Lactococcus lactis Diversity in Undefined Mixed Dairy Starter Cultures as Revealed by Comparative Genome Analyses and Targeted Amplicon Sequencing of epsD.
ABSTRACT: Undefined mesophilic mixed (DL) starter cultures are used in the production of continental cheeses and contain unknown strain mixtures of Lactococcus lactis and leuconostocs. The choice of starter culture affects the taste, aroma, and quality of the final product. To gain insight into the diversity of Lactococcus lactis strains in starter cultures, we whole-genome sequenced 95 isolates from three different starter cultures. Pan-genomic analyses, which included 30 publically available complete genomes, grouped the strains into 21 L. lactis subsp. lactis and 28 L. lactis subsp. cremoris lineages. Only one of the 95 isolates grouped with previously sequenced strains, and the three starter cultures showed no overlap in lineage distributions. The culture diversity was assessed by targeted amplicon sequencing using purR, a core gene, and epsD, present in 93 of the 95 starter culture isolates but absent in most of the reference strains. This enabled an unprecedented discrimination of starter culture Lactococcus lactis and revealed substantial differences between the three starter cultures and compositional shifts during the cultivation of cultures in milk.IMPORTANCE In contemporary cheese production, standardized frozen seed stock starter cultures are used to ensure production stability, reproducibility, and quality control of the product. The dairy industry experiences significant disruptions of cheese production due to phage attacks, and one commonly used countermeasure to phage attack is to employ a starter rotation strategy, in which two or more starters with minimal overlap in phage sensitivity are used alternately. A culture-independent analysis of the lactococcal diversity in complex undefined starter cultures revealed large differences between the three starter cultures and temporal shifts in lactococcal composition during the production of bulk starters. A better understanding of the lactococcal diversity in starter cultures will enable the development of more robust starter cultures and assist in maintaining the efficiency and stability of the production process by ensuring the presence of key bacteria that are important to the characteristics of the product.
Project description:Lactococcus lactis is one of the most important bacteria in dairy fermentations, being used in the production of cheese and buttermilk. The processes are vulnerable to phage attacks, and undefined mixtures of lactococcal strains are often used to reduce the risk of bacteriophage caused fermentation failure. Other preventive measures include culture rotation to prevent phage build-up and phage monitoring. Phage diversity, rather than quantity, is the largest threat to fermentations using undefined mixed starter cultures. We have developed a method for culture independent diversity analysis of lytic bacteriophages of the 936 group, the phages most commonly found in dairies. Using, as a target, a highly variable region of the portal protein gene, we demonstrate an unprecedented diversity and the presence of new 936 phages in samples taken from cheese production. The method should be useful to the dairy industry and starter culture manufacturers in their efforts to reduce phage problems.
Project description:Lactococcus lactis is a Gram-positive bacterium widely used as a starter culture for the production of different dairy products, especially a large variety of cheeses. Infection of lactococcal starter cultures by bacteriophages is one of the major causes of fermentation failure and often leads to production halt. Lactococcal bacteriophages belonging to the c2, 936, and P335 species are the most commonly isolated in dairy plants and have been extensively investigated in the past three decades. Information regarding bacteriophages belonging to less commonly isolated species is, on the other hand, less extensive, although these phages can also contribute to starter culture infection. Here, we report the nucleotide sequence of the newly isolated L. lactis phage CHPC971, belonging to the rare 1706 species of lactococcal phages. We investigated the nature of the host receptor recognized by the phage and collected evidence that strongly suggests that it binds to a specific sugar moiety in the cell wall pellicle of its host. An in silico analysis of the genome of phage CHPC971 identified the hypothetical genes involved in receptor binding.IMPORTANCE Gathering information on how lactococcal bacteriophages recognize their host and proliferate in the dairy environment is of vital importance for the establishment of proper starter culture rotation plans and to avoid fermentation failure and consequent great economic losses for dairy industries. We provide strong evidence on the type of receptor recognized by a newly isolated 1706-type lactococcal bacteriophage, increasing knowledge of phage-host interactions relevant to dairying. This information can help to prevent phage infection events that, so far, are hard to predict and avoid.
Project description:Dairy fermentations constitute a perfect "breeding ground" for bacteriophages infecting starter cultures, particularly strains of Lactococcus lactis. In modern fermentations, these phages typically belong to one of three groups, i.e., the 936, P335, and c2 phage groups. Traditional production methods present fewer chemical and physical barriers to phage proliferation compared to modern production systems, while the starter cultures used are typically complex, variable, and undefined. In the current study, a variety of cheese whey, animal-derived rennet, and vat swab samples from artisanal cheeses produced in Sicily were analysed for the presence of lactococcal phages to assess phage diversity in such environments. The complete genomes of 18 representative phage isolates were sequenced, allowing the identification of 10 lactococcal 949 group phages, six P087 group phages, and two members of the 936 group phages. The genetic diversity of these isolates was examined using phylogenetic analysis as well as a focused analysis of the receptor binding proteins, which dictate specific interactions with the host-encoded receptor. Thermal treatments at 63 °C and 83 °C indicate that the 949 phages are particularly sensitive to thermal treatments, followed by the P087 and 936 isolates, which were shown to be much less sensitive to such treatments. This difference may explain the relatively low frequency of isolation of the so-called "rare" 949 and P087 group phages in modern fermentations.
Project description:Lactococcus lactis subsp. cremoris strains are used globally for the production of fermented dairy products, particularly hard cheeses. Believed to be of plant origin, L. lactis strains that are used as starter cultures have undergone extensive adaptation to the dairy environment, partially through the acquisition of extrachromosomal DNA in the form of plasmids that specify technologically important phenotypic traits. Here, we present a detailed analysis of the eight plasmids of L. lactis UC509.9, an Irish dairy starter strain. Key industrial phenotypes were mapped, and genes that are typically associated with lactococcal plasmids were identified. Four distinct, plasmid-borne bacteriophage resistance systems were identified, including two abortive infection systems, AbiB and AbiD1, thereby supporting the observed phage resistance of L. lactis UC509.9. AbiB escape mutants were generated for phage sk1, which were found to carry mutations in orf6, which encodes the major capsid protein of this phage.
Project description:Lactococcus lactis is a lactic acid bacterium widely used as a starter culture in the manufacture of dairy products, especially a wide variety of cheeses. Improved industrial strains would help to manufacture better food products that can meet the industry's and consumer's demands with respect to e.g. quality, taste, texture and shelf life. Bacteriophage infection of L. lactis starter cultures represents one of the main causes of fermentation failure and consequent economic losses for the dairy industry. In this study, however, we aim at employing bacteriophages for beneficial purposes. We developed an experimental setup to assess whether phage-mediated horizontal gene transfer could be used to enhance the genetic characteristics of L. lactis strains in accordance with the European law regarding the use of genetically modified organisms (GMOs) in the food industry. Although we could not show the transfer of chromosomal DNA we did successfully transduce two dissimilar plasmids from L. lactis strain MG1363 to one of its derivatives employing three different lactococcal bacteriophages.
Project description:Despite being potentially highly useful for characterizing the biodiversity of phages, metagenomic studies are currently not available for dairy bacteriophages, partly due to the lack of a standard procedure for phage extraction. We optimized an extraction method that allows the removal of the bulk protein from whey and milk samples with losses of less than 50% of spiked phages. The protocol was applied to extract phages from whey in order to test the notion that members of Lactococcus lactis 936 (now Sk1virus), P335, c2 (now C2virus) and Leuconostoc phage groups are the most frequently encountered in the dairy environment. The relative abundance and diversity of phages in eight and four whey mixtures from dairies using undefined mesophilic mixed-strain cultures containing Lactococcus lactis subsp. lactis biovar diacetylactis and Leuconostoc species (i.e., DL starter cultures) and defined cultures, respectively, were assessed. Results obtained from transmission electron microscopy and high-throughput sequence analyses revealed the dominance of Lc. lactis 936 phages (order Caudovirales, family Siphoviridae) in dairies using undefined DL starter cultures and Lc. lactis c2 phages (order Caudovirales, family Siphoviridae) in dairies using defined cultures. The 936 and Leuconostoc phages demonstrated limited diversity. Possible coinduction of temperate P335 prophages and satellite phages in one of the whey mixtures was also observed.IMPORTANCE The method optimized in this study could provide an important basis for understanding the dynamics of the phage community (abundance, development, diversity, evolution, etc.) in dairies with different sizes, locations, and production strategies. It may also enable the discovery of previously unknown phages, which is crucial for the development of rapid molecular biology-based methods for phage burden surveillance systems. The dominance of only a few phage groups in the dairy environment signifies the depth of knowledge gained over the past decades, which served as the basis for designing current phage control strategies. The presence of a correlation between phages and the type of starter cultures being used in dairies might help to improve the selection and/or design of suitable, custom, and cost-efficient phage control strategies.
Project description:In southern Italy, some artisanal farms produce mozzarella and caciocavallo cheeses by using natural whey starter (NWS), whose microbial diversity is responsible for the characteristic flavor and texture of the final product. We studied the microbial community of NWS cultures of cow's milk (NWSc) for the production of caciocavallo and buffalo's milk (NWSb) for the production of mozzarella, both from artisanal farms. Bacterial identification at species and strain level was based on an integrative strategy, combining culture-dependent (sequencing of the 16S rDNA, species/subspecies-specific Polymerase Chain Reaction (PCR) and clustering by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) and culture-independent (next-generation sequencing analysis, NGS) approaches. Results obtained with both approaches showed the occurrence of five species of lactic acid bacteria in NWSb (<i>Lactococcus</i> <i>lactis</i> subsp. <i>lactis</i>, <i>Lactobacillus</i> <i>fermentum</i>, <i>Streptococcus</i> <i>thermophilus, Lactobacillus</i> <i>delbrueckii</i>, and <i>Lactobacillus helveticus</i>) and five species in NWSc (<i>Lc.</i> <i>lactis</i> subsp. <i>lactis</i>, <i>Enterococcus</i> <i>faecium</i>, and <i>S.</i> <i>thermophilus</i>, <i>Lb. helveticus</i>, and <i>Lb. delbrueckii</i>), with the last two found only by the NGS analysis. Moreover, RAPD profiles, performed on <i>Lc. lactis</i> subsp. <i>lactis</i> different isolates from both NWSs, showed nine strains in NWSb and seven strains in NWSc, showing a microbial diversity also at strain level. Characterization of the microbiota of natural whey starters aims to collect new starter bacteria to use for tracing microbial community during the production of artisanal cheeses, in order to preserve their quality and authenticity, and to select new Lactic Acid Bacteria (LAB) strains for the production of functional foods.
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:Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed.IMPORTANCEStreptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations.
Project description:Lactococcus lactis is widely used as a starter in the manufacture of cheese and fermented milk. Its main role is the production of lactic acid, but also contributes to the sensory attributes of cheese. Unfortunately, the diversity of suitable strains to be commercialized as dairy starters is limited. In this work, we have applied adaptive evolution under cell envelope stress (AE-CES) as means to provide evolved L. lactis strains with distinct physiological and metabolic traits. A total of seven strains, three of industrial origin and four wild nisin Z-producing L. lactis, were exposed to subinhibitory concentrations of Lcn972, a bacteriocin that triggers the cell envelope stress response in L. lactis. Stable Lcn972 resistant (Lcn972R) mutants were obtained from all of them and two mutants per strain were further characterized. Minimal inhibitory Lcn972 concentrations increased from 4- to 32-fold compared to their parental strains and the Lcn972R mutants retained similar growth parameters in broth. All the mutants acidified milk to a pH below 5.3 with the exception of one that lost the lactose plasmid during adaptation and was unable to grow in milk, and two others with slower acidification rates in milk. While in general phage susceptibility was unaltered, six mutants derived from three nisin Z producers became more sensitive to phage attack. Loss of a putative plasmid-encoded anti-phage mechanism appeared to be the reason for phage susceptibility. Otherwise, nisin production in milk was not compromised. Different inter- and intra-strain-dependent phenotypes were observed encompassing changes in cell surface hydrophobicity and in their autolytic profile with Lcn972R mutants being, generally, less autolytic. Resistance to other antimicrobials revealed cross-protection mainly to cell wall-active antimicrobials such as lysozyme, bacitracin, and vancomycin. Finally, distinct and shared non-synonymous mutations were detected in the draft genome of the Lcn972R mutants. Depending on the parental strain, mutations were found in genes involved in stress response, detoxification modules, cell envelope biogenesis and/or nucleotide metabolism. As a whole, the results emphasize the different strategies by which each strain becomes resistant to Lcn972 and supports the feasibility of AE-CES as a novel platform to introduce diversity within industrial L. lactis dairy starters.