Survey of clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) systems in multiple sequenced strains of Klebsiella pneumoniae

Abstract

BackgroundIn recent years the emergence of multidrug resistant Klebsiella pneumoniae strains has been an increasingly common event. This opportunistic species is one of the five main bacterial pathogens that cause hospital infections worldwide and multidrug resistance has been associated with the presence of high molecular weight plasmids. Plasmids are generally acquired through horizontal transfer and therefore is possible that systems that prevent the entry of foreign genetic material are inactive or absent. One of these systems is CRISPR/Cas. However, little is known regarding the clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) system in K. pneumoniae. The adaptive immune system CRISPR/Cas has been shown to limit the entry of foreign genetic elements into bacterial organisms and in some bacteria it has been shown to be involved in regulation of virulence genes. Thus in this work we used bioinformatics tools to determine the presence or absence of CRISPR/Cas systems in available K. pneumoniae genomes.ResultsThe complete CRISPR/Cas system was identified in two out of the eight complete K. pneumoniae genomes sequences and in four out of the 44 available draft genomes sequences. The cas genes in these strains comprises eight cas genes similar to those found in Escherichiacoli, suggesting they belong to the type I-E group, although their arrangement is slightly different. As for the CRISPR sequences, the average lengths of the direct repeats and spacers were 29 and 33 bp, respectively. BLAST searches demonstrated that 38 of the 116 spacer sequences (33%) are significantly similar to either plasmid, phage or genome sequences, while the remaining 78 sequences (67%) showed no significant similarity to other sequences. The region where the CRISPR/Cas systems were located is the same in all the Klebsiella genomes containing it, it has a syntenic architecture, and is located among genes encoding for proteins likely involved in metabolism and resistance to antibiotics.ConclusionsThe CRISPR/Cas system is not widely distributed in K. pneumoniae genomes, those present most likely belong to type I-E with few differences from the arrangement of the cse3 gene and most of the spacers have not been are not described yet. Given that the CRISPR/Cas system is scarcely distributed among K. pneumoniae genomes it is not clear whether it is involved in either immunity against foreign genetic material or virulence. We consider that this study represents a first step to understand the role of CRISPR/Cas in K. pneumoniae.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1285-7) contains supplementary material, which is available to authorized users.

Survey on the CRISPR arrays in Lactobacillus helveticus genomes

“…Moreover, the proportion of L. helveticus spacer sequences that did not show similarity to other sequences contained in GenBank database was about 61%, as observed by Ostria‐Hernandez et al . () for K. pneumoniae . This result could be a consequence of the vast amount of uncharacterized genetic elements in nature as well as a lack of genome and phage sequences belonging to the L. helveticus species (Zago et al .…”

“…Also, Ostria‐Hernandez et al . () found that 13% of spacer sequences retrieved in CRIPSR loci from Klebsiella pneumoniae was similar to sequences of phages, whereas only 8 and 5% from plasmids and Klebsiella genome sequences respectively. Similarly, some of the spacer sequences in L. helveticus were like more than one region of the same phage or genome sequence (data not shown).…”

“…Specifically, three different type I subtypes were retrieved: subtype I-E, identified in twelve genomes, subtype I-C (four genomes), and subtype I-B (six genomes). Type I is a system type detected frequently in pathogenic bacteria and in many enterobacteria (Makarova et al 2011), whereas type II system is known 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 515 3 BT AA15A 1 BM A BH BI BJ BK BN BO BP BF BG BQ AA15A 2 BC BD BE ATCC10386 A to occur preferentially in LAB, especially within lactobacilli (Makarova et al 2015;Sanozky-Dawes et al 2015;Crawley et al 2018). Type I system is characterized by the cas3 signature protein, which encodes a single-stranded DNA (ssDNA)-stimulated superfamily 2 helicase, in addition to the presence of the conserved Cas1 and Cas2 proteins.…”

“…An important step in understanding the role of CRISPR was the identification of the conserved genes located very close to them, named cas. The number of cas genes may vary from 4 to over 20 and the product of these genes, the Cas proteins, provide the enzymatic machinery required for the acquisition of spacers as well as for invader element marking (Ostria-Hernandez et al 2015). CRISPR systems might play a critical role in the adaptation of the host to its surroundings and explain the persistence of particular bacterial strains in ecosystems where phages are present.…”

Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones ofKlebsiella pneumoniae

“…CRISPR arrays were identified from genome assemblies using the CRISPR Recognition Tool v1.2 96 and genomes with >3 putative arrays were investigated manually to check for spurious identifications and/or identifications of single arrays split over multiple assembly contigs. Nucleotide sequences for the previously described Kp cas genes 97 were extracted from the NTUH--K2044 reference chromosome (accession: NC_012731.1). Genomes were screened for novel cas genes by HMM domain search using the domain profiles developed by Burstein and colleagues 98 (HMMER v3.1b2, bit score ≥200 99 ).…”