Mutations Selected After Exposure to Bacteriocin Lcn972 Activate a Bce-Like Bacitracin Resistance Module in Lactococcus lactis.

Resistance against antimicrobial peptides (AMPs) is often mediated by detoxification modules that rely on sensing the AMP through a BceAB-like ATP-binding cassette (ABC) transporter that subsequently activates a cognate two-component system (TCS) to mount the cell response. Here, the Lactococcus lactis ABC transporter YsaDCB is shown to constitute, together with TCS-G, a detoxification module that protects L. lactis against bacitracin and the bacteriocin Lcn972, both AMPs that inhibit cell wall biosynthesis. Initially, increased expression of ysaDCB was detected by RT-qPCR in three L. lactis resistant to Lcn972, two of which were also resistant to bacitracin. These mutants shared, among others, single-point mutations in ysaB coding for the putative Bce-like permease. These results led us to investigate the function of YsaDCB ABC-transporter and study the impact of these mutations. Expression in trans of ysaDCB in L. lactis NZ9000, a strain that lacks a functional detoxification module, enhanced resistance to both AMPs, demonstrating its role as a resistance factor in L. lactis. When the three different ysaB alleles from the mutants were expressed, all of them outperformed the wild-type transporter in resistance against Lcn972 but not against bacitracin, suggesting a distinct mode of protection against each AMP. Moreover, P ysaD promoter fusions, designed to measure the activation of the detoxification module, revealed that the ysaB mutations unlock transcriptional control by TCS-G, resulting in constitutive expression of the ysaDCB operon. Finally, deletion of ysaD was also performed to get an insight into the function of this gene. ysaD encodes a secreted peptide and is part of the ysaDCB operon. YsaD appears to modulate signal relay between the ABC transporter and TCS-G, based on the different response of the P ysaD promoter fusions when it is not present. Altogether, the results underscore the unique features of this lactococcal detoxification module that warrant further research to advance in our overall understanding of these important resistance factors in bacteria.

Complete Genome Sequences of 28 Lactococcal Bacteriophages Isolated from Failed Dairy Fermentation Processes.

Lactococcus lactis is a Gram-positive lactic acid bacterium commonly used in the dairy industry for the production of fermented foods such as buttermilk and a wide variety of cheeses. Here, we report the complete genome sequences of 28 bacteriophages infecting different L. lactis industrial starter strains isolated from dairy plants throughout the world.

A Specific Sugar Moiety in the Lactococcus lactis Cell Wall Pellicle Is Required for Infection by CHPC971, a Member of the Rare 1706 Phage Species.

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.

A comparative genomics approach for identifying host-range determinants in Streptococcus thermophilus bacteriophages.

Comparative genomics has proven useful in exploring the biodiversity of phages and understanding phage-host interactions. This knowledge is particularly useful for phages infecting Streptococcus thermophilus, as they constitute a constant threat during dairy fermentations. Here, we explore the genetic diversity of S. thermophilus phages to identify genetic determinants with a signature for host specificity, which could be linked to the bacterial receptor genotype. A comparative genomic analysis was performed on 142 S. thermophilus phage genomes, 55 of which were sequenced in this study. Effectively, 94 phages were assigned to the group cos (DT1), 36 to the group pac (O1205), six to the group 5093, and six to the group 987. The core genome-based phylogeny of phages from the two dominating groups and their receptor binding protein (RBP) phylogeny corresponded to the phage host-range. A role of RBP in host recognition was confirmed by constructing a fluorescent derivative of the RBP of phage CHPC951, followed by studying the binding of the protein to the host strain. Furthermore, the RBP phylogeny of the cos group was found to correlate with the host genotype of the exocellular polysaccharide-encoding operon. These findings provide novel insights towards developing strategies to combat phage infections in dairies.

Adaptive Evolution of Industrial Lactococcus lactis Under Cell Envelope Stress Provides Phenotypic Diversity.

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.

Cell Wall Glycans Mediate Recognition of the Dairy Bacterium Streptococcus thermophilus by Bacteriophages.

Receptors on the cell surfaces of bacterial hosts are essential during the infection cycle of bacteriophages. To date, the phage receptors of the industrial relevant dairy starter bacterium Streptococcus thermophilus remain elusive. Thus, we set out to identify cell surface structures that are involved in host recognition by dairy streptococcal phages. Five industrial S. thermophilus strains sensitive to different phages (pac type, cos type, and the new type 987), were selected to generate spontaneous bacteriophage-insensitive mutants (BIMs). Of these, approximately 50% were deselected as clustered regularly interspaced short palindromic repeat (CRISPR) mutants, while the other pool was further characterized to identify receptor mutants. On the basis of genome sequencing data, phage resistance in putative receptor mutants was attributed to nucleotide changes in genes encoding glycan biosynthetic pathways. Superresolution structured illumination microscopy was used to visualize the interactions between S. thermophilus and its phages. The phages were either regularly distributed along the cells or located at division sites of the cells. The cell wall structures mediating the latter type of phage adherence were further analyzed via phenotypic and biochemical assays. Altogether, our data suggested that phage adsorption to S. thermophilus is mediated by glycans associated with the bacterial cell surface. Specifically, the pac-type phage CHPC951 adsorbed to polysaccharides anchored to peptidoglycan, while the 987-type phage CHPC926 recognized exocellular polysaccharides associated with the cell surface.IMPORTANCEStreptococcus thermophilus is widely used in starter cultures for cheese and yoghurt production. During dairy fermentations, infections of bacteria with bacteriophages result in acidification failures and a lower quality of the final products. An understanding of the molecular factors involved in phage-host interactions, in particular, the phage receptors in dairy bacteria, is a crucial step for developing better strategies to prevent phage infections in dairy plants.

Investigation of the bacteriophage community in induced lysates of undefined mesophilic mixed-strain DL-cultures using classical and metagenomic approaches.

To investigate the notion that starter cultures can be a reservoir of bacteriophages (phages) in the dairy environment, strains of three DL-starters (undefined mesophilic mixed-strain starters containing Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc species) were selected and induced by mitomycin C, and the whole starters were induced spontaneously as well as by mitomycin C. Frequency of induction of 17%, 26% and 12% was estimated among the isolates of the three starters, with majority of the induced phages mostly showing morphological similarity to known P335 phages, and with a fraction of them showing atypical features. Sequences of P335 quasi-species phages were found to be the most frequent entities in almost all metaviromes derived from the induced lysates. However, sequences of Sk1virus phages (previously 936 phages) were emerged as the predominant entities following spontaneous induction of one of the starters, suggesting a phage-carrier state. Sequences of other phages such as 949, 1706, C2virus (previously c2 phages) and Leuconostoc species could also be observed but with a lower relative frequency. Taken together, the majority of the P335 quasi-species phages could represent the induced viral community of the starters and the remaining phage groups mainly represent the background ambient viral community.

Polysaccharide production by lactic acid bacteria: from genes to industrial applications.

The ability to produce polysaccharides with diverse biological functions is widespread in bacteria. In lactic acid bacteria (LAB), production of polysaccharides has long been associated with the technological, functional and health-promoting benefits of these microorganisms. In particular, the capsular polysaccharides and exopolysaccharides have been implicated in modulation of the rheological properties of fermented products. For this reason, screening and selection of exocellular polysaccharide-producing LAB has been extensively carried out by academia and industry. To further exploit the ability of LAB to produce polysaccharides, an in-depth understanding of their biochemistry, genetics, biosynthetic pathways, regulation and structure-function relationships is mandatory. Here, we provide a critical overview of the latest advances in the field of glycosciences in LAB. Surprisingly, the understanding of the molecular processes involved in polysaccharide synthesis is lagging behind, and has not accompanied the increasing commercial value and application potential of these polymers. Seizing the natural diversity of polysaccharides for exciting new applications will require a concerted effort encompassing in-depth physiological characterization of LAB at the systems level. Combining high-throughput experimentation with computational approaches, biochemical and structural characterization of the polysaccharides and understanding of the structure-function-application relationships is essential to achieve this ambitious goal.

Novel Variants of Streptococcus thermophilus Bacteriophages Are Indicative of Genetic Recombination among Phages from Different Bacterial Species.

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.

Enhancing the Sweetness of Yoghurt through Metabolic Remodeling of Carbohydrate Metabolism in Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

UNLABELLED: Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus are used in the fermentation of milk to produce yoghurt. These species normally metabolize only the glucose moiety of lactose, secreting galactose and producing lactic acid as the main metabolic end product. We used multiple serial selection steps to isolate spontaneous mutants of industrial strains of S. thermophilus and L. delbrueckii subsp. bulgaricus that secreted glucose rather than galactose when utilizing lactose as a carbon source. Sequencing revealed that the S. thermophilus strains had mutations in the galKTEM promoter, the glucokinase gene, and genes encoding elements of the glucose/mannose phosphotransferase system (PTS). These strains metabolize galactose but are unable to phosphorylate glucose internally or via the PTS. The L. delbrueckii subsp. bulgaricus mutants had mutations in genes of the glucose/mannose PTS and in the pyruvate kinase gene. These strains cannot grow on exogenous glucose but are proficient at metabolizing internal glucose released from lactose by ß-galactosidase. The resulting strains can be combined to ferment milk, producing yoghurt with no detectable lactose, moderate levels of galactose, and high levels of glucose. Since glucose tastes considerably sweeter than either lactose or galactose, the sweetness of the yoghurt is perceptibly enhanced. These strains were produced without the use of recombinant DNA technology and can be used for the industrial production of yoghurt with enhanced intrinsic sweetness and low residual levels of lactose. IMPORTANCE: Based on a good understanding of the physiology of the lactic acid bacteria Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, we were able, by selecting spontaneously occurring mutants, to change dramatically the metabolic products secreted into the growth medium. These mutants consume substantially more of the lactose, metabolize some of the galactose, and secrete the remaining galactose and most of the glucose back into the milk. This allows production of yoghurt with very low lactose levels and enhanced natural sweetness, because humans perceive glucose as sweeter than either lactose or galactose.

The art of strain improvement of industrial lactic acid bacteria without the use of recombinant DNA technology.

The food industry is constantly striving to develop new products to fulfil the ever changing demands of consumers and the strict requirements of regulatory agencies. For foods based on microbial fermentation, this pushes the boundaries of microbial performance and requires the constant development of new starter cultures with novel properties. Since the use of ingredients in the food industry is tightly regulated and under close scrutiny by consumers, the use of recombinant DNA technology to improve microbial performance is currently not an option. As a result, the focus for improving strains for microbial fermentation is on classical strain improvement methods. Here we review the use of these techniques to improve the functionality of lactic acid bacteria starter cultures for application in industrial-scale food production. Methods will be described for improving the bacteriophage resistance of specific strains, improving their texture forming ability, increasing their tolerance to stress and modulating both the amount and identity of acids produced during fermentation. In addition, approaches to eliminating undesirable properties will be described. Techniques include random mutagenesis, directed evolution and dominant selection schemes.

Identification of Lactococcus lactis genes required for bacteriophage adsorption.

The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and phi 645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that phi645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that phi 645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and phi 645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and phi 645 that showed no homology in the C-terminal part.

Characterisation of technologically proficient wild Lactococcus lactis strains resistant to phage infection.

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.

Identification of the host determinant of two prolate-headed phages infecting Lactococcus lactis.

A gene responsible for host determination was identified in two prolate-headed bacteriophages of the c2 species infecting strains of Lactococcus lactis. The identification of the host determinant gene was based on low DNA sequence homology in a specific open reading frame (ORF) between prolate-headed phages with different host ranges. When a host carrying this ORF from one phage on a plasmid was infected with another phage, we obtained phages with an altered host range at a frequency of 10(-6) to 10(-7). Sequencing of phage DNA originating from 10 independent single plaques confirmed that a genetic recombination had taken place at different positions between the ORF on the plasmid and the infecting phage. The adsorption of the recombinant phages to their bacterial hosts had also changed to match the phage origin of the ORF. Consequently, it is concluded that this ORF codes for the host range determinant.

Bacteriophage resistance of a deltathyA mutant of Lactococcus lactis blocked in DNA replication.

The thyA gene, which encodes thymidylate synthase (TS), of Lactococcus lactis CHCC373 was sequenced, including the upstream and downstream regions. We then deleted part of thyA by gene replacement. The resulting strain, MBP71 deltathyA, was devoid of TS activity, and in media without thymidine, such as milk, there was no detectable dTTP pool in the cells. Hence, DNA replication was abolished, and acidification by MBP71 was completely unaffected by the presence of nine different phages tested at a multiplicity of infection (MOI) of 0.1. Nonreplicating MBP71 must be inoculated at a higher level than CHCC373 to achieve a certain pH within a specified time. For a pH of 5.2 to be reached in 6 h, the inoculation level of MBP71 must be 17-fold higher than for CHCC373. However, by adding a limiting amount of thymidine this could be lowered to just 5-fold the normal amount, while acidification was unaffected with MBP71 up to an MOI of 0.01. It was found that nonreplicating MBP71 produced largely the same products as CHCC373, though the acetaldehyde production of the former was higher.