title: Comparative genomic analysis identifies structural features of CRISPR-Cas systems in Riemerella anatipestifer.
abstract: Riemerella anatipestifer infection is a contagious disease that has resulted in major economic losses in the duck industry worldwide. This study attempted to characterize CRISPR-Cas systems in the disease-causing agent, Riemerella anatipestifer (R. anatipestifer). The CRISPR-Cas system provides adaptive immunity against foreign genetic elements in prokaryotes and CRISPR-cas loci extensively exist in the genomes of archaea and bacteria. However, the structure characteristics of R. anatipestifer CRISPR-Cas systems remains to be elucidated due to the limited availability of genomic data.
  
  
  
  To identify the structure and components associated with CRISPR-Cas systems in R. anatipestifer, we performed comparative genomic analysis of CRISPR-Cas systems in 25 R. anatipestifer strains using high-throughput sequencing. The results showed that most of the R. anatipestifer strains (20/25) that were analyzed have two CRISPR loci (CRISPR1 and CRISPR2). CRISPR1 was shown to be flanked on one side by cas genes, while CRISPR2 was designated as an orphan. The other analyzed strains harbored only one locus, either CRISPR1 or CRISPR2. The length and content of consensus direct repeat sequences, as well as the length of spacer sequences associated with the two loci, differed from each other. Only three cas genes (cas1, cas2 and cas9) were located upstream of CRISPR1. CRISPR1 was also shown to be flanked by a 107 bp-long putative leader sequence and a 16 nt-long anti-repeat sequence. Combined with analysis of spacer organization similarity and phylogenetic tree of the R. anatipestifer strains, CRISPR arrays can be divided into different subgroups. The diversity of spacer organization was observed in the same subgroup. In general, spacer organization in CRISPR1 was more divergent than that in CRISPR2. Additionally, only 8 % of spacers (13/153) were homologous with phage or plasmid sequences. The cas operon flanking CRISPR1 was observed to be relatively conserved based on multiple sequence alignments of Cas amino acid sequences. The phylogenetic analysis associated with Cas9 showed Cas9 sequence from R. anatipestifer was closely related to that of Bacteroides fragilis and formed part of the subtype II-C subcluster.
  
  
  
  Our data revealed for the first time the structural features of R. anatipestifer CRISPR-Cas systems. The illumination of structural features of CRISPR-Cas system may assist in studying the specific mechanism associated with CRISPR-mediated adaptive immunity and other biological functions in R. anatipestifer.
authors: Research Center of Avian Diseases, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China.
authors: De-Kang
authors: DK
authors: Zhu
authors: Research Center of Avian Diseases, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China.
authors: Xue-Qin
authors: XQ
authors: Yang
authors: Research Center of Avian Diseases, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China.
authors: Yang
authors: Y
authors: He
authors: Research Center of Avian Diseases, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China.
authors: Wang-Shu
authors: WS
authors: Zhou
authors: Research Center of Avian Diseases, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China.
authors: Xiao-Heng
authors: XH
authors: Song
authors: Research Center of Avian Diseases, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China.
authors: Jiang-Bo
authors: JB
authors: Wang
authors: Research Center of Avian Diseases, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China.
authors: Yu
authors: Y
authors: Zhang
authors: Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China.|Institute of Preventive Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, China.
authors: Ma-Feng
authors: MF
authors: Liu
authors: Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China.|Institute of Preventive Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, China.
authors: Ming-Shu
authors: MS
authors: Wang
authors: Research Center of Avian Diseases, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China.
authors: Ren-Yong
authors: RY
authors: Jia
authors: Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China.|Institute of Preventive Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, China.
authors: Shun
authors: S
authors: Chen
authors: Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China.|Institute of Preventive Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, China.
authors: Kun-Feng
authors: KF
authors: Sun
authors: Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China.|Institute of Preventive Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, China.
authors: Qiao
authors: Q
authors: Yang
authors: Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China.|Institute of Preventive Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, China.
authors: Ying
authors: Y
authors: Wu
authors: Research Center of Avian Diseases, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China.
authors: Xiao-Yue
authors: XY
authors: Chen
authors: Research Center of Avian Diseases, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, China. chenganchun@vip.163.com.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, Sichuan, China. chenganchun@vip.163.com.
authors: An-Chun
authors: AC
authors: Cheng
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D055028:Comparative Genomic Hybridization; D014644:Genetic Variation; D023281:Genomics; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D060494:Riemerella / Q000235:genetics* / Q000472:pathogenicity
keywords: CRISPR-Cas system; Riemerella anatipestifer; cas gene; phylogenetic analysis; repeat sequence; spacer sequence