Escherichia coli CRISPR arrays from early life fecal samples preferentially target prophages.

CRISPR-Cas systems are defense mechanisms against phages and other nucleic acids that invade bacteria and archaea. In Escherichia coli, it is generally accepted that CRISPR-Cas systems are inactive in laboratory conditions due to a transcriptional repressor. In natural isolates, it has been shown that CRISPR arrays remain stable over the years and that most spacer targets (protospacers) remain unknown. Here, we re-examine CRISPR arrays in natural E. coli isolates and investigate viral and bacterial genomes for spacer targets using a bioinformatics approach coupled to a unique biological dataset. We first sequenced the CRISPR1 array of 1769 E. coli isolates from the fecal samples of 639 children obtained during their first year of life. We built a network with edges between isolates that reflect the number of shared spacers. The isolates grouped into 34 modules. A search for matching spacers in bacterial genomes showed that E. coli spacers almost exclusively target prophages. While we found instances of self-targeting spacers, those involving a prophage and a spacer within the same bacterial genome were rare. The extensive search for matching spacers also expanded the library of known E. coli protospacers to 60%. Altogether, these results favor the concept that E. coli's CRISPR-Cas is an antiprophage system and highlight the importance of reconsidering the criteria use to deem CRISPR-Cas systems active.

Host-encoded, cell surface-associated exopolysaccharide required for adsorption and infection by lactococcal P335 phage subtypes.

Lactococcus lactis and Lactococcus cremoris compose commercial starter cultures widely used for industrial dairy fermentations. Some lactococcal strains may produce exopolysaccharides (EPS), which have technological applications, including texture production and phage resistance. Two distinct gene clusters associated with EPS production, designated 6073-like and 7127-like, were identified on plasmids in lactococcal strains. Infectivity of two subsets of P335 group phages, distinguished based on their single-component baseplate/receptor-binding protein nucleotide sequences, was correlated to the presence of a host-encoded 6073-like or 7127-like eps gene cluster. Furthermore, phages belonging to these subsets differentially adsorbed to lactococcal strains harboring the respective eps gene cluster. Loss of the respective EPS-encoding plasmid from a fully phage-sensitive strain resulted in loss of phage adsorption and resistance to the phage. Transmission electron microscopy (TEM) showed that the EPS produced by strains encoding the 6073-like or 7127-like eps gene clusters are cell-surface associated, which, coupled with phage plaquing and adsorption data, shows that specific capsular EPS are involved in host recognition by certain P335 phage subgroups. To our knowledge, this is the first description of the involvement of EPS produced via the Wzx/Wzy-dependent pathway in phage sensitivity of L. lactis or L. cremoris. This study also shows strains that do not appear to be phage-related based on plaque formation may still be related by phage adsorption and indicates that optimal formulation of phage-robust cultures should take into account the EPS type of individual strains.

Expanding natural transformation to improve beneficial lactic acid bacteria.

Nowadays, the growing human population exacerbates the need for sustainable resources. Inspiration and achievements in nutrient production or human/animal health might emanate from microorganisms and their adaptive strategies. Here, we exemplify the benefits of lactic acid bacteria (LAB) for numerous biotechnological applications and showcase their natural transformability as a fast and robust method to hereditarily influence their phenotype/traits in fundamental and applied research contexts. We described the biogenesis of the transformation machinery and we analyzed the genome of hundreds of LAB strains exploitable for human needs to predict their transformation capabilities. Finally, we provide a stepwise rational path to stimulate and optimize natural transformation with standard and synthetic biology techniques. A comprehensive understanding of the molecular mechanisms driving natural transformation will facilitate and accelerate the improvement of bacteria with properties that serve broad societal interests.

Functional Study of the Type II-A CRISPR-Cas System of Streptococcus agalactiae Hypervirulent Strains.

Nearly all strains of Streptococcus agalactiae, the leading cause of invasive infections in neonates, encode a type II-A clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. Interestingly, S. agalactiae strains belonging to the hypervirulent Sequence Type 17 (ST17) contain significantly fewer spacers in their CRISPR locus than other lineages, which could be the result of a less functional CRISPR-Cas system. Here, we revealed one large deletion in the ST17 cas promoter region and we evaluated its impact on the transcription of cas genes as well as the functionalities of the CRISPR-Cas system. We demonstrated that Cas9 interference is functional and that the CRISPR-Cas system of ST17 strains can still acquire new spacers, despite the absence of a regular cas promoter. We demonstrated that a promoter sequence upstream of srn036, a small RNA partially overlapping the antisense tracrRNA, is responsible for the ST17 CRISPR-Cas adaptation and interference activities.

Dairy lactococcal and streptococcal phage-host interactions: an industrial perspective in an evolving phage landscape.

Almost a century has elapsed since the discovery of bacteriophages (phages), and 85 years have passed since the emergence of evidence that phages can infect starter cultures, thereby impacting dairy fermentations. Soon afterward, research efforts were undertaken to investigate phage interactions regarding starter strains. Investigations into phage biology and morphology and phage-host relationships have been aimed at mitigating the negative impact phages have on the fermented dairy industry. From the viewpoint of a supplier of dairy starter cultures, this review examines the composition of an industrial phage collection, providing insight into the development of starter strains and cultures and the evolution of phages in the industry. Research advances in the diversity of phages and structural bases for phage-host recognition and an overview of the perpetual arms race between phage virulence and host defense are presented, with a perspective toward the development of improved phage-resistant starter culture systems.

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

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.

Author Correction: A mutation in the methionine aminopeptidase gene provides phage resistance in Streptococcus thermophilus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants.

The number and diversity of known CRISPR-Cas systems have substantially increased in recent years. Here, we provide an updated evolutionary classification of CRISPR-Cas systems and cas genes, with an emphasis on the major developments that have occurred since the publication of the latest classification, in 2015. The new classification includes 2 classes, 6 types and 33 subtypes, compared with 5 types and 16 subtypes in 2015. A key development is the ongoing discovery of multiple, novel class 2 CRISPR-Cas systems, which now include 3 types and 17 subtypes. A second major novelty is the discovery of numerous derived CRISPR-Cas variants, often associated with mobile genetic elements that lack the nucleases required for interference. Some of these variants are involved in RNA-guided transposition, whereas others are predicted to perform functions distinct from adaptive immunity that remain to be characterized experimentally. The third highlight is the discovery of numerous families of ancillary CRISPR-linked genes, often implicated in signal transduction. Together, these findings substantially clarify the functional diversity and evolutionary history of CRISPR-Cas.

A mutation in the methionine aminopeptidase gene provides phage resistance in Streptococcus thermophilus.

Streptococcus thermophilus is a lactic acid bacterium widely used by the dairy industry for the manufacture of yogurt and specialty cheeses. It is also a Gram-positive bacterial model to study phage-host interactions. CRISPR-Cas systems are one of the most prevalent phage resistance mechanisms in S. thermophilus. Little information is available about other host factors involved in phage replication in this food-grade streptococcal species. We used the model strain S. thermophilus SMQ-301 and its virulent phage DT1, harboring the anti-CRISPR protein AcrIIA6, to show that a host gene coding for a methionine aminopeptidase (metAP) is necessary for phage DT1 to complete its lytic cycle. A single mutation in metAP provides S. thermophilus SMQ-301 with strong resistance against phage DT1. The mutation impedes a late step of the lytic cycle since phage adsorption, DNA replication, and protein expression were not affected. When the mutated strain was complemented with the wild-type version of the gene, the phage sensitivity phenotype was restored. When this mutation was introduced into other S. thermophilus strains it provided resistance against cos-type (Sfi21dt1virus genus) phages but replication of pac-type (Sfi11virus genus) phages was not affected. The mutation in the gene coding for the MetAP induces amino acid change in a catalytic domain conserved across many bacterial species. Introducing the same mutation in Streptococcus mutans also provided a phage resistance phenotype, suggesting the wide-ranging importance of the host methionine aminopeptidase in phage replication.

Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins.

CRISPR-Cas systems are bacterial anti-viral systems, and bacterial viruses (bacteriophages, phages) can carry anti-CRISPR (Acr) proteins to evade that immunity. Acrs can also fine-tune the activity of CRISPR-based genome-editing tools. While Acrs are prevalent in phages capable of lying dormant in a CRISPR-carrying host, their orthologs have been observed only infrequently in virulent phages. Here we identify AcrIIA6, an Acr encoded in 33% of virulent Streptococcus thermophilus phage genomes. The X-ray structure of AcrIIA6 displays some features unique to this Acr family. We compare the activity of AcrIIA6 to those of other Acrs, including AcrIIA5 (also from S. thermophilus phages), and characterize their effectiveness against a range of CRISPR-Cas systems. Finally, we demonstrate that both Acr families from S. thermophilus phages inhibit Cas9-mediated genome editing of human cells.

CRISPR: A Useful Genetic Feature to Follow Vaginal Carriage of Group B Streptococcus.

Clustered regularly interspaced short palindromic repeats (CRISPR) and Cas (CRISPR-associated proteins) play a critical role in adaptive immunity against mobile genetic elements, especially phages, through their ability to acquire novel spacer sequences. Polarized spacer acquisition results in spacer polymorphism and temporal organization of CRISPR loci, making them attractive epidemiological markers. Group B Streptococcus (GBS), a genital commensal for 10 to 30% of healthy women and a major neonatal pathogen, possesses a ubiquitous and functional CRISPR1 locus. Our aim was to assess the CRISPR1 locus as an epidemiological marker to follow vaginal carriage of GBS in women. This study also allowed us to observe the evolution of the CRISPR1 locus in response to probable phage infection occurring in vivo. We followed carriage of GBS among 100 women over an 11-year period, with a median duration of approximately 2 years. The CRISPR1 locus was highly conserved over time. The isolates that show the same CRISPR1 genotype were collected from 83% of women. There was an agreement between CRISPR genotyping and other typing methods [MLVA (multilocus variable number of tandem repeat Analysis) and MLST (multilocus sequence typing)] for 94% of the cases. The CRISPR1 locus of the isolates from 18 women showed modifications, four of which acquired polarized spacer, highlighting the in vivo functionality of the system. The novel spacer of one isolate had sequence similarity with phage, suggesting that phage infection occurred during carriage. These findings improve our understanding of CRISPR-Cas evolution in GBS and provide a glimpse of host-phage dynamics in vivo.

An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9.

The CRISPR-Cas system owes its utility as a genome-editing tool to its origin as a prokaryotic immune system. The first demonstration of its activity against bacterial viruses (phages) is also the first record of phages evading that immunity 1 . This evasion can be due to point mutations 1 , large-scale deletions 2 , DNA modifications 3 , or phage-encoded proteins that interfere with the CRISPR-Cas system, known as anti-CRISPRs (Acrs) 4 . The latter are of biotechnological interest, as Acrs can serve as off switches for CRISPR-based genome editing 5 . Every Acr characterized to date originated from temperate phages, genomic islands, or prophages 4-8 , and shared properties with the first Acr discovered. Here, with a phage-oriented approach, we have identified an unrelated Acr in a virulent phage of Streptococcus thermophilus. In challenging a S. thermophilus strain CRISPR-immunized against a set of virulent phages, we found one that evaded the CRISPR-encoded immunity >40,000× more often than the others. Through systematic cloning of its genes, we identified an Acr solely responsible for the abolished immunity. We extended our findings by demonstrating activity in another S. thermophilus strain, against unrelated phages, and in another bacterial genus immunized using the heterologous SpCas9 system favoured for genome editing. This Acr completely abolishes SpCas9-mediated immunity in our assays.

Natural DNA Transformation Is Functional in Lactococcus lactis subsp. cremoris KW2.

Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis subsp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a small amount of ComX, the deletion of mecA, clpC, or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species.IMPORTANCE Lactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis subsp. cremoris KW2 by the overproduction of the master competence regulator ComX. The developed procedure enables a flexible approach to modify the chromosome with single point mutation, sequence insertion, or sequence replacement. These results represent an important step for the genetic engineering of L. lactis that will facilitate the design of strains optimized for industrial applications. This will also help to discover natural regulatory mechanisms controlling competence in the genus Lactococcus.

The CRISPR-Cas app goes viral.

If biology laboratories were smartphones, CRISPR-Cas would be the leading app. Nowadays, technology users rely on apps to communicate, get directions, entertain, and more. Likewise, many life scientists now rely on CRISPR-Cas systems to study the interactions between microbes and their viruses, to track strains as well as to modify and modulate genomes. Considering their high level of polymorphism, CRISPR arrays can increase the resolution of a microbial typing scheme. As dynamic systems, they allow the identification and the tracking of specific sequences, which is highly valuable for epidemiological studies. As a defense mechanism, they offer an opportunity to generate virus-resistant strains or even to construct strains refractory to the acquisition of specific genes. And last but not least, as customizable and transferable tools, CRISPR-Cas systems are particularly promising to fight multi-drug resistant bacteria through the engineering of phages.

A decade of discovery: CRISPR functions and applications.

This year marks the tenth anniversary of the identification of the biological function of CRISPR-Cas as adaptive immune systems in bacteria. In just a decade, the characterization of CRISPR-Cas systems has established a novel means of adaptive immunity in bacteria and archaea and deepened our understanding of the interplay between prokaryotes and their environment, and CRISPR-based molecular machines have been repurposed to enable a genome editing revolution. Here, we look back on the historical milestones that have paved the way for the discovery of CRISPR and its function, and discuss the related technological applications that have emerged, with a focus on microbiology. Lastly, we provide a perspective on the impacts the field has had on science and beyond.

Lactobacillus herbarum sp. nov., a species related to Lactobacillus plantarum.

Strain TCF032-E4 was isolated from a traditional Chinese fermented radish. It shares >99% 16S rRNA sequence identity with L. plantarum, L. pentosus and L. paraplantarum. This strain can ferment ribose, galactose, glucose, fructose, mannose, mannitol, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, lactose, melibiose, trehalose and gentiobiose. It cannot ferment sucrose, which can be used by L. pentosus, L. paraplantarum, L. fabifermentans, L. xiangfangensis and L. mudanjiangensis, as well as most of the L. plantarum strains (88.7%). TCF032-E4 cannot grow at temperature above 32 °C. This strain shares 78.2-83.6% pheS (phenylalanyl-tRNA synthetase alpha subunit) and 89.5-94.9% rpoA (RNA polymerase alpha subunit) sequence identity with L. plantarum, L. pentosus, L. paraplantarum, L. fabifermentans, L. xiangfangensis and L. mudanjiangensis. These results indicate that TCF032-E4 represents a distinct species. This hypothesis was further confirmed by whole-genome sequencing and comparison with available genomes of related species. The draft genome size of TCF032-E4 is approximately 2.9 Mb, with a DNA G+C content of 43.5 mol%. The average nucleotide identity (ANI) between TCF032-E4 and related species ranges from 79.0 to 81.1%, the highest ANI value being observed with L. plantarum subsp. plantarum ATCC 14917T. A novel species, Lactobacillus herbarum sp. nov., is proposed with TCF032-E4T ( = CCTCC AB2015090T = DSM 100358T) as the type strain.

An updated evolutionary classification of CRISPR-Cas systems.

The evolution of CRISPR-cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR-cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR-Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized.

Draft Genome Sequence of Lactobacillus sp. Strain TCF032-E4, Isolated from Fermented Radish.

Here, we report the draft genome sequence of Lactobacillus sp. strain TCF032-E4 (= CCTCC AB2015090 = DSM 100358), isolated from a Chinese fermented radish. The total length of the 57 contigs is about 2.9 Mb, with a G+C content of 43.5 mol% and 2,797 predicted coding sequences (CDSs).

Analysis of the type II-A CRISPR-Cas system of Streptococcus agalactiae reveals distinctive features according to genetic lineages.

CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) are found in 90% of archaea and about 40% of bacteria. In this original system, CRISPR arrays comprise short, almost unique sequences called spacers that are interspersed with conserved palindromic repeats. These systems play a role in adaptive immunity and participate to fight non-self DNA such as integrative and conjugative elements, plasmids, and phages. In Streptococcus agalactiae, a bacterium implicated in colonization and infections in humans since the 1960s, two CRISPR-Cas systems have been described. A type II-A system, characterized by proteins Cas9, Cas1, Cas2, and Csn2, is ubiquitous, and a type I-C system, with the Cas8c signature protein, is present in about 20% of the isolates. Unlike type I-C, which appears to be non-functional, type II-A appears fully functional. Here we studied type II-A CRISPR-cas loci from 126 human isolates of S. agalactiae belonging to different clonal complexes that represent the diversity of the species and that have been implicated in colonization or infection. The CRISPR-cas locus was analyzed both at spacer and repeat levels. Major distinctive features were identified according to the phylogenetic lineages previously defined by multilocus sequence typing, especially for the sequence type (ST) 17, which is considered hypervirulent. Among other idiosyncrasies, ST-17 shows a significantly lower number of spacers in comparison with other lineages. This characteristic could reflect the peculiar virulence or colonization specificities of this lineage.

Rough and smooth morphotypes isolated from Lactobacillus farciminis CNCM I-3699 are two closely-related variants.

This study focused on a pleomorphic strain Lactobacillus farciminis CNCM I-3699 known as probiotic for animal applications. On plating, this strain was characterized by the presence of rough and smooth morphotypes depending on experimental conditions. Dominant smooth (S) form, bright white, having smooth edges with moist, ropy, and creamy along with rough (R) form, pale white, having irregular edges and a dry and granular aspect were always obtained from the parent strain under aerobic culture conditions. In anaerobic conditions, only S form growth was observed. Biochemical dosage of capsular exopolysaccharides showed a significant difference between S and R forms (p<0.01), in agreement with a ropy or non ropy phenotype for the S or R form, respectively. These differences were confirmed by transmission electronic microscopy. The auto-aggregation profile revealed major differences in cultural behaviors. The R morphotype presented a highly auto-aggregative ability contrary to the S morphotype. However, biochemical and molecular analyses revealed that R and S morphotypes: 1) shared the same sugar fermentation pattern; 2) belonged to L. farciminis species using 16S rDNA sequencing; 3) had identical PFGE patterns using NotI and ApaI endonucleases; and 4) had identical CRISPR loci but different from those of other L. farciminis strains. Furthermore, the novelty and uniqueness of CRISPR spacer sequences in CNCM I-3699 provides a genetic support for the development of a molecular tracking tool for CNCM I-3699 and its variants. In conclusion, L. farciminis CNCM I-3699 is a pleomorphic strain giving reproducibly rise to two phenotypically distinct morphotypes R and S. This phenomenon may explain survival and growth abilities in in vitro fluctuating aerobic-anaerobic conditions along with modulation of exopolysaccharide synthesis and autoaggregation profile.