Novel Genus of Phages Infecting Streptococcus thermophilus: Genomic and Morphological Characterization

ABSTRACT Streptococcus thermophilus is a lactic acid bacterium commonly used for the manufacture of yogurt and specialty cheeses. Virulent phages represent a major risk for milk fermentation processes worldwide, as they can inactivate the added starter bacterial cells, leading to low-quality fermented dairy products. To date, four genetically distinct groups of phages infecting S. thermophilus have been described. Here, we describe a fifth group. Phages P738 and D4446 are virulent siphophages that infect a few industrial strains of S. thermophilus. The genomes of phages P738 and D4446 were sequenced and found to contain 34,037 and 33,656 bp as well as 48 and 46 open reading frames, respectively. Comparative genomic analyses revealed that the two phages are closely related to each other but display very limited similarities to other S. thermophilus phages. In fact, these two novel S. thermophilus phages share similarities with streptococcal phages of nondairy origin, suggesting that they emerged recently in the dairy environment. IMPORTANCE Despite decades of research and adapted antiphage strategies such as CRISPR-Cas systems, virulent phages are still a persistent risk for the milk fermentation industry worldwide, as they can cause manufacturing failures and alter product quality. Phages P738 and D4446 are novel virulent phages that infect the food-grade Gram-positive bacterial species Streptococcus thermophilus. These two related viruses represent a fifth group of S. thermophilus phages, as they are significantly distinct from other known S. thermophilus phages. Both phages share similarities with phages infecting nondairy streptococci, suggesting their recent emergence and probable coexistence in dairy environments. These findings highlight the necessity of phage surveillance programs as the phage population evolves in response to the application of antiphage strategies.

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Biology and Genome Sequence of Streptococcus mutans Phage M102AD

Applied and Environmental Microbiology ◽

10.1128/aem.07726-11 ◽

2012 ◽

Vol 78 (7) ◽

pp. 2264-2271 ◽

Cited By ~ 12

Author(s):

Allan L. Delisle ◽

Ming Guo ◽

Natalia I. Chalmers ◽

Gerard J. Barcak ◽

Geneviève M. Rousseau ◽

...

Keyword(s):

Streptococcus Mutans ◽

Genome Sequence ◽

Gc Content ◽

Genomic Analysis ◽

Streptococcus Thermophilus ◽

Open Reading Frames ◽

Comparative Genomic ◽

Content Type ◽

Close Relationship ◽

Virion Structure

ABSTRACTM102AD is the new designation for aStreptococcus mutansphage described in 1993 as phage M102. This change was necessitated by the genome analysis of anotherS. mutansphage named M102, which revealed differences from the genome sequence reported here. Additional host range analyses confirmed thatS. mutansphage M102AD infects only a few serotype c strains. Phage M102AD adsorbed very slowly to its host, and it cannot adsorb to serotype e and f strains ofS. mutans. M102AD adsorption was blocked by c-specific antiserum. Phage M102AD also adsorbed equally well to heat-treated and trypsin-treated cells, suggesting carbohydrate receptors. Saliva and polysaccharide production did not inhibit plaque formation. The genome of this siphophage consisted of a linear, double-stranded, 30,664-bp DNA molecule, with a GC content of 39.6%. Analysis of the genome extremities indicated the presence of a 3′-overhangcossite that was 11 nucleotides long. Bioinformatic analyses identified 40 open reading frames, all in the same orientation. No lysogeny-related genes were found, indicating that phage M102AD is strictly virulent. No obvious virulence factor gene candidates were found. Twelve proteins were identified in the virion structure by mass spectrometry. Comparative genomic analysis revealed a close relationship betweenS. mutansphages M102AD and M102 as well as withStreptococcus thermophilusphages. This study also highlights the importance of conducting research with biological materials obtained from recognized microbial collections.

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An Evolutionary Link between Natural Transformation and CRISPR Adaptive Immunity

mBio ◽

10.1128/mbio.00309-12 ◽

2012 ◽

Vol 3 (5) ◽

Cited By ~ 52

Author(s):

Peter Jorth ◽

Marvin Whiteley

Keyword(s):

Gene Transfer ◽

Horizontal Gene Transfer ◽

Bacterial Diversity ◽

Natural Transformation ◽

Bacterial Species ◽

Comparative Genomic ◽

Content Type ◽

Immune Systems ◽

Adaptive Immune ◽

Adaptive Immune Systems

ABSTRACTNatural transformation by competent bacteria is a primary means of horizontal gene transfer; however, evidence that competence drives bacterial diversity and evolution has remained elusive. To test this theory, we used a retrospective comparative genomic approach to analyze the evolutionary history ofAggregatibacter actinomycetemcomitans, a bacterial species with both competent and noncompetent sister strains. Through comparative genomic analyses, we reveal that competence is evolutionarily linked to genomic diversity and speciation. Competence loss occurs frequently during evolution and is followed by the loss of clustered regularly interspaced short palindromic repeats (CRISPRs), bacterial adaptive immune systems that protect against parasitic DNA. Relative to noncompetent strains, competent bacteria have larger genomes containing multiple rearrangements. In contrast, noncompetent bacterial genomes are extremely stable but paradoxically susceptible to infective DNA elements, which contribute to noncompetent strain genetic diversity. Moreover, incomplete noncompetent strain CRISPR immune systems are enriched for self-targeting elements, which suggests that the CRISPRs have been co-opted for bacterial gene regulation, similar to eukaryotic microRNAs derived from the antiviral RNA interference pathway.IMPORTANCEThe human microbiome is rich with thousands of diverse bacterial species. One mechanism driving this diversity is horizontal gene transfer by natural transformation, whereby naturally competent bacteria take up environmental DNA and incorporate new genes into their genomes. Competence is theorized to accelerate evolution; however, attempts to test this theory have proved difficult. Through genetic analyses of the human periodontal pathogenAggregatibacter actinomycetemcomitans, we have discovered an evolutionary connection between competence systems promoting gene acquisition and CRISPRs (clustered regularly interspaced short palindromic repeats), adaptive immune systems that protect bacteria against genetic parasites. We show that competentA. actinomycetemcomitansstrains have numerous redundant CRISPR immune systems, while noncompetent bacteria have lost their CRISPR immune systems because of inactivating mutations. Together, the evolutionary data linking the evolution of competence and CRISPRs reveals unique mechanisms promoting genetic heterogeneity and the rise of new bacterial species, providing insight into complex mechanisms underlying bacterial diversity in the human body.

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A Stringent Analysis of Polyphosphate Dynamics inEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.00070-19 ◽

2019 ◽

Vol 201 (9) ◽

Author(s):

Michael Downey

Keyword(s):

Escherichia Coli ◽

Infection Control ◽

Bacterial Species ◽

Stringent Response ◽

Industrial Applications ◽

Bacterial Cells ◽

Inescherichia Coli ◽

Content Type ◽

Polyphosphate Accumulation ◽

Inorganic Phosphates

ABSTRACTDuring stress, bacterial cells activate a conserved pathway called the stringent response that promotes survival. Polyphosphates are long chains of inorganic phosphates that modulate this response in diverse bacterial species. In this issue, Michael J. Gray provides an important correction to the model of how polyphosphate accumulation is regulated during the stringent response inEscherichia coli(M. J. Gray, J. Bacteriol, 201:e00664-18, 2019,https://doi.org/10.1128/JB.00664-18). With other recent publications, this study provides a revised framework for understanding how bacterial polyphosphate dynamics might be exploited in infection control and industrial applications.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01320-17 ◽

2017 ◽

Vol 83 (20) ◽

Cited By ~ 11

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.

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Identification of isolated or mixed strains from long reads: a challenge met on Streptococcus thermophilus using a MinION sequencer

Microbial Genomics ◽

10.1099/mgen.0.000654 ◽

2021 ◽

Vol 7 (11) ◽

Author(s):

Grégoire Siekaniec ◽

Emeline Roux ◽

Téo Lemane ◽

Eric Guédon ◽

Jacques Nicolas

Keyword(s):

Memory Management ◽

Bacterial Species ◽

Streptococcus Thermophilus ◽

Bacterial Strains ◽

Content Type ◽

Long Reads ◽

Detailed Classification ◽

Long Read ◽

Identification Of Bacteria

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.

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Involvement of the Major Capsid Protein and Two Early-Expressed Phage Genes in the Activity of the Lactococcal Abortive Infection Mechanism AbiT

Applied and Environmental Microbiology ◽

10.1128/aem.01755-12 ◽

2012 ◽

Vol 78 (19) ◽

pp. 6890-6899 ◽

Cited By ~ 14

Author(s):

Simon J. Labrie ◽

Denise M. Tremblay ◽

Maxim Moisan ◽

Manuela Villion ◽

Alfonso H. Magadán ◽

...

Keyword(s):

Capsid Protein ◽

Major Capsid Protein ◽

Control Strategies ◽

Fermented Milk ◽

Genomic Region ◽

Comparative Genomic ◽

Abortive Infection ◽

Content Type ◽

Milk Fermentation ◽

Phage Morphogenesis

ABSTRACTThe dairy industry uses the mesophilic, Gram-positive, lactic acid bacterium (LAB)Lactococcus lactisto produce an array of fermented milk products. Milk fermentation processes are susceptible to contamination by virulent phages, but a plethora of phage control strategies are available. One of the most efficient is to use LAB strains carrying phage resistance systems such as abortive infection (Abi) mechanisms. Yet, the mode of action of most Abi systems remains poorly documented. Here, we shed further light on the antiviral activity of the lactococcal AbiT system. Twenty-eight AbiT-resistant phage mutants derived from the wild-type AbiT-sensitive lactococcal phages p2, bIL170, and P008 were isolated and characterized. Comparative genomic analyses identified three different genes that were mutated in these virulent AbiT-insensitive phage derivatives:e14(bIL170 [e14bIL170]),orf41(P008 [orf41P008]), andorf6(p2 [orf6p2] and P008 [orf6P008]). The genese14bIL170andorf41P008are part of the early-expressed genomic region, but bioinformatic analyses did not identify their putative function.orf6is found in the phage morphogenesis module. Antibodies were raised against purified recombinant ORF6, and immunoelectron microscopy revealed that it is the major capsid protein (MCP). Coexpression inL. lactisof ORF6p2and ORF5p2, a protease, led to the formation of procapsids. To our knowledge, AbiT is the first Abi system involving distinct phage genes.

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Enzymatic Synthesis of Bioinformatically Predicted Microcin C-Like Compounds Encoded by Diverse Bacteria

mBio ◽

10.1128/mbio.01059-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 16

Author(s):

Olga Bantysh ◽

Marina Serebryakova ◽

Kira S. Makarova ◽

Svetlana Dubiley ◽

Kirill A. Datsenko ◽

...

Keyword(s):

Yersinia Pseudotuberculosis ◽

Streptococcus Thermophilus ◽

Trna Synthetase ◽

Open Reading Frames ◽

Peptide Substrates ◽

Content Type ◽

E Coli ◽

Gram Positive Bacteria ◽

Synechococcus Sp

ABSTRACT The Trojan horse Escherichia coli antibiotic microcin C (McC) consists of a heptapeptide attached to adenosine through a phosphoramidate linkage. McC is synthesized by the MccB enzyme, which terminally adenylates the ribosomally synthesized heptapeptide precursor MccA. The peptide part is responsible for McC uptake; it is degraded inside the cell to release a toxic nonhydrolyzable aspartyl-adenylate. Bionformatic analysis reveals that diverse bacterial genomes encoding mccB homologues also contain adjacent short open reading frames that may encode MccA-like adenylation substrates. Using chemically synthesized predicted peptide substrates and recombinant cognate MccB protein homologs, adenylated products were obtained in vitro for predicted MccA peptide-MccB enzyme pairs from Helicobacter pylori, Streptococcus thermophilus, Lactococcus johnsonii, Bartonella washoensis, Yersinia pseudotuberculosis, and Synechococcus sp. Some adenylated products were shown to inhibit the growth of E. coli by targeting aspartyl-tRNA synthetase, the target of McC. IMPORTANCE Our results prove that McC-like adenylated peptides are widespread and are encoded by both Gram-negative and Gram-positive bacteria and by cyanobacteria, opening ways for analyses of physiological functions of these compounds and for creation of microcin C-like antibiotics targeting various bacteria.

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Bactopia: a Flexible Pipeline for Complete Analysis of Bacterial Genomes

mSystems ◽

10.1128/msystems.00190-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Robert A. Petit ◽

Timothy D. Read

Keyword(s):

Open Source ◽

Genome Analysis ◽

Bacterial Species ◽

Bacterial Genome ◽

Complete Analysis ◽

Comparative Genomic ◽

Data Sets ◽

Bacterial Genomes ◽

Data Set ◽

Content Type

ABSTRACT Sequencing of bacterial genomes using Illumina technology has become such a standard procedure that often data are generated faster than can be conveniently analyzed. We created a new series of pipelines called Bactopia, built using Nextflow workflow software, to provide efficient comparative genomic analyses for bacterial species or genera. Bactopia consists of a data set setup step (Bactopia Data Sets [BaDs]), which creates a series of customizable data sets for the species of interest, the Bactopia Analysis Pipeline (BaAP), which performs quality control, genome assembly, and several other functions based on the available data sets and outputs the processed data to a structured directory format, and a series of Bactopia Tools (BaTs) that perform specific postprocessing on some or all of the processed data. BaTs include pan-genome analysis, computing average nucleotide identity between samples, extracting and profiling the 16S genes, and taxonomic classification using highly conserved genes. It is expected that the number of BaTs will increase to fill specific applications in the future. As a demonstration, we performed an analysis of 1,664 public Lactobacillus genomes, focusing on Lactobacillus crispatus, a species that is a common part of the human vaginal microbiome. Bactopia is an open source system that can scale from projects as small as one bacterial genome to ones including thousands of genomes and that allows for great flexibility in choosing comparison data sets and options for downstream analysis. Bactopia code can be accessed at https://www.github.com/bactopia/bactopia. IMPORTANCE It is now relatively easy to obtain a high-quality draft genome sequence of a bacterium, but bioinformatic analysis requires organization and optimization of multiple open source software tools. We present Bactopia, a pipeline for bacterial genome analysis, as an option for processing bacterial genome data. Bactopia also automates downloading of data from multiple public sources and species-specific customization. Because the pipeline is written in the Nextflow language, analyses can be scaled from individual genomes on a local computer to thousands of genomes using cloud resources. As a usage example, we processed 1,664 Lactobacillus genomes from public sources and used comparative analysis workflows (Bactopia Tools) to identify and analyze members of the L. crispatus species.

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Novel Phage Group Infecting Lactobacillus delbrueckii subsp. lactis, as Revealed by Genomic and Proteomic Analysis of Bacteriophage Ldl1

Applied and Environmental Microbiology ◽

10.1128/aem.03413-14 ◽

2014 ◽

Vol 81 (4) ◽

pp. 1319-1326 ◽

Cited By ~ 18

Author(s):

Eoghan Casey ◽

Jennifer Mahony ◽

Horst Neve ◽

Jean-Paul Noben ◽

Fabio Dal Bello ◽

...

Keyword(s):

Burst Size ◽

Bacterial Species ◽

Lactobacillus Delbrueckii ◽

Comparative Genomic ◽

Phage Group ◽

Content Type ◽

Large Head ◽

Electron Microscopy Analysis ◽

Microscopy Analysis

ABSTRACTLdl1 is a virulent phage infecting the dairy starterLactobacillus delbrueckiisubsp.lactisLdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infectingLactobacillus delbrueckii.In vitropropagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 ± 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that ofLactobacillus plantarumphage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognizedL. delbrueckiiphage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.

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Genomics of the Uncultivated, Periodontitis-Associated BacteriumTannerellasp. BU045 (Oral Taxon 808)

mSystems ◽

10.1128/msystems.00018-18 ◽

2018 ◽

Vol 3 (3) ◽

Cited By ~ 6

Author(s):

Clifford J. Beall ◽

Alisha G. Campbell ◽

Ann L. Griffen ◽

Mircea Podar ◽

Eugene J. Leys

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Chronic Periodontitis ◽

Bacterial Species ◽

The United States ◽

Oral Microbiome ◽

Rrna Gene ◽

Bacterial Cells ◽

Data Set ◽

Content Type

ABSTRACTDespite decades of research into the human oral microbiome, many species remain uncultivated. The technique of single-cell whole-genome amplification and sequencing provides a means of deriving genome sequences for species that can be informative on biological function and suggest pathways to cultivation.Tannerella forsythiahas long been known to be highly associated with chronic periodontitis and to cause periodontitis-like symptoms in experimental animals, andTannerellasp. BU045 (human oral taxon 808) is an uncultivated relative of this organism. In this work, we extend our previous sequencing of theTannerellasp. BU063 (human oral taxon 286) genome by sequencing amplified genomes from 11 cells ofTannerellasp. BU045, including 3 genomes that are at least 90% complete.Tannerellasp. BU045 is more closely related toTannerellasp. BU063 than toT. forsythiaby gene content and average nucleotide identity. However, two independent data sets of association with periodontitis, one based on 16S rRNA gene abundance and the other based on gene expression in a metatranscriptomic data set, show thatTannerellasp. BU045 is more highly associated with disease thanTannerellasp. BU063. Comparative genomics shows genes and functions that are shared or unique to the different species, which may direct further research of the pathogenesis of chronic periodontitis.IMPORTANCEPeriodontitis (gum disease) affects 47% of adults over 30 in the United States (P. I. Eke, B. A. Dye, L. Wei, G. O. Thornton-Evans, R. J. Genco, et al., J Dent Res 91:914–920, 2012), and it cost between $39 and $396 billion worldwide in 2015 (A. J. Righolt, M. Jevdjevic, W. Marcenes, and S. Listl, J Dent Res, 17 January 2018, https://doi.org/10.1177/0022034517750572). Many bacteria associated with the disease are known only by the DNA sequence of their 16S rRNA gene. In this publication, amplification and sequencing of DNA from single bacterial cells are used to obtain nearly complete genomes ofTannerellasp. BU045, a species of bacteria that is more prevalent in patients with periodontitis than in healthy patients. Comparing the complete genome of this bacterium to genomes of related bacterial species will help to better understand periodontitis and may help to grow this organism in pure culture, which would allow a better understanding of its role in the mouth.

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