Use of Real-Time Quantitative PCR for the Analysis of LC3 Prophage Stability in Lactococci (original) (raw)
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Effects of Diverse Environmental Conditions on LC3 Prophage Stability in Lactococcus lactis
Applied and Environmental Microbiology, 2005
The effects of various growth conditions on spontaneous LC3 prophage induction in Lactococcus lactis subsp. cremoris IMN-C1814 was analyzed with a half fraction of a 4 4 factorial experimental design. The four factors included in the study were nutrient availability, acidity, osmolarity, and temperature, each applied at four levels. These environmental factors are related to the fermentation processes in the dairy industry, in which bacteriophage attacks on sensitive starter strains are a constant threat to successful fermentation processes. The frequency of spontaneous LC3 induction was determined by quantitative analyses of restored DNA attachment sites (attB) on the bacterial chromosomes in a population of lysogenic cells. Statistical analysis revealed that all four environmental factors tested affected LC3 prophage stability and that the environmental factors were involved in interactions (interactions exist when the effect of one factor depends on the level of another factor). The spontaneous LC3 induction frequency varied from 0.08 to 1.76%. In general, the induction frequency remained at the same rate or decreased when level 1 to 3 of the four environmental factors was applied. At level 4, which generally gave the least favorable growth conditions, the induction frequency was either unchanged, decreased, or increased, depending on the type of stress. It appeared that the spontaneous induction frequency was independent of the growth behavior of the host. It was the environmental growth conditions that were the decisive factor in induction frequency.
Journal of Applied Microbiology, 2004
Aims: To characterize a group of closely related Lactococcus lactis subsp. lactis casein starter strains used commercially, which differ in their sensitivity to bacteriophages isolated from the same industrial environment. Methods and Results: Nine strains of L. lactis, six of which had been used as starter cultures for lactic casein manufacture, were shown to be closely related by pulsed-field gel electrophoresis and total DNA profiles. Nineteen phages which propagated on one or more of these starter strains were isolated from industrial casein whey samples. The phages were all small isometric-headed and could be divided into five groups on the basis of host range on the nine strains. Most of the phages did not give a PCR product with primers designed to detect the two most common lactococcal small isometric phage species (936 and P335). The hosts could be divided into six groups depending on their phage sensitivity. Plasmids encoding genes for the cell envelope associated PI-type proteinase, lactose metabolism and specificity subunits of a type I restriction/modification system were identified. Conclusions: This work demonstrates how isolates of the same starter strain may come to be regarded as separate cultures because of their different origins, and how these closely related strains may differ in some of their industrially relevant characteristics. Significance and Impact of the Study: This situation may be very common among lactococci used as dairy starter cultures, and implies that the dairy industry worldwide depends on a small number of different strains.
FEMS Microbiology Letters, 2000
We present here a new and general approach for monitoring the life cycles of temperate bacteriophages which establish lysogeny by inserting their genomes site-specifically into the bacterial host chromosome. The method is based on quantitative amplification of specific DNA sites involved in various cut-and-join events during the life cycles of the phages (i.e. the cos, attP, attB, attL and attR sites) with the use of sequence-specific primers. By comparing the amounts of these specific DNA sites at different intervals, we were able to follow the development of the lytic and lysogenic life cycles of the temperate lactococcal bacteriophage PLC3 after infection of its bacterial host Lactococcus lactis ssp. cremoris IMN-C18. ß : S 0 3 7 8 -1 0 9 7 ( 0 0 ) 0 0 4 2 1 -3
International Journal of Food Microbiology, 2003
The aim of this work was to establish whether Lactococcus lactis strains isolated from spontaneous dairy fermentations exhibited useful milk-processing capabilities and resistance to bacteriophage infection in order to be used as components in starter formulations. The 33 out of 100 isolates of L. lactis, originated from farmhouse cheeses, were found to be resistant to a collection of 34 phages belonging to the c2 and 936 groups. Six of the isolates were discarded as potential starters because they were lysogenic and other five because they produced tyramine. Plasmid and chromosomal profiles of the 22 remaining isolates allowed their classification into 16 different strains. All of these were good lactic acid producers from lactose, moderately proteolytic and, in eight cases, diacetyl production from citrate was observed. The mechanism(s) leading to the phenotype of phage resistance was identified for all the strains used in this study. Inhibition of adsorption was the most frequent one, although genetic determinants for some abortive infection systems were also detected (abiB, abiG and abiI). Frequently, more than one mechanism was present in the same strain. One of the strains, L. lactis IPLA542, was selected as a model starter for pilot fermentations. It clotted milk normally both in the absence and in the presence of phage at concentrations that completely abolished the process when promoted by a phage-susceptible strain. D
Screening for and characterization of Lactococcus lactis bacteriophages with high thermal resistance
International Dairy Journal, 2009
Fifty-six Lactococcus lactis phage isolates collected from different German dairies and obtained from a starter culture manufacturer were tested for their heat resistance. About 40% of these isolates resisted treatment at 80 C for 5 min when they were heated in milk. The most resistant phage isolate, P1532, was collected from sour cream. Plaque-formation was still detectable even after heating at 97 C for 5 min. The second heat-resistant one, P680, showed some plaque-forming ability after heating at 95 C for 5 min. Kinetic parameters for the thermal inactivation of these two resistant phages were determined for temperatures ranging from 70 to 97 C. The inactivation of phage P1532 in skim milk and in buffer medium were found to follow first-order kinetics and did not exhibit tailing, whereas in the inactivation curves of phage P680 tailing was observed. The D-value of P1532 at pasteurization temperature of 72 C was calculated as 112 min.
2011
CHAPTER The potential of lacticin 3147, enterocin AS-48, lacticin 481, variacin, and sakacin P for food biopreservation. 1 CHAPTER Genetic response to bacteriophage infection in Lactococcus lactis reveals a four-strand approach involving induction of membrane stress proteins, Dalanylation of the cell wall, maintenance of proton motive force and energy conservation. 50 CHAPTER Novel conjugative plasmids from the natural isolate Lactococcus lactis subsp. cremoris DPC3758: a repository of genes for the potential improvement of dairy starters. 96 CHAPTER Plasmids of raw milk cheese isolate L. lactis subsp. lactis biovar. diacetylactis DPC3901 suggest a plant-based origin for the strain 154 CHAPTER The presence of pMRC01 promotes greater cell permeability and autolysis in lactococcal starter cultures. 204 CHAPTER Microarray-based targeting of the abortive infection mechanism of pMRC01 during bacteriophage infection of the lactococcal host. 233 CHAPTER General Conclusions 262 Acknowledgements 272 iv DECLARATION I hereby certify that this material, which I submit for assessment on the programme of study leading to the award of Ph.D. is entirely my own work and has not been
Food Biotechnology, 2010
The dairy industry utilizes Lactococcus lactis strains as starter cultures to manufacture fermented products. Bacteriophages specific for the cultures are an important and persistent problem for the industry. The development of phage-insensitive strains is one approach to handle the problem. We have identified loci that were spontaneously mutated in Lactococcus lactis ssp. cremoris MG1363 during its natural conversion to the phage-resistant phenotype as a challenge to sk1 phage. Whole genome analysis by PCR fingerprinting technique pinpointed genes encoding proteins involved in cell wall metabolism, transmembrane and membrane associated proteins, transcriptional regulators, prophage components, and enzymes of basal metabolism.
Journal of Applied Microbiology, 1997
The lytic activity induced by the lactococcal bacteriophage POOl was isolated from phage lysates of Lactococcus lactis by a four-step purification procedure. Two proteins lytic for L. lactis were identified with molecular weights of 28 kDA and 8 kDa, respectively. The N-terminal amino acid sequences of the two proteins were determined and degenerated oligonucleotide probes corresponding to these sequences were synthesized. DNA hybridization experiments with phage POOI-DNA and lactococcal DNA revealed that both proteins were apparently encoded by a single lysin gene located on the phage POOl genome. This was confirmed by alignment of the determined N-terminal amino acid sequences with nucleotide sequences which were deduced from cloned Lactococcus bacteriophage lysin genes.
Comparative Analyses of Prophage-Like Elements Present in Two Lactococcus lactis Strains
Applied and Environmental Microbiology, 2007
In this study, we describe the genetic organizations of six and five apparent prophage-like elements present in the genomes of the Lactococcus lactis subsp. cremoris strains MG1363 and SK11, respectively. Phylogenetic investigation as well bioinformatic analyses indicates that all 11 prophages belong to subdivisions of the lactococcal P335 group of temperate bacteriophages.