The Clustered Regularly Interspaced Short Palindromic Repeat System and Argonaute: An Emerging Bacterial Immunity System for Defense Against Natural Transformation?

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR) systems and prokaryotic Argonaute proteins (Agos) have been shown to defend bacterial and archaeal cells against invading nucleic acids. Indeed, they are important elements for inhibiting horizontal gene transfer between bacterial and archaeal cells. The CRISPR system employs an RNA-guide complex to target invading DNA or RNA, while Agos target DNA using single stranded DNA or RNA as guides. Thus, the CRISPR and Agos systems defend against exogenous nucleic acids by different mechanisms. It is not fully understood how antagonization of these systems occurs during natural transformation, wherein exogenous DNA enters a host cell as single stranded DNA and is then integrated into the host genome. In this review, we discuss the functions and mechanisms of the CRISPR system and Agos in cellular defense against natural transformation.

Keywords: Argonaute proteins; Clustered regularly interspaced short palindromic repeat-Cas; bacterial immunity system; natural transformation; ssDNA.

Figure 1

Natural transformation in the Gram-negative bacterium Vibrio cholerae (Seitz and Blokesch, 2013; Seitz et al., 2014; Ellison et al., 2018) and the Gram-positive bacterium Streptococcus pneumoniae (Laurenceau et al., 2013, 2015; Balaban et al., 2014; Muschiol et al., 2015). (A) Natural transformation of V. cholerae. Extracellular DNA is bound to the pilus and transported across the OM with the participation of the periplasmic protein ComEA. The pilus is composed of PilA (the main fiber subunit), the PilB and PilT ATPases, PilQ (a secretin pore), PliC (the motor), and other proteins (Matthey and Blokesch, 2016; McCallum et al., 2019). (B) Natural transformation of S. pneumoniae. DNA is bound to a pseudopilus and is transported across the cell wall with the participation of the membrane protein ComEA. The pseudopilus is mostly composed of ComGC (the major pilin), ComGB (a membrane protein), and ComGA (an ATPase; Muschiol et al., 2015). One strand is degraded by the EndA nuclease, while the other strand is transported into the cytoplasm by ComEC, which also occurs in V. cholerae. (C) ssDNA integration into the genome. DprA binds internalized ssDNA and recruits the recombinase RecA that integrates ssDNA into the host genome with the help of ComM. (D) The replication and expression of the integrated DNA in the cell. After replication of the genome, the host cell contains two genome types. The integrated DNA is immediately expressed in the host cell and the expression product is propagated by both non-genetic and genetic inheritance mechanisms after cell division. tDNA, transforming DNA; M, membrane; OM, outer membrane; IM, inner membrane; PG, peptidoglycan.

Figure 2

A proposed model of type II clustered regularly interspaced short palindromic repeat (CRISPR) system inhibition of natural transformation. CRISPRs consist of short repeats separated by non-repetitive spacers that are obtained from previous invading sequences. (I) CRISPR loci are transcribed as long CRISPR RNA (crRNA) precursors and then cleaved into small mature crRNAs, (II) during natural transformation, heterologous DNA enters the cell as ssDNA that is then targeted by the CRISPR system, (III) after replication in the transformant chromosome, a dsDNA that contains a “protospacer” and PAM sequence is produced, and (IV) the complex of crRNAs and Cas proteins recognize and cleave the complementary “protospacer” in the transformed chromosomes.

Figure 3

A model of TtAgo-mediated DNA-guided plasmid inhibition. (I) a plasmid enters the cell as ssDNA through natural transformation. At this stage, the ssDNA is resistant to apo-TtAgo, (II) after replication, the dsDNA plasmid is chopped by apo-TtAgo, (III) the fragmented dsDNA is then bound by TtAgo and one strand of the DNA is released from TtAgo through an unknown mechanism, and (IV) the siDNA-loaded TtAgo targets complementary DNA (ssDNA and dsDNA) and cleaves it.