A Novel Bacteriophage Exclusion (BREX) System Encoded by the pglX Gene in Lactobacillus casei Zhang

Abstract

The bacteriophage exclusion (BREX) system is a novel prokaryotic defense system against bacteriophages. To our knowledge, no study has systematically characterized the function of the BREX system in lactic acid bacteria. Lactobacillus casei Zhang is a probiotic bacterium originating from koumiss. By using single-molecule real-time sequencing, we previously identified N⁶-methyladenine (m6A) signatures in the genome of L. casei Zhang and a putative methyltransferase (MTase), namely, pglX This work further analyzed the genomic locus near the pglX gene and identified it as a component of the BREX system. To decipher the biological role of pglX, an L. casei Zhang pglX mutant (ΔpglX) was constructed. Interestingly, m6A methylation of the 5'-ACRCAG-3' motif was eliminated in the ΔpglX mutant. The wild-type and mutant strains exhibited no significant difference in morphology or growth performance in de Man-Rogosa-Sharpe (MRS) medium. A significantly higher plasmid acquisition capacity was observed for the ΔpglX mutant than for the wild type if the transformed plasmids contained pglX recognition sites (i.e., 5'-ACRCAG-3'). In contrast, no significant difference was observed in plasmid transformation efficiency between the two strains when plasmids lacking pglX recognition sites were tested. Moreover, the ΔpglX mutant had a lower capacity to retain the plasmids than the wild type, suggesting a decrease in genetic stability. Since the Rebase database predicted that the L. casei PglX protein was bifunctional, as both an MTase and a restriction endonuclease, the PglX protein was heterologously expressed and purified but failed to show restriction endonuclease activity. Taken together, the results show that the L. casei Zhang pglX gene is a functional adenine MTase that belongs to the BREX system.IMPORTANCELactobacillus casei Zhang is a probiotic that confers beneficial effects on the host, and it is thus increasingly used in the dairy industry. The possession of an effective bacterial immune system that can defend against invasion of phages and exogenous DNA is a desirable feature for industrial bacterial strains. The bacteriophage exclusion (BREX) system is a recently described phage resistance system in prokaryotes. This work confirmed the function of the BREX system in L. casei and that the methyltransferase (pglX) is an indispensable part of the system. Overall, our study characterizes a BREX system component gene in lactic acid bacteria.

Keywords: BREX; Lactobacillus casei Zhang; MTase; bacteriophage exclusion system; methyltransferase; pglX.

FIG 1

Schematic diagram showing the multigene bacteriophage exclusion (BREX) loci in Lactobacillus casei Zhang. The classic type I BREX cassette includes brxA (LCAZH_2059), brxB (LCAZH_2058), brxC (LCAZH_2057), pglX (LCAZH_2056) (a putative N⁶-adenine-specific MTase), pglZ (LCAZH_2053), and brxL (LCAZH_2052). The identified BREX system had an insertion of two genes between pglX and pglZ, namely, int (LCAZH_2055) (a putative phage integrase) and LCAZH_2054 (a putative DNA MTase).

FIG 2

(a) Scatterplot of the modification quality value (QV) versus per-strand coverage in Lactobacillus casei Zhang ΔpglX. (b to e) Gram-stained cells and colony morphology of L. casei Zhang wild-type (b and d) and ΔpglX mutant (c and e) strains. Cell morphology was viewed at a ×1,000 magnification. (f to h) Growth curves of L. casei Zhang wild-type and ΔpglX mutant strains in MRS broth. Shown are changes in the OD₆₀₀ (f), viable counts (g), and pHs (h). Error bars represent standard deviations.

FIG 3

(a to c) Plasmid transformation efficiencies of Lactobacillus casei Zhang wild-type and mutant strains. Zhang is the wild type, and Zhang ΔpglX is the mutant strain. Statistical analysis was performed using Student’s unpaired two-tailed t test. Double asterisks represent significant differences (P < 0.01). (d and e) Stability of plasmids pMSP3535 (d) and pTRKH2 (e) in Lactobacillus casei Zhang wild-type and ΔpglX mutant strains. Error bars represent standard deviations.

FIG 4

Plasmid transformation efficiencies of Lactobacillus casei Zhang wild-type and mutant strains. Zhang is the wild type, and Zhang ΔpglX is the mutant strain. Error bars represent standard deviations.

FIG 5

Plasmid transformation efficiencies of the Lactobacillus casei Zhang wild-type strain with plasmid pSec:Leiss:Nuc (Zhang-pleiss), the mutant strain with plasmid pSec:Leiss:Nuc (Zhang ΔpglX-pleiss), and the complemented strain (Zhang ΔpglX-pglX). Error bars represent standard deviations. Statistical analysis was performed using Student’s unpaired two-tailed t test. Double asterisks represent significant differences (P < 0.01).

FIG 6

(a) SDS-PAGE analysis of overexpressed PglX protein. Lane M, protein marker; lane DE3/pglX, crude extract of pET-pglX-transformed E. coli DE3; lane PglX, purified PglX protein. (b) Endonuclease activity of purified PglX protein λ DNA. Lane M, λ-HindIII digestion DNA marker; lane B, λ DNA digested with buffer only; lane C, λ DNA digested with the crude extract of pET-pglX-transformed E. coli DE3; lane P, λ DNA digested with recombinant PglX protein.