Life Sciences 231 (2019) 116531 Contents lists available at ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie Analysis of CRISPR/Cas system of Proteus and the factors affected the T functional mechanism ⁎ Daofeng Qua, Shiyao Lua, Peng Wanga, Mengxue Jianga, Songqiang Yib, Jianzhong Hana, a School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang 310018, China b Jiangxi Animal Husbandry Technology Extension Station, Nanchang 330046, China ARTICLE INFO ABSTRACT Keywords: Background: The Proteus is one of the most common human and animal pathogens. Clustered regularly inter- Proteus spaced short palindromic repeats and CRISPR-associated proteins (CRISPR/Cas) are inheritable genetic elements CRISPR found in a variety of archaea and bacteria in the evolution, providing immune function against foreign invasion. Spacer Objectives: To analyze the characteristics and functions of the CRISPR/Cas system in Proteus genomes, as well as Cas the internal and external factors affecting the system. Mobile genetic element Methods: CRISPR loci were identified and divided into groups based on the repeat sequence in96 Proteus strains by identification. Compared the RNA secondary structure and minimum free energy of CRISPR loci through bioinformatics, the evolution of cas genes, and the effects of related elements were also discussed. Results: 85 CRISPR loci were identified and divided into six groups based on the sequence of repeats, andthe more stable the secondary structure of RNA, the smaller the minimum free energy, the fewer base mutations in the repeat, the more stable the CRISPR and the more complete the evolution of the system. In addition, Cas1 gene can be a symbol to distinguish species to some extent. Of all the influencing factors, CRISPR/Cas had the greatest impact on plasmids. Conclusions: This study examined the diversity of CRISPR/Cas system in Proteus and found statistically sig- nificant positive/negative correlations between variety factors (the RNA stability, free energy, etc.)andthe CRISPR locus, which played a vital role in regulating the CRISPR/Cas system. 1. Introduction intestinal infections [5]. The CRISPR (Clustered Regularly Interspaced Short Palindromic Gram-negative Enterobacteriaceae bacteria widely distributes in Repeats)/Cas system which comprises genomic (CRISPR) and pro- nature and has a wide range of hosts. There are parasitic or symbiotic, teomic (Cas) components are found in 75% bacteria and archaea, and epiphytic and saprophytic phenomena in humans, animals, and plants mediate an adaptive immune response against invading viruses and they can easily be found in soil or water [27]. Some strains, like [4,7,20]. The genomic component is a DNA loci containing short Escherichia coli and Proteus are important sources for the study of ge- fragments of targeted nucleic acid sequences (spacers) interspaced by netics and molecular biology. short repeated sequences (repeats) [17]. The spacer sequences can be Proteus includes five species, which are Proteus vulgaris, Proteus either foreign or self-origin [30]. The length of the repeat sequences mirabilis, Proteus viscous, Proteus pneumoniae, and Proteus hausmannii varies between 25 and 40 nt, whereas the length of the spacer se- [8]. Common proteobacteria and Proteus mirabilis are closely related to quences varies between 21 and 71 nt [34]. As mentioned above, some the clinic. Proteus food poisoning is one of the common food poisoning spacers show high homology with foreign nucleic acids, but the origin in China, the proteobacteria which caused food poisoning are mainly of a significant percentage of spacers remains unknown [2]. caused by P. vulgaris and P. mirabilis [6]. Escherichia coli, the re- The objective of this study was to gain the further insights into the presentative strain of the genus Escherichia, is the most important and character of CRISPRs in Proteus species by analyzing a collection of 96 abundant type of bacteria in the intestine of humans and animals [15]. unique strains. In this research, we characterized the CRISPR content It is generally not pathogenic, and is a resident bacterium in human and and the presence of the mobile elements, regulators, etc., to explore the animal intestines, under certain conditions, exactly, it can lead to putative link between them. ⁎ Corresponding author. E-mail address: [email protected] (J. Han). https://doi.org/10.1016/j.lfs.2019.06.006 Received 24 March 2019; Received in revised form 28 May 2019; Accepted 3 June 2019 Available online 05 June 2019 0024-3205/ © 2019 Elsevier Inc. All rights reserved. D. Qu, et al. Life Sciences 231 (2019) 116531 2. Materials and methods integrated, the statistical correlation between the data and CRISPR was analyzed by using Principle Component Analysis (PCA). 2.1. Strains collection 3. Results We chose 96 Proteus strains and 50 Escherichia coli strains (for contrast) from the National Center for Biotechnology Information 3.1. Geographical comparison of CRISPR alleles (https://www.ncbi.nlm.nih.gov/genome/), and downloaded complete genomes and bioinformation of these strains with default parameters We selected all Proteus and Escherichia coli complete genomes (Table S1). CRISPR loci and cas genes were searched in CRISPRs da- available from the NCBI database, totaling 146 strains (Table S1). tabase (http://crispr.i2bc.paris-saclay.fr/crispr/)[28] and CRISPR According to CRISPR database and Guo et al. [14], confirmed CRISPR Finder [13], then we obtained the flanking sequence, repeat and spacer sites should contain at least two different spacers. The number of nucleotide sequence of these strains. CRISPR loci varied from 1 to 5 depending on the strains. Most strains have CRISPR loci and cas genes. In Proteus, only 43 strains contain 2.2. Identification and analysis of CRISPR CRISPR/Cas system. Statistical analysis results showed that, in Proteus, the number of direct repeats was between 3 and 17, and the number of The classification of confirmed CRISPR loci were divided intosix spacers ranged from 2 to 16; and in Escherichia coli, the number of groups [21] based on six different repeats, named CRISPR1~6. The spacers ranged from 2 to 21, and the number of repeats was between 3 typical repeats and terminal repeats of CRISPR were analyzed through and 22 (typically 6, 7;3, 4, Table S2). multiple sequence alignment using ClustalX [31], and these six con- firmed CRISPR loci were visualized with Weblogo (http://weblogo. 3.2. The profiles of Proteus CRISPRs berkeley.edu/logo.cgi). These repeat sequences were regarded as the specific gene signature for CRISPR. Secondary structure prediction of The CRISPR loci were assigned into six groups based on the repeat the most frequent sequence of each repeat was performed by RNAfold sequence similarity, since the direct repeat length of CRISPR loci was (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi)[9], similar within each locus by multiple sequence alignment analysis. It and the Minimum Free Energy of the RNA was obtained with current was indicated that CRISPR1, CRISPR 5 and CRISPR6 were the most limits of 7500 nt for section function calculations and 10,000 nt for common confirmed loci in all strains; the number of each repeat was MFE-only predictions. 180, 147 and 60. These groups were taken into account in the current classification of CRISPR/Cas system (Table 1). 2.3. Analysis of spacers In order to better understand the features of these CRISPR groups, we used Weblogo to analyze the differences between repeats including We collected the number and nucleotide base pairs of spacers in all terminal repeats, repeat variants and typical repeats in the same strains and made a statistical correlation between spacers and the re- CRISPR group, so that we can see individual nucleotide base mutations peats, and IS finder, INTEGRALL, CRISPRTarget were used to analyze in six different CRISPR groups (Fig. 1). It was described the results of spacers (Biswas, Gagnon, 2013, [12]). To identify the spacer sequences CRISPR, which showed that CRISPR5 and CRISPR6 had less mutation matching sequences from mobile elements, the spacers were subjected and high frequency. From the analysis of the diversity of base muta- to the standard BLASTN search (e-value threshold, 1.10−5) in Genbank, tions, the implications of these findings confirmed which CRISPR's genetic mobile elements were defined by identifying homologous se- structure was stable in these six CRISPR groups, and the fewer base quences with an e-value < 1.10−5 and < 10% difference in sequence mutations in the CRISPR repeat sequence, the more stable the CRISPR length [32]. and the more complete the evolution of the system. Previous researches have indicated that CRISPR repeats may form 2.4. Phylogenetic tree of Cas1 gene in Proteus and Escherichia coli stable hairpin-like secondary structures (classical stem-loop) due to the partially palindromic nature [3], which contains a large and a small We used Cygwin64 terminal to obtain the nucleotide sequence of cas loop at both ends of each repeats of CRISPR [22]. The RNA secondary gene from 10,000 bp upstream to 10,000 bp downstream in the CRISPR structure and minimum free energy (MFE) was detected for typical loci. The MEGA7.0 program was used to estimate nucleotide diversity direct repeat sequences of each group through the RNAfold Web Server and evolutionary distances as well as to build phylogenetic trees by the (Fig. 1). From the short review above, key findings emerged that in neighbor-joining method using the Jukes-Cantor distances. The cas these 6 CRISPR groups, we showed that, their RNA secondary structures gene has 45 families; Proteus cas gene belongs to I-E and I-F types. The almost have two rings at each end and a stem in the middle, except cas gene of the publicly available Proteus genome was chosen to con- CRISPR4 which only had a circle. The stem length in other Group was struct the Proteus cas gene tree in order to identify the Proteus cas gene, from 3 to 7, which appeared highly conservative.