A bioinformatic approach to identify confirmed and probable CRISPR-Cas systems in the Acinetobacter calcoaceticus- Acinetobacter baumannii complex genomes

Abstract

Introduction: The Acinetobacter calcoaceticus-Acinetobacter baumannii complex, or Acb complex, consists of six species: Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacter seifertii, and Acinetobacter lactucae. A. baumannii is the most clinically significant of these species and is frequently related to healthcare-associated infections (HCAIs). Clustered regularly interspaced short palindromic repeat (CRISPR) arrays and associated genes (cas) constitute bacterial adaptive immune systems and function as variable genetic elements. This study aimed to conduct a genomic analysis of Acb complex genomes available in databases to describe and characterize CRISPR systems and cas genes.

Methods: Acb complex genomes available in the NCBI and BV-BRC databases, the identification and characterization of CRISPR-Cas systems were performed using CRISPRCasFinder, CRISPRminer, and CRISPRDetect. Sequence types (STs) were determined using the Oxford scheme and ribosomal multilocus sequence typing (rMLST). Prophages were identified using PHASTER and Prophage Hunter.

Results: A total of 293 genomes representing six Acb species exhibited CRISPR-related sequences. These genomes originate from various sources, including clinical specimens, animals, medical devices, and environmental samples. Sequence typing identified 145 ribosomal multilocus sequence types (rSTs). CRISPR-Cas systems were confirmed in 26.3% of the genomes, classified as subtypes I-Fa, I-Fb and I-Fv. Probable CRISPR arrays and cas genes associated with CRISPR-Cas subtypes III-A, I-B, and III-B were also detected. Some of the CRISPR-Cas systems are associated with genomic regions related to Cap4 proteins, and toxin-antitoxin systems. Moreover, prophage sequences were prevalent in 68.9% of the genomes. Analysis revealed a connection between these prophages and CRISPR-Cas systems, indicating an ongoing arms race between the bacteria and their bacteriophages. Furthermore, proteins associated with anti-CRISPR systems, such as AcrF11 and AcrF7, were identified in the A. baumannii and A. pittii genomes.

Discussion: This study elucidates CRISPR-Cas systems and defense mechanisms within the Acb complex, highlighting their diverse distribution and interactions with prophages and other genetic elements. This study also provides valuable insights into the evolution and adaptation of these microorganisms in various environments and clinical settings.

Keywords: Acinetobacter baumannii; Acinetobacter calcoaceticus–Acinetobacter baumannii complex; CRISPR systems; cas genes; prophages.

Figure 1

Flowchart for the search for CRISPR–Cas systems. Flowchart indicating data collection for Acb complex genomes, detection and analysis of the CRISPR–Cas systems, resistance and virulence genes, and prophage-associated sequences.

Figure 2

Identification of CRISPR–Cas systems through bioinformatics tools. Characteristics of the CRISPR–Cas systems detected in Acb complex chromosomes showing the SR consensus sequence and its variations. (A) Subtypes of CRISPR–Cas systems identified in A. baumannii chromosomes. (B) Subtypes of CRISPR–Cas systems identified in A. nosocomialis chromosomes. (C) Subtypes of CRISPR–Cas systems identified in A. pittii chromosomes. (D) Subtypes of CRISPR–Cas systems identified in A. calcoaceticus chromosomes. A graphic of the sequences was made with WebLogo (https://weblogo.berkeley.edu/., Crooks et al., 2004).

Figure 3

Identification of SSs in CRISPR–Cas systems. The number of spacers is indicated on each confirmed array. Shared SSs between genomes are highlighted in light green boxes, and unique spacers are shown in purple boxes. SSs that appear multiple times in the array and are shared between genomes are represented by light blue boxes. Unique SSs that appear multiple times in the array are shown in navy blue. Visualization was performed with the iTol program (Letunic and Bork, 2021).

Figure 4

CRISPR-flanking genomic regions of the Acb complex. This figure shows the regions associated with the arrays, highlighting the regions encoding Cap4, CAPPSs, transposons, and toxin–antitoxin systems.

Figure 5

Correlation between spacers and intact prophages in the genomes. Briefly, the correlations and interactions between intact prophages and spacers targeting them are described. The purple circles represent the genomes with CRISPR–Cas systems, and the colored circles represent the intact prophages identified among the studied genomes. The networks show the interactions among the genomes that carry spacers associated with the identified prophages. The analysis and visualization of the networks were carried out with Gephi 0.10.1 (https://gephi.org/, Bastian et al., 2009).

Figure 6

Anti-CRISPR proteins encoded in the genomes of the Acb complex. Identification of the AcrF11 protein in the LAC4, 7835, 3207, and 9201 genomes. The position of the protein coincides with the position of the PHAGE_Acinet_YMC11/11/R3177 in the genomes of the Acb complex.