scholarly journals Unleashing Natural Competence in Lactococcus lactis by Induction of the Competence Regulator ComX

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Joyce Mulder ◽  
Michiel Wels ◽  
Oscar P. Kuipers ◽  
Michiel Kleerebezem ◽  
Peter A. Bron

ABSTRACT In biotechnological workhorses like Streptococcus thermophilus and Bacillus subtilis, natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis, natural competence has not been established to date. However, in silico analysis of the complete genome sequences of 43 L. lactis strains revealed complete late competence gene sets in 2 L. lactis subsp. cremoris strains (KW2 and KW10) and at least 10 L. lactis subsp. lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon and elicited a state of natural competence in this strain. In contrast, comX expression in L. lactis NZ9000, which was predicted to encode an incomplete competence gene set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147 abolished the comX-driven natural competence, underlining the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence in these strains also, expanding this phenotype to other L. lactis strains of both subspecies. IMPORTANCE Specific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

2017 ◽  
Author(s):  
Joyce Mulder ◽  
Michiel Wels ◽  
Oscar P. Kuipers ◽  
Michiel Kleerebezem ◽  
Peter A. Bron

AbstractIn biotechnological work horses likeStreptococcus thermophilusandBacillus subtilisnatural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starterLactococcus lactisnatural competence has not been established to date. However,in silicoanalysis of complete genome sequences of 43L. lactisstrains revealed complete late-competence gene-sets in 2L. lactissubspeciescremorisstrains (KW2 and KW10) and 8L. lactissubspecieslactisstrains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulatorcomXinL. lactissubsp.lactisKF147 resulted in the induction of expression of the canonical competence regulon, and elicited a state of natural competence in this strain. By contrast,comXexpression inL. lactisNZ9000, predicted to encode an incomplete competence gene-set, failed to induce natural competence. Moreover, mutagenesis of thecomEA-ECoperon in strain KF147, abolished thecomXdriven natural competence, underpinning the involvement of the competence machinery. Finally, introduction of nisin-induciblecomXexpression intonisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence also in these strains, expanding this phenotype to otherL. lactisstrains of both subspecies.Significance statementSpecific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the speciesLactococcus lactisare very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end-product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria, and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.


2016 ◽  
Vol 83 (5) ◽  
Author(s):  
Paula Szymczak ◽  
Thomas Janzen ◽  
Ana Rute Neves ◽  
Witold Kot ◽  
Lars H. Hansen ◽  
...  

ABSTRACT 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. IMPORTANCE Streptococcus 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.


mSystems ◽  
2021 ◽  
Vol 6 (4) ◽  
Author(s):  
Jennifer Mahony

Food fermentations rely on the application of robust bacterial starter cultures, the majority of which are represented by members of the lactic acid bacteria including Lactococcus lactis and Streptococcus thermophilus . Bacteriophage (or phage) proliferation remains one of the most significant threats to the fermentation industry.


Viruses ◽  
2018 ◽  
Vol 11 (1) ◽  
pp. 7 ◽  
Author(s):  
Vinícius da Silva Duarte ◽  
Sabrina Giaretta ◽  
Stefano Campanaro ◽  
Laura Treu ◽  
Andrea Armani ◽  
...  

Streptococcus thermophilus is considered one of the most important species for the dairy industry. Due to their diffusion in dairy environments, bacteriophages can represent a threat to this widely used bacterial species. Despite the presence of a CRISPR-Cas system in the S. thermophilus genome, some lysogenic strains harbor cryptic prophages that can increase the phage-host resistance defense. This characteristic was identified in the dairy strain S. thermophilus M17PTZA496, which contains two integrated prophages 51.8 and 28.3 Kb long, respectively. In the present study, defense mechanisms, such as a lipoprotein-encoding gene and Siphovirus Gp157, the last associated to the presence of a noncoding viral DNA element, were identified in the prophage M17PTZA496 genome. The ability to overexpress genes involved in these defense mechanisms under specific stressful conditions, such as phage attack, has been demonstrated. Despite the addition of increasing amounts of Mitomycin C, M17PTZA496 was found to be non-inducible. However, the transcriptional activity of the phage terminase large subunit was detected in the presence of the antagonist phage vB_SthS-VA460 and of Mitomycin C. The discovery of an additional immune mechanism, associated with bacteriophage-insensitive strains, is of utmost importance, for technological applications and industrial processes. To our knowledge, this is the first study reporting the capability of a prophage integrated into the S. thermophilus genome expressing different phage defense mechanisms. Bacteriophages are widespread entities that constantly threaten starter cultures in the dairy industry. In cheese and yogurt manufacturing, the lysis of Streptococcus thermophilus cultures by viral attacks can lead to huge economic losses. Nowadays S. thermophilus is considered a well-stablished model organism for the study of natural adaptive immunity (CRISPR-Cas) against phage and plasmids, however, the identification of novel bacteriophage-resistance mechanisms, in this species, is strongly desirable. Here, we demonstrated that the presence of a non-inducible prophage confers phage-immunity to an S. thermophilus strain, by the presence of ltp and a viral noncoding region. S. thermophilus M17PTZA496 arises as an unconventional model to study phage resistance and potentially represents an alternative starter strain for dairy productions.


2012 ◽  
Vol 78 (14) ◽  
pp. 4914-4922 ◽  
Author(s):  
Bodil M. Kristensen ◽  
Sunita Sinha ◽  
John D. Boyce ◽  
Anders M. Bojesen ◽  
Joshua C. Mell ◽  
...  

ABSTRACTGallibacterium anatisis a pathogen of poultry. Very little is known about its genetics and pathogenesis. To enable the study of gene function inG. anatis, we have established methods for transformation and targeted mutagenesis. The genusGallibacteriumbelongs to thePasteurellaceae, a group with several naturally transformable members, includingHaemophilus influenzae. Bioinformatics analysis identifiedG. anatishomologs of theH. influenzaecompetence genes, and natural competence was induced inG. anatisby the procedure established forH. influenzae: transfer from rich medium to the starvation medium M-IV. This procedure gave reproducibly high transformation frequencies withG. anatischromosomal DNA and with linearized plasmid DNA carryingG. anatissequences. Both DNA types integrated into theG. anatischromosome by homologous recombination. Targeted mutagenesis gave transformation frequencies of >2 × 10−4transformants CFU−1. Transformation was also efficient with circular plasmid containing noG. anatisDNA; this resulted in the establishment of a self-replicating plasmid. Nine diverseG. anatisstrains were found to be naturally transformable by this procedure, suggesting that natural competence is common and the M-IV transformation procedure widely applicable for this species. TheG. anatisgenome is only slightly enriched for the uptake signal sequences identified in other pasteurellaceaen genomes, butG. anatisdid preferentially take up its own DNA over that ofEscherichia coli. Transformation by electroporation was not effective for chromosomal integration but could be used to introduce self-replicating plasmids. The findings described here provide important tools for the genetic manipulation ofG. anatis.


Author(s):  
Maria Tereza Pereira ◽  
Elsa Helena Walter de Santana ◽  
Joice Sifuentes dos Santos

Produtos lácteos fermentados contêm bactérias ácido lácticas (BAL), naturalmente presentes ou adicionadas na matriz láctea como culturas iniciadoras (starters), contribuindo com aroma, textura, valor nutricional e segurança microbiológica. Lactobacillus spp., Streptococcus spp., Lactococcus spp. e Leuconostoc spp. são utilizados como culturas starters em laticínios. As BAL podem ser classificadas em mesofílicas (ex Lactococcus lactis) e termofílicas (ex Streptococcus thermophilus), e de acordo com seus metabólitos de fermentação em homofermentativas (ácido lático) e heterofermentativas (ácido lático, dióxido de carbono, diacetil e outros compostos flavorizantes). Entre as BAL há um grupo de bactérias lácticas que não fazem parte da cultura láctica (non starter lactic acid bacteria - NSLAB), que são oriundas do leite cru, do ambiente de ordenha ou da indústria formando biofilmes. As NSLAB são representadas por espécies heterofermentativas de lactobacilos mesofílicos como Lactobacillus casei spp., L. paracasei spp., L. rhamnosus spp. e L. plantarum spp., e ainda por Pediococcus spp., Leuconostoc spp. e Micrococcus spp. NSLAB termoduricas como Bacillus spp. também são relatadas. As NSLAB em queijos podem ajudar a desenvolver sabor e aroma, porém também são associadas aos defeitos em queijos e leites fermentados. Problemas como odores estranhos, sabor amargo ou muito ácido, perda de viscosidade, perda de coloração, estufamento e formação de gás são associados com a presença e contaminação por NSLAB. Assim, as BAL são importantes micro-organismos na indústria láctea, garantindo sabores e aromas aos derivados. Já a presença de NSLAB podem ser associados com defeitos em queijos e leites fermentados, sendo um problema na indústria beneficiadora.   Palavras-chave: Característica Sensorial. Leites Fermentados. Queijo. Textura.                       Abstract Fermented dairy products contain acid bacteria (BAL) naturally present or added to the dairy matrix as starter cultures (starters), contributing to aroma, texture, nutritional value and microbiological safety. Lactobacillus spp., Streptococcus spp., Lactococcus spp. and Leuconostoc spp. are used as starter dairy crops. As BAL it can be classified as mesophilic (ex: Lactococcus lactis) and thermophilic (ex: Streptococcus thermophilus), and agree with its fermentation metabolites in homofermentative (lactic acid) and heterofermentative (lactic acid, carbon dioxide, diacetyl and other flavorings). Among the BAL, there is a group of lactic bacteria that are not part of the dairy culture (non-initiating lactic acid bacteria - NSLAB) that originate from raw milk, the milking environment or the biofilm-forming industry. NSLAB is represented by heterofermentative species of mesophilic lactobacilli such as Lactobacillus casei spp., L. paracasei spp., L. rhamnosus spp. and L. plantarum spp., and also by Pediococcus spp., Leuconostoc spp. and Micrococcus spp. Termoduric NSLAB such as Bacillus spp. are also related. NSLAB in cheeses may help develop flavor and aroma, and they are also associated with defects in fermented cheeses and milks. Problems such as strange odors, bitter or very acidic taste, loss of viscosity, loss of color, establishment and gas training are associated with the presence and contamination by NSLAB. Thus,  BALs are important microorganisms in the dairy industry, contributing to the dairy flavors and aromas. The presence of NSLAB, on the other hand, can be associated with defects in fermented milk and cheese, being a problem in the processing industry.   Keywords: Cheese. Fermented Milk. Sensory Characteristic. Texture.


2021 ◽  
Vol 7 (11) ◽  
Author(s):  
Grégoire Siekaniec ◽  
Emeline Roux ◽  
Téo Lemane ◽  
Eric Guédon ◽  
Jacques Nicolas

This study aimed to provide efficient recognition of bacterial strains on personal computers from MinION (Nanopore) long read data. Thanks to the fall in sequencing costs, the identification of bacteria can now proceed by whole genome sequencing. MinION is a fast, but highly error-prone sequencing device and it is a challenge to successfully identify the strain content of unknown simple or complex microbial samples. It is heavily constrained by memory management and fast access to the read and genome fragments. Our strategy involves three steps: indexing of known genomic sequences for a given or several bacterial species; a request process to assign a read to a strain by matching it to the closest reference genomes; and a final step looking for a minimum set of strains that best explains the observed reads. We have applied our method, called ORI, on 77 strains of Streptococcus thermophilus . We worked on several genomic distances and obtained a detailed classification of the strains, together with a criterion that allows merging of what we termed ‘sibling’ strains, only separated by a few mutations. Overall, isolated strains can be safely recognized from MinION data. For mixtures of several non-sibling strains, results depend on strain abundance.


2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Paula Szymczak ◽  
Sérgio Raposo Filipe ◽  
Gonçalo Covas ◽  
Finn Kvist Vogensen ◽  
Ana Rute Neves ◽  
...  

ABSTRACTReceptors 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 bacteriumStreptococcus thermophilusremain elusive. Thus, we set out to identify cell surface structures that are involved in host recognition by dairy streptococcal phages. Five industrialS. thermophilusstrains sensitive to different phages (pactype,costype, 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 betweenS. thermophilusand 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 toS. thermophilusis mediated by glycans associated with the bacterial cell surface. Specifically, thepac-type phage CHPC951 adsorbed to polysaccharides anchored to peptidoglycan, while the 987-type phage CHPC926 recognized exocellular polysaccharides associated with the cell surface.IMPORTANCEStreptococcus thermophilusis 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.


2014 ◽  
Vol 80 (14) ◽  
pp. 4341-4349 ◽  
Author(s):  
Stuart Ainsworth ◽  
Jennifer Mahony ◽  
Douwe van Sinderen

ABSTRACTLactococcus lactissubsp.cremorisstrains are used globally for the production of fermented dairy products, particularly hard cheeses. Believed to be of plant origin,L. lactisstrains 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 ofL. lactisUC509.9, 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 ofL. lactisUC509.9. AbiB escape mutants were generated for phage sk1, which were found to carry mutations inorf6, which encodes the major capsid protein of this phage.


2013 ◽  
Vol 115 (8) ◽  
pp. 1169-1186 ◽  
Author(s):  
Muhammad Saeed ◽  
Faqir Muhammad Anjum ◽  
Moazzam Rafiq Khan ◽  
Muhammad Issa Khan ◽  
Muhammad Nadeem

PurposeWhey products have conventionally been professed as a means of reducing ingredient costs. The authentic benefits of adding whey products are the enhanced worth resulting from flavor, texture and nutritional improvements as well as nutraceutical or health‐enhancing payback. Therefore, the present study aims to isolate and characterize suitable starter cultures for the production of wheyghurt drink.Design/methodology/approachKeeping in view all the benefits of yoghurt technology this study was planned to isolate the starter cultures and optimize the conditions for the production of wheyghurt drink. The starter cultures (Lactobacillus delbruceckii ssp. Bulgaricus and Streptococcus thermophilus) were isolated from the yoghurt and further characterized on the basis of their morphological and biochemical characteristics. The wheyghurt drink prepared from starter cultures with varying starter culture concentrations (1, 1.5, 2 and 2.5 percent) was analyzed for the physicochemical and sensory characteristics to explore the potential of wheyghurt drink.FindingsDuring storage, color, flavor, taste and overall acceptability were affected significantly. But the interaction between treatments and storage was found non‐significant to all the sensory parameters. At zero day maximum score (7.40) for overall acceptability was recorded for T3 and minimum score (5.60) was awarded to T4. After five, ten and 15 days of storage, judges observed a slight decline in overall acceptability in all wheyghurt drink samples. Hence it was concluded that wheyghurt drink sample T3 obtained maximum scores regarding the organoleptic evaluation and remained the best.Practical implicationsThe key to growth is a continuous evaluation and modification of the product to match consumer expectations. Currently there are many apparent benefits that result from incorporating selected whey products into yogurt formulas. The starter cultures for the production of fermented whey products are not presently produced in Pakistan and are imported for industrial use. The use of LAB as starter culture may help to improve the quality and shelf life of the whey products.Originality/valueThe research is useful for food manufacturers in order to develop functional food products for consumers. Understanding consumer needs and preferences is critical to successful product development and enhancing marketing values of a product. Nutritionally improved foods, such as wheyghurt over the conventional counterpart, will be highly successful in the marketplace. Consumers will prefer such kinds of foods because they are more conscious about their health and such foods provide them with what they desire, i.e. health benefits with good nutrition.


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