one C3-T1 att site was present in the C3 chromosome. No homology was detected between DNA from C3-T1ℓ and DNA from lytic phages commonly isolated from cheese wheys. Key words: lactococcal phages, temperate phages, lysogeny, evolution.

Plasmid content and bacteriocin production by five strains ofLactococcus lactisisolated from semi-hard homemade cheese

2006 ◽  
Vol 52 (11) ◽  
pp. 1110-1120 ◽  
Author(s):  
Milan Kojic ◽  
Ivana Strahinic ◽  
Djordje Fira ◽  
Branko Jovcic ◽  
Ljubisa Topisirovic
Keyword(s):  
Lactococcus Lactis ◽  
Bacteriocin Production ◽  
Plasmid Curing ◽  
Structural Genes ◽  
Rolling Circle ◽  
Plasmid Content ◽  
Plasmid Maintenance ◽  
Lactococcus Lactis Subsp ◽  
Natural Isolates ◽  
Large Plasmids

In this study, the plasmid content and bacteriocin production of natural isolates of lactococci were investigated. Five bacteriocin producing lactococcal strains (Lactococcus lactis subsp. lactis BGMN1-2, BGMN1-3, BGMN1-5, BGMN1-6, and BGMN2-7) were isolated as nonstarter microflora of semi-hard homemade cheese and characterized. All isolates contained a number of plasmids. It was shown that lcnB structural genes for bacteriocin lactococcin B were located on large plasmids in all isolates. In the strains BGMN1-3 and BGMN1-5 proteinase prtP genes collocated with lcnB. Furthermore, these strains produced two additional bacteriocins (LsbA and LsbB) with genes responsible for their production and immunity located on the small rolling circle-replicating plasmid pMN5. Using deletion experiments of pMN5, minimal replicon of the plasmid and involvement of a bacteriocin locus in plasmid maintenance were identified. In addition, plasmid curing experiments showed that genes for catabolism or transport of 10 carbohydrates in the strain BGMN1-5 were plasmid located.Key words: lactococci, natural isolates, bacteriocin, plasmid curing.

scholarly journals Replication of Staphylococcal Multiresistance Plasmids

2000 ◽  
Vol 182 (8) ◽  
pp. 2170-2178 ◽  
Author(s):  
Neville Firth ◽  
Sumalee Apisiridej ◽  
Tracey Berg ◽  
Brendon A. O'Rourke ◽  
Steve Curnock ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Heavy Metal Resistance ◽  
Metal Resistance ◽  
Replication Initiation ◽  
Conjugative Plasmid ◽  
Rolling Circle ◽  
Segregational Stability ◽  
Structural And Functional Properties ◽  
Resistance Plasmids ◽  
Genes Encoding

ABSTRACT Based on structural and functional properties, three groups of large staphylococcal multiresistance plasmids have been recognized, viz., the pSK1 family, pSK41-like conjugative plasmids, and β-lactamase–heavy-metal resistance plasmids. Here we describe an analysis of the replication functions of a representative of each of these plasmid groups. The replication initiation genes from theStaphylococcus aureus plasmids pSK1, pSK41, and pI9789::Tn552 were found to be related to each other and to the Staphylococcus xylosus plasmid pSX267 and are also related to rep genes of several plasmids from other gram-positive genera. Nucleotide sequence similarity between pSK1 and pI9789::Tn552 extended beyond theirrep genes, encompassing upstream divergently transcribed genes, orf245 and orf256, respectively. Our analyses revealed that genes encoding proteins related to the deducedorf245 product are variously represented, in several types of organization, on plasmids possessing six seemingly evolutionarily distinct types of replication initiation genes and including both theta-mode and rolling-circle replicons. Construction of minireplicons and subsequent functional analysis demonstrated that orf245is required for the segregational stability of the pSK1 replicon. In contrast, no gene equivalent to orf245 is evident on the conjugative plasmid pSK41, and a minireplicon encoding only the pSK41 rep gene was found to exhibit a segregational stability approaching that of the parent plasmid. Significantly, the results described establish that many of the large multiresistance plasmids that have been identified in clinical staphylococci, which were formerly presumed to be unrelated, actually utilize an evolutionarily related theta-mode replication system.

scholarly journals A molecular characterization ofClostridium difficileisolates from humans, animals and their environments

1993 ◽  
Vol 111 (2) ◽  
pp. 257-264 ◽  
Author(s):  
G. O'Neill ◽  
J. E. Adams ◽  
R. A. Bowman ◽  
T. V. Riley
Keyword(s):  
Fragment Length Polymorphism ◽  
Hospital Environment ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Direct Transmission ◽  
Chromosomal Dna ◽  
Veterinary Clinic ◽  
Enhanced Chemiluminescence ◽  
Typing Methods ◽  
Rflp Typing

SummaryIt is generally accepted that most patients withClostridium difficile-associated diarrhoea acquire the organism from the environment. Recently we demonstrated that household pets may constitute a significant reservoir ofC. difficilethrough gastrointestinal carriage in up to 39% of cats and dogs. These findings suggested that direct transmission from household pets, or contamination of the environment by them, may be a factor in the pathogenesis ofC. difficile-associated diarrhoea. To investigate this possibility, we examined isolates ofC. difficilefrom humans, pets and the environment by restriction enzyme analysis (REA) and restriction fragment length polymorphism (RFLP) typing using enhanced chemiluminescence. Both REA and RFLP typing methods usedHindIII digests of chromosomal DNA. A total of 116 isolates ofC. difficilefrom pets (26), veterinary clinic environmental sites (33), humans (37) and hospital environmental sites (20) was examined. REA was far more discriminatory than RFLP typing and for all isolates there were 34 REA types versus 6 RFLP types. There was good correlation between the REA types found in isolates from pets and from the veterinary clinic environment, and between isolates from humans and from those found in the hospital environment. There was, however, no correlation between REA type ofC. difficilefound in pets and isolates of human origin. We conclude that there may still be a risk of humans acquiringC. difficilefrom domestic pets as these findings may be the result of geographical variation.

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