MspGI Restriction–Modification System and Its Flanking Genes of Microbacterium sp. Gd 4-13 ^†

Abstract

Bacteria have defense systems against the penetration of foreign DNA (phages and plasmids). Some defense systems protect bacteria from phage penetration into the cell, while others block phage replication. Antiphage defense systems are often located next to already known defense systems (RM system, CRISP-Cas system, and others). In this work, the genetic sequence flanking the restriction–modification system of Microbacterium sp. strain Gd 4-13 was analyzed. The genes of the endonuclease HNH, hypothetical proteins, recombinase, and helicase/ATPase have been identified. Their combination and some analogy with the DISARM, BREX, and Druantia systems allow us to hypothesize that, next to the MspGI restriction–modification system, there is another defense system, and together they create an island of antiphage defense in the bacteria in vivo.

1. Introduction

Bacteria and archaea are frequently attacked by phages and, as a result, have developed multiple sophisticated lines of active defense called the “immune system” of prokaryotes [1,2,3].

Restriction–modification (RM) systems provide basic protection from the penetration of foreign DNA [4,5,6,7], and their genes are found in more than 96% of bacterial genomes and more than 99% of archaea genomes [5,8]. RM system enzymes (REase and MTase) recognize a specific nucleotide sequence (recognition site) in DNA. Restriction endonuclease (REase) cleaves the DNA [5,6,7].

The other constituent of the RM system is methyltransferase (MTase). MTases methylate recognition site nucleotide residues (adenine or cytosine) immediately after replication. If the recognition site is not methylated, REase introduces a double-strand break into the DNA. Thus, the cleavage of foreign DNA occurs in the bacterium without affecting its own DNA because foreign DNA is not methylated. All RM systems require Mg²⁺ as a cofactor [6]. Currently, based on the subunit structure, substrate specificity, enzyme’s need for cofactors, and nature of DNA cleavage, four types of RM systems are distinguished [5,6].

The Type I RM system is a complex oligomeric complex consisting of three subunits, HsdS, HsdM, and HsdR, in the ratio R₂M₂S₁. AdoMet, Mg²⁺, and ATP are required for activity [7,9]. The HsdS ensures recognition of certain DNA regions, determining the specificity of the RM system and serves as the core to which other subunits bind. The HsdM subunit binds to the methylation cofactor AdoMet and methylates the adenine residues within the restriction site. HsdR cleaves phosphodiester bonds in DNA and exhibits helicase activity. This complex recognizes an asymmetric sequence, consisting of 3–4 bp at the 5’ end and 4–5 bp at the 3’-end specific for recognition by REase, separated by 6–8 bp, insignificant for recognition (for example EcoBI 5′-TGAN8TGCT-3′). DNA cleavage occurs at a distance of 40 bp to several kb from the recognizable sequence, approximately in the middle of the two recognition sites [10,11]. Type I REases have a remarkable ability to alter sequence specificity through domain shuffling and rearrangement [12].

The Type II RM system [5] consists of MTase and REase, which are independent proteins. REase hydrolyzes DNA at strictly defined positions within or near restriction sites. This feature of this type of RM system has contributed to the widespread use of REases in genetic engineering. Type II REases, the ‘work horses’ of modern molecular biology, are used daily in laboratories for DNA analysis and gene cloning [13]. The Type II RM system does not require ATP or other energy sources to function. MTases function as monomers and methylate cytosine or adenine. In the Type II RM system, there are several subgroups, and some RM systems may belong to several of them simultaneously [5].

Type III RM systems include two subunits (Res and Mod) that combine to form a heterotetramer (Res2Mod2) [7] with endonuclease and methyltransferase activities. The Res subunit exhibits helicase activity and requires ATP hydrolysis to function [10]. Unlike the Type I RM system, DNA hydrolysis requires the interaction of two enzyme complexes. They recognize two identical recognition sites located in opposite orientations. Sites may be unmethylated or methylated along one chain [7]. The Mod subunit can function separately from Res by performing MTase function. At the same time, the nuclease activity of the Res subunit appears only when it is in complex with Mod [5].

The RM system Type IV cleaves only modified DNA containing methylated, hydroxymethylated or glycosyl-hydroxymethylated bases. GTP hydrolysis is required for DNA cleavage. There is no methyltransferase activity. Nuclease activity is allosterically activated by AdoMet. The restriction site is asymmetrical and consists of two parts separated by a space. REase cleaves DNA within or away from specific sequences. [14].

Recently, new systems for protecting bacteria from foreign DNA have been discovered and described. These include the BREX (Bacteriophage Exclusion) and DISARM (defense island system associated with restriction–modification). They are multigene complexes similar to the Type I and Type III RM systems [15,16].

BREX, a novel phage resistance system, is widely distributed in microbial genomes [15]. BREX comprises six genes that confer resistance to a wide range of phages, including virulent and temperate phages. BREX contains a putative Lon-like protease, alkaline phosphatase domain protein, putative RNA-binding protein, DNA methylase, ATPase domain protein, and a protein of unknown function. The BREX system allows the phage to be adsorbed but blocks its replication. Unlike restriction–modification systems, phage DNA does not appear to be cleaved or degraded by BREX, suggesting a novel mechanism of defense [15].

The DISARM system is widespread among bacteria and archaea. It comprises five genes, including DNA methylase, and four other genes designated as helicase domain, phospholipase D domain (PLD), DUF1998 domain, and a gene of unknown function [16]. DISARM is a new type of multigene restriction–modification module that expands the arsenal of defense systems that prokaryotes are known to possess at their disposal against their viruses. DISARM limits phages proliferation.

Nine new systems for protecting bacteria and archaea from foreign DNA have been described [17]. These defense systems were discovered next to already known systems such as the RM system, the CRISP-Cas system, and the abortive infection (Abi) system. These comprise between one and five genes and span between 2 kb and 12 kb of genomic DNA. Some genes are repeated in DISARM and BREX, such as Helicase, HNH-endonuclease, ATPase, and the proteins of unknown function.

The genes comprising the new systems encode many protein domains that are commonly present in antiviral systems such as CRISPR-Cas and RNA interference (RNAi), including helicases, nucleases, and nucleic acid binding domains, as well as many domains of unknown function and atypical domains. These three systems contain membrane-bound proteins, as evidenced by the presence of multiple transmembrane helicases [17].

The discovery and mechanistic understanding of antiphage defense systems led to the development of important biotechnological tools. For example, the discovery of restriction enzymes has revolutionized genetic engineering. The close proximity of different protective systems on DNA suggests their joint activity in protecting bacterial cells from foreign DNA.

In this study, we analyzed the sequences of genes flanking the MspGI RM system to identify the genes involved in the antiphage defense system of Microbacterium sp. Gd 4-13, which, together with the RM system, forms a defense island.

2. Results

Microbacterium sp. Gd 4-13 was obtained from permafrost deposits on the Yamal Peninsula. The sample dates back up to 34 thousand years. Draft genome sequencing of the nucleotide sequence of this microorganism has been carried out [18]. Analysis of the nucleotide sequences of the genome Microbacterium sp. Gd 3-14 showed the presence of an RM system. It includes two enzymes, REase and MTase, and belongs to the Type II RM system.

The arrangement of REase and MTase genes relative to each other in the genome of Microbacterium sp. Gd 3-14 is shown in Figure 1.

As shown in Figure 1, the end of the MTase gene overlaps with four nucleotides at the beginning of the REase gene.

Analysis of the sequence of genes flanking the MspGI RM system (Figure 2) showed that downstream from the Rease there are four bp, whereafter the genes of the endonuclease HNH, the hypothetical protein (hp), and recombinase are located. Upstream of the MTase gene at a distance of 3.5 kb, a gene was discovered, which was identified by the BLAST program first as a helicase and then as an AAA family ATPase. Therefore, in this study, we indicate both the names of this gene (helicase/ATPase). The membrane protein is located upstream of the helicase/ATPase gene. Between the helicase/ATPase and MTase genes, three short open reading frames (ORF) (120–200 aa) are defined as hypothetical proteins. Their functions remain unknown.

The alignment of the genes of the hypothetical proteins in BLAST revealed that one (hp1) is a protein domain found in the genome of Microbacterium sp. Gd 4-13, whose function is unknown. The second hp (hp2) showed 74% homology with hypoxanthine phosphoribosyltransferase of the microorganism Curtobacterium sp. MCSS17_005. The third hp (hp3) did not show homology with any known protein.

The close proximity of these three hp genes to helicase/ATPase suggests an analogy with the Druantia Type I system, which is included in the list of the nine new defense systems. This system also contains three proteins of unknown function that are located adjacent to the helicase [17].

Two more hypothetical proteins, one between the helicase/ATPase and the membrane protein and the other between HNH endonuclease and recombinase, also did not show homology with any known protein (Figure 2, hp are marked with asterisks).

The presence of cytosine MTase and the close proximity of HNH endonuclease and helicase/ATPase indicate the possible presence of an antiphage defense system in Microbacterium sp. Gd 4-13, next to the MspGI RM system similar to Class I DISARM or BREX (Figure 2). The presence of three hp genes and a membrane protein is similar to Druantia Type I and Zory [17].

3. Discussion

Analysis of the genes flanking the MspGI RM system revealed the presence of genes considered to be antiphage defense systems. For some of the nine defense systems, it has been shown that deletion or mutation of one of the genes leads to the inactivation of the protective properties of the system [17].

This is also true for the classical RM system. If we transform a bacterium by MTase gene, then the cell will be viable (in the absence of the paired REase) but will no longer protect the cell from the penetration of foreign DNA. Whereas a bacterial cell transformed by the REase gene is not viable, because this protein is toxic to it and only the presence of MTase protects the cell from the destructive action of REase. Probably other antiphage defense systems work the same way, some genes protect the DNA host, while others are aimed at destroying foreign DNA. Removing any gene from a defense system results in inactivation of the protective function of that defense system.

The association with multiple other restriction systems may reflect the general tendency of defense systems to cluster in defense islands, or, alternatively, might suggest that the function of the core DISARM genes can be combined with additional RM modules and provide a synergistic defensive advantage [16].

Thus, the MspGI RM system is flanked by genes, the set of which does not completely replicate any system of protection against foreign DNA. However, the close proximity of these genes to the MspGI RM system and some analogies with the DISARM, BREX, and Druantia systems suggest that the described set of genes, together with the RM system, may represent an antiphage defense island in Microbacterium sp. Gd 4-13. Therefore, it is necessary to test this experimentally.

4. Materials and Methods

4.1. Isolation Bacteria Microbacterium sp. Gd. 4-13

The Microbacterium sp. strain Gd 4-13 was isolated from the Gydan Peninsula cryogenic permafrost marine sediment sample obtained from a 4.0 m depth with radiocarbon dating of approximately 34,300 years.

4.2. Cultivation of Microorganisms and Growth Conditions

Isolation of cultures from samples was carried out by cumulative cultivation at 4 and 20 °C on standard nutrient media R2A and 1/2 TSB (Difco Laboratories, Detroit, USA), LB, and SOC with the addition of NaCl in the concentration range of 100–250 g/L.

4.3. Determination of Taxonomic Affiliation of a Strain Containing the MspGI RM System

Determination of taxonomic affiliation was based on homology of the 16S rRNA sequence with nucleotide sequences available in the RDP (then http://rdp.cme.msu.edu, accessed until 27 April 2018), now available and accessed on 15 August 2024 via https://www.canr.msu.edu/cme/resources and https://sourceforge.net/projects/rdp-classifier, and GenBank (https://www.ncbi.nlm.nih.gov/genbank) databases using the BLAST program.

Genome sequence analysis of Microbacterium sp. Gd 4-13 technologies was conducted using the following programs accessed on 15 August 2024: https://www.ncbi.nlm.nih.gov/nucleotide (NCBI Reference Sequence: NZ_QEIJ01000002.1) and https://blast.ncbi.nlm.nih.gov/Blast.cgi.