- buchneri in order to make better use of the species in a wide range of applicable industrial settings.. - All isolates harbor at least one CRISPR-Cas system, and many contain putative prophage regions, some of which are targeted by the host ’ s own DNA-encoded spacer sequences.. - Keywords: Lactobacillus buchneri , Comparative genomics, Lactic acid bacteria, CRISPR-Cas systems, Fermentation, Spoilage, Food microbiology. - buchneri CD034 revealed the presence of enzymes re- quired to convert lactic acid to acetic acid and CO 2 in the presence of oxygen, or 1,2-propanediol anaerobically, a unique metabolic feature protecting against acidification of the cytoplasm in the presence of large amounts of lactate [5]. - buchneri useful in the aerobic stabilization of silage, effectively. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Full list of author information is available at the end of the article. - It has been previously reported that lactic acid bacteria are highly adapted to specific ecological niches, and have small genomes compared to other bacteria as a conse- quence of a process called genome reduction, resulting in the maintenance of a minimal number of essential genes required for niche-specific survival [18]. - buchneri, we sequenced and assembled draft ge- nomes of eight phenotypically distinct strains previously identified by Daughtry et al. - To generate an overview of the strains’ genomic similarity, we aligned the newly-assembled draft genomes with the two publicly-available closed reference genomes, NRRL B-30929 and CD034. - CRISPR-Cas systems copy a short segment of DNA from the invading nucleic acid sequence and integrate it into the CRISPR locus as a template to prevent future attacks, called a spacer. - Despite the wide range of phenotypes observed across these strains, we found that they share significant iden- tity in terms of protein coding potential, as well as a high degree of similarity across their CRISPR-Cas systems, re- vealing identical repeat sequences and unique genotypic signatures constructed through the presence of shared ancestral spacers.. - Whole-genome assembly was performed on each of the eight strains, revealing draft genome sizes between 2.49 Mb and 2.76 Mb (Table 1). - Nethery et al. - 36.5 kb) appears to be unique to NRRL B-30929, whereas most of the coding sequences in the putative prophage II region. - Of the 4060 total coding sequences, 1904 were shared by all strains, comprising the core-genome. - The non-core genes, termed accessory-genome, is composed of 2156 total coding sequences, likely contributing to the major phenotypic differences between strains as de- scribed by Daughtry et al. - Interest- ingly, however, the third largest orthologous group, which encodes ~ 9% of the total core-genome, contains proteins of unknown function. - Functional core-genome groups containing the least number of coding sequences belong to the ‘cell motility’, ‘mobilome’, and ‘secondary metabolite biosynthesis’ groups. - prophages, transposons’ group showed the lowest pro- portion between the number of core-genes vs the num- ber of pan-genes, with only 5 sequences in the core- genome versus 137 in the pan-genome, illustrating ex- ceptional diversity even across these highly related strains. - To bolster our understanding of the environmental interaction between these strains and invasive nucleic acids, we analyzed their CRISPR-Cas systems in detail.. - Location and identification of CRISPR-Cas systems were not hindered by the highly fragmented genome assem- blies, and loci were successfully assigned a canonical type and subtype using standard tools and references (37, 38). - Across the 10 strains analyzed, we found Table 1 Whole-genome assembly statistics for each of the eight sequenced Lactobacillus buchneri isolates. - CRISPR-Cas systems belonging to both II-A and I-E ca- nonical subtypes [37]. - Two of the three repeats match the consensus repeat of CRISPR 1. - While CD034 does have a type II-A locus, none of the identified spacers share significant identity with any type II-A spacer sequences from the other isolates.. - A total of 16 protospacers were identified in the human gut metagenome, Lactobacillus plasmids, and various food metagenome samples, as well as within the ge- nomes of Lactobacillus parabuchneri FAM21731, LA1184, NRRL B-30929, and CD034 (Fig. - In all CRISPR-Cas systems except for type III, a conserved protospacer-adjacent motif (PAM) sequence is required for successful acquisition of new spacers and for inter- ference [40–43]. - 90% identity to corresponding spacers across 5 isolates were used in the analysis, yielding a pre- dicted PAM of 5. - AAAA – 3′, two nucleotides down- stream of the protospacer (Fig. - The three identified proto- spacers in the genome of Lactobacillus parabuchneri FAM21731 are clustered within a ~ 23 kb putative pro- phage region (Fig. - Curi- ously, the two spacers found to match sequences in the LA1184 genome are self-targeting: they are encoded by LA1184’s own CRISPR 1 locus. - Of the two protospacers found in the genome of NRRL B-30929, one is self- targeting: encoded by NRRL B-30929 spacer 9, and one is encoded by LA1184 spacer 15. - The protospacers found in the genome of CD034 are matched by LA1181 spacer 25, which targets a phage tape measure protein, and LA1167 spacer 9 which targets a hypothetical protein ~ 20 kb upstream.. - LA1184 spacer 6 shows 100% identity with the matching protospacer sequence, but a single nucleotide polymorphism (SNP) exists in the PAM: AGAA. - The protospacer matching LA1184 spacer 13 has the proper PAM (AAAA) but contains three consecutive SNPs on the 3′ end of the protospacer sequence in what is called the seed sequence [41]. - Regarding NRRL B-30929 spacer 9, there are two SNPs in the middle of the protospacer sequence, as well as a single SNP in the PAM: ATAA.. - buchneri, as well as its preva- lence in the food industry, there is a relatively low number of publicly available genome sequences. - buchneri, a significant addition to the number of genomes available in the NCBI Genbank [46].. - 2.76 Mb, are typical of the 1.8 to 3.3 Mb range reportedly found in lactic acid bacteria [18]. - a Repeats are highly conserved across all isolates within CRISPR-Cas types II-A and I-E. - b Some degree of shared evolutionary history is represented by the conservation of at least the first two ancestral spacers (on the right) in the type II-A spacer alignment. - b The predicted PAM sequence for this type II-A CRISPR-Cas system, 5. - c Protospacers of bacterial origin cluster together in putative prophage regions of the genome. - With only half of identified coding sequences con- served across all ten strains, there is a considerable level of genomic diversity represented in the accessory- genome. - When each coding sequence is assigned to a COG, we see a large number of pan-genome sequences assigned to the ‘mobilome: prophages, transposons’ cat- egory versus the number in the core-genome, with more than half identified as transposons. - Interestingly, we see that ~ 9% of the core- genome is comprised of sequences with a currently unknown function, providing candidates for future func- tional studies. - In all strains analyzed, a type II-A CRISPR-Cas system was found. - The small number of unique spacers and conserved repeat sequences in CRISPR 2, along with the differences in amino acid iden- tity between the cas sequences of the two loci seem to indi- cate this locus remained functional post-duplication and has begun diverging from CRISPR 1 over evolutionary time.. - Assuming a fully functional CRISPR-Cas system, the expression of self-targeting spacers should result in host eradication, however, a variety of escape mechanisms have been observed [48, 49]. - Two such escape mechanisms in- clude mutating of the protospacer sequence or mutation of the PAM, which can lower binding efficiency of the. - CRISPR-Cas machinery to the target, reducing or inhibiting nucleic acid cleavage [41]. - In the self-targeting spacers de- scribed here, we saw SNPs in the PAM sequence, SNPs in the protospacer, or both. - SNPs in the PAM could prevent the initial binding of the Cas9 effector complex, while mis- matches between the protospacer and RNA guide could prevent the conformational change required by the CRISPR-Cas effector complex for target cleavage [50]. - Al- though individual phages may escape CRISPR-Cas targeting through random mutation, ultimately, they cannot avoid the random acquisition of spacers accrued by all cells of a bacterial population [51, 52]. - An alternative phage defense mechanism against CRISPR-Cas targeting has been ob- served: the expression of CRISPR-Cas inhibitory proteins:. - These small phage-encoded proteins have been implicated in many cases in which self-targeting spacers have been observed, effectively inhibiting death of the host cell by prohibiting DNA cleavage by the CRISPR- Cas effector complex [53–55]. - Anti-CRISPR proteins could play a role in the interaction between the host prophage re- gions and CRISPR-Cas machinery and is an attractive area for further study.. - buchneri’s CRISPR-Cas systems could be exploited in the food biotechnology sector to treat and reduce contamination and spoilage by this organism, po- tentially preventing industrial loss and promoting robust bioprocessing. - The CRISPR-Cas systems described here should undergo functional testing to explore their utility in engineering endogenous phage resistance into strains to protect starter cultures and promote the ensiling process. - Whole-genome sequencing of 7 pheno- typically diverse strains found in spoiled, fermented cucumber in concert with the ATCC type strain, ATCC 4005, were deposited in the NCBI Genbank, significantly increasing the number of publicly available L. - A single type II-A CRISPR-Cas system was found in each of these strains, with the exception of LA1167, which was found to contain a second type II-A. - CRIPSR-Cas locus just downstream of the primary locus.. - Resulting samples were sent to the High- Throughput Sequencing and Genotyping Unit of the Roy J.. - R script distributed in the Roary package at https://github.com/sanger-pathogens/Roary/blob/master/. - The phylogenetic tree and gene presence/absence coverage were produced using the roary_plots.py script, also available in the public Roary dis- tribution https://github.com/sanger-pathogens/Roary/tree/. - CRISPR-Cas loci were identified, visualized, and aligned using CRISPRviz [38], and type was determined using the canonical definitions defined by Koonin et al. - The PAM sequence was bioinformatically pre- dicted using the procedure and CRISPRutils software package previously described by Nethery et al. - The subsequent comparative genomic analyses and writing of the manuscript was conducted by MAN, with input from all authors. - 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Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, et al.. - Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, et al.. - An updated evolutionary classification of CRISPR-Cas systems. - Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al.. - Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner B, et al. - Westra ER, van Erp PB, Kunne T, Wong SP, Staals RH, Seegers CL, et al.. - Predicting and visualizing features of CRISPR-Cas systems. - Outcomes and characterization of chromosomal self-targeting by native CRISPR-Cas systems in Streptococcus thermophilus. - Unravelling the structural and mechanistic basis of CRISPR-Cas systems. - van Houte S, Ekroth AK, Broniewski JM, Chabas H, Ashby B, Bondy-Denomy J, et al. - Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. - Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, et al. - Pawluk A, Staals RH, Taylor C, Watson BN, Saha S, Fineran PC, et al.. - 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