scholarly journals A Cryptic Non-Inducible Prophage Confers Phage-Immunity on the Streptococcus thermophilus M17PTZA496

Viruses ◽  
2018 ◽  
Vol 11 (1) ◽  
pp. 7 ◽  
Author(s):  
Vinícius da Silva Duarte ◽  
Sabrina Giaretta ◽  
Stefano Campanaro ◽  
Laura Treu ◽  
Andrea Armani ◽  
...  
Keyword(s):  
Mitomycin C ◽  
Defense Mechanisms ◽  
Large Subunit ◽  
Bacterial Species ◽  
Dairy Industry ◽  
Model Organism ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Economic Losses ◽  
Important Species

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.

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
Keyword(s):  
Lactococcus Lactis ◽  
Genetic Manipulation ◽  
Bacterial Species ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Natural Competence ◽  
Content Type ◽  
Competence Gene ◽  
Gene Sets ◽  
Novel Traits

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.

scholarly journals Unleashing natural competence inLactococcus lactisby induction of the competence regulator ComX

2017 ◽  
Author(s):  
Joyce Mulder ◽  
Michiel Wels ◽  
Oscar P. Kuipers ◽  
Michiel Kleerebezem ◽  
Peter A. Bron
Keyword(s):  
Lactococcus Lactis ◽  
Genetic Manipulation ◽  
Bacterial Species ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Natural Competence ◽  
Competence Gene ◽  
Gene Sets ◽  
Novel Traits ◽  
Derivatives Of

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.

scholarly journals Detection and Characterization of Streptococcus thermophilus Bacteriophages by Use of the Antireceptor Gene Sequence

2005 ◽  
Vol 71 (10) ◽  
pp. 6096-6103 ◽  
Author(s):  
Ana G. Binetti ◽  
Beatriz Del Río ◽  
M. Cruz Martín ◽  
Miguel A. Álvarez
Keyword(s):  
Starter Culture ◽  
Dairy Industry ◽  
Pcr Amplification ◽  
Variable Region ◽  
Streptococcus Thermophilus ◽  
Resistance Mechanisms ◽  
Starter Cultures ◽  
Phage Types ◽  
Specific Pcr

ABSTRACT In the dairy industry, the characterization of Streptococcus thermophilus phage types is very important for the selection and use of efficient starter cultures. The aim of this study was to develop a characterization system useful in phage control programs in dairy plants. A comparative study of phages of different origins was initially performed based on their morphology, DNA restriction profiles, DNA homology, structural proteins, packaging mechanisms, and lifestyles and on the presence of a highly conserved DNA fragment of the replication module. However, these traditional criteria were of limited industrial value, mainly because there appeared to be no correlation between these variables and host ranges. We therefore developed a PCR method to amplify VR2, a variable region of the antireceptor gene, which allowed rapid detection of S. thermophilus phages and classification of these phages. This method has a significant advantage over other grouping criteria since our results suggest that there is a correlation between typing profiles and host ranges. This association could be valuable for the dairy industry by allowing a rational starter rotation system to be established and by helping in the selection of more suitable starter culture resistance mechanisms. The method described here is also a useful tool for phage detection, since specific PCR amplification was possible when phage-contaminated milk was used as a template (detection limit, 105 PFU ml−1).

scholarly journals Functional strain redundancy and persistent phage infection in Swiss hard cheese starter cultures

2021 ◽  
Author(s):  
Vincent Somerville ◽  
Hélène Berthoud ◽  
Remo S. Schmidt ◽  
Hans-Peter Bachmann ◽  
Yi Hélène Meng ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Evolutionary Dynamics ◽  
Temporal Dynamics ◽  
Negative Impact ◽  
Bacterial Species ◽  
Starter Culture ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Lactobacillus Delbrueckii ◽  
Shotgun Metagenomics

AbstractUndefined starter cultures are poorly characterized bacterial communities from environmental origin used in cheese making. They are phenotypically stable and have evolved through domestication by repeated propagation in closed and highly controlled environments over centuries. This makes them interesting for understanding eco-evolutionary dynamics governing microbial communities. While cheese starter cultures are known to be dominated by a few bacterial species, little is known about the composition, functional relevance, and temporal dynamics of strain-level diversity. Here, we applied shotgun metagenomics to an important Swiss cheese starter culture and analyzed historical and experimental samples reflecting 82 years of starter culture propagation. We found that the bacterial community is highly stable and dominated by only a few coexisting strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis. Genome sequencing, metabolomics analysis, and co-culturing experiments of 43 isolates show that these strains are functionally redundant, but differ tremendously in their phage resistance potential. Moreover, we identified two highly abundant Streptococcus phages that seem to stably coexist in the community without any negative impact on bacterial growth or strain persistence, and despite the presence of a large and diverse repertoire of matching CRISPR spacers. Our findings show that functionally equivalent strains can coexist in domesticated microbial communities and highlight an important role of bacteria-phage interactions that are different from kill-the-winner dynamics.

scholarly journals In Silico Prediction of Horizontal Gene Transfer Events in Lactobacillus bulgaricus and Streptococcus thermophilus Reveals Protocooperation in Yogurt Manufacturing

2009 ◽  
Vol 75 (12) ◽  
pp. 4120-4129 ◽  
Author(s):  
Mengjin Liu ◽  
Roland J. Siezen ◽  
Arjen Nauta
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Gene Cluster ◽  
In Silico ◽  
Bacterial Species ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Lactobacillus Helveticus ◽  
Lactobacillus Bulgaricus ◽  
Genetic Level

ABSTRACT Lactobacillus bulgaricus and Streptococcus thermophilus, used in yogurt starter cultures, are well known for their stability and protocooperation during their coexistence in milk. In this study, we show that a close interaction between the two species also takes place at the genetic level. We performed an in silico analysis, combining gene composition and gene transfer mechanism-associated features, and predicted horizontally transferred genes in both L. bulgaricus and S. thermophilus. Putative horizontal gene transfer (HGT) events that have occurred between the two bacterial species include the transfer of exopolysaccharide (EPS) biosynthesis genes, transferred from S. thermophilus to L. bulgaricus, and the gene cluster cbs-cblB(cglB)-cysE for the metabolism of sulfur-containing amino acids, transferred from L. bulgaricus or Lactobacillus helveticus to S. thermophilus. The HGT event for the cbs-cblB(cglB)-cysE gene cluster was analyzed in detail, with respect to both evolutionary and functional aspects. It can be concluded that during the coexistence of both yogurt starter species in a milk environment, agonistic coevolution at the genetic level has probably been involved in the optimization of their combined growth and interactions.

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

2016 ◽  
Vol 83 (5) ◽  
Author(s):  
Paula Szymczak ◽  
Thomas Janzen ◽  
Ana Rute Neves ◽  
Witold Kot ◽  
Lars H. Hansen ◽  
...  
Keyword(s):  
Genetic Recombination ◽  
Genetic Relationships ◽  
Bacterial Species ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Sequencing Analysis ◽  
Content Type ◽  
Novel Variants ◽  
Positive Results

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.

Genomic impact of CRISPR immunization against bacteriophages

2013 ◽  
Vol 41 (6) ◽  
pp. 1383-1391 ◽  
Author(s):  
Rodolphe Barrangou ◽  
Anne-Claire Coûté-Monvoisin ◽  
Buffy Stahl ◽  
Isabelle Chavichvily ◽  
Florian Damange ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Draft Genome ◽  
Model Organism ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Host Chromosome ◽  
Small Indels ◽  
The Impact

CRISPR (clustered regularly interspaced short palindromic repeats) together with cas (CRISPR-associated) genes form the CRISPR–Cas immune system, which provides sequence-specific adaptive immunity against foreign genetic elements in bacteria and archaea. Immunity is acquired by the integration of short stretches of invasive DNA as novel ‘spacers’ into CRISPR loci. Subsequently, these immune markers are transcribed and generate small non-coding interfering RNAs that specifically guide nucleases for sequence-specific cleavage of complementary sequences. Among the four CRISPR–Cas systems present in Streptococcus thermophilus, CRISPR1 and CRISPR3 have the ability to readily acquire new spacers following bacteriophage or plasmid exposure. In order to investigate the impact of building CRISPR-encoded immunity on the host chromosome, we determined the genome sequence of a BIM (bacteriophage-insensitive mutant) derived from the DGCC7710 model organism, after four consecutive rounds of bacteriophage challenge. As expected, active CRISPR loci evolved via polarized addition of several novel spacers following exposure to bacteriophages. Although analysis of the draft genome sequence revealed a variety of SNPs (single nucleotide polymorphisms) and INDELs (insertions/deletions), most of the in silico differences were not validated by Sanger re-sequencing. In addition, two SNPs and two small INDELs were identified and tracked in the intermediate variants. Overall, building CRISPR-encoded immunity does not significantly affect the genome, which allows the maintenance of important functional properties in isogenic CRISPR mutants. This is critical for the development and formulation of sustainable and robust next-generation starter cultures with increased industrial lifespans.

scholarly journals Functional strain redundancy and persistent phage infection in Swiss hard cheese starter cultures

2021 ◽  
Author(s):  
Vincent Somerville ◽  
Hélène Berthoud ◽  
Remo S. Schmidt ◽  
Hans- Peter Bachmann ◽  
Yi Hélène Meng ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Evolutionary Dynamics ◽  
Temporal Dynamics ◽  
Negative Impact ◽  
Bacterial Species ◽  
Starter Culture ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Lactobacillus Delbrueckii ◽  
Shotgun Metagenomics

AbstractUndefined starter cultures are poorly characterized bacterial communities from environmental origin used in cheese making. They are phenotypically stable and have evolved through domestication by repeated propagation in closed and highly controlled environments over centuries. This makes them interesting for understanding eco-evolutionary dynamics governing microbial communities. While cheese starter cultures are known to be dominated by a few bacterial species, little is known about the composition, functional relevance, and temporal dynamics of strain-level diversity. Here, we applied shotgun metagenomics to an important Swiss cheese starter culture and analyzed historical and experimental samples reflecting 82 years of starter culture propagation. We found that the bacterial community is highly stable and dominated by only a few coexisting strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis. Genome sequencing, metabolomics analysis, and co-culturing experiments of 43 isolates show that these strains are functionally redundant, but differ tremendously in their phage resistance potential. Moreover, we identified two highly abundant Streptococcus phages that seem to stably coexist in the community without any negative impact on bacterial growth or strain persistence, and despite the presence of a large and diverse repertoire of matching CRISPR spacers. Our findings show that functionally equivalent strains can coexist in domesticated microbial communities and highlight an important role of bacteria-phage interactions that are different from kill-the-winner dynamics.

Molecular Detection of Bacterial Pathogens Directly from the Nasal Swabs and Lung Tissue of Sheep and Goats

2019 ◽  
Vol 15 (02) ◽  
pp. 22-25
Author(s):  
Sunaina Thakur ◽  
Subhash Verma ◽  
Prasenjit Dhar ◽  
Mandeep Sharma
Keyword(s):  
Pasteurella Multocida ◽  
Direct Detection ◽  
Bacterial Species ◽  
Economic Losses ◽  
Isolation And Identification ◽  
Sheep And Goats ◽  
Histophilus Somni ◽  
Nasal Swabs ◽  
Mycoplasma Spp

Respiratory infections of sheep and goats cause heavy morbidity and mortality, leading to huge economic losses. Conventional methods of diagnosis that include isolation and identification of incriminating microbes are time-consuming and fraught with logistic challenges. Direct detection of incriminating microbes using molecular tools is gaining popularity in clinical, microbiological settings. In this study, a total of 50 samples (44 nasal swabs and 6 lung tissues) from sheep and goats were screened for the detection of different bacterial species by in vitro amplification of genus or species-specific genes. Histophilus somni was detected in 2% goat samples, Trueperella pyogenes in 20% goat nasal swabs, whereas 22% goat nasal swab samples were found positive for Mycoplasma spp. None of the samples from sheep was detected positive for H. somni, T. pyogenes, Mycoplasma spp. Similarly, all samples, irrespective, whether from sheep or goats, showed negative results for Pasteurella multocida, Mannheimia haemolytica, and Corynebacterium pseudotuberculosis.

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