A Cytosine Methyltransferase Modulates the Cell Envelope Stress Response in the Cholera Pathogen [corrected]

Abstract

DNA methylation is a key epigenetic regulator in all domains of life, yet the effects of most bacterial DNA methyltransferases on cellular processes are largely undefined. Here, we used diverse techniques, including bisulfite sequencing, transcriptomics, and transposon insertion site sequencing to extensively characterize a 5-methylcytosine (5mC) methyltransferase, VchM, in the cholera pathogen, Vibrio cholerae. We have comprehensively defined VchM's DNA targets, its genetic interactions and the gene networks that it regulates. Although VchM is a relatively new component of the V. cholerae genome, it is required for optimal V. cholerae growth in vitro and during infection. Unexpectedly, the usually essential σE cell envelope stress pathway is dispensable in ∆vchM V. cholerae, likely due to its lower activation in this mutant and the capacity for VchM methylation to limit expression of some cell envelope modifying genes. Our work illuminates how an acquired DNA methyltransferase can become integrated within complex cell circuits to control critical housekeeping processes.

Fig 1. VchM methyltransferase activity promotes V. cholerae growth and pathogenicity.

(A) Mice were co-infected with wildtype V. cholerae (either C6706 or O395) and a differentially marked methyltransferase mutant, which could be distinguished by blue/white screening. The competitive index was calculated from the ratio of mutant (LacZ⁺) to WT (LacZ^-) cells recovered after infection divided by the corresponding ratio of the inoculum. WT represents a control competition between LacZ⁺ and LacZ^- V. cholerae C6706. **p-value <0.01. (B) Mice were singly infected either with wt or ∆vchM V. cholerae C6706 and the total number of CFUs recovered per small intestine is shown. **p-value <0.01. (C) Differentially marked strains (LacZ^-, ∆hsdM, ∆vca0447, and either ∆vchM, a vchM active site mutant (C109A), or a deletion mutant into which the wt gene had been reintroduced (wt revertant)) were co-cultured in LB with wt cells and the competitive indices were calculated as in (A). **p-value <0.01. (D) The growth rate of wt and ∆vchM cells in LB was monitored over time using OD 600 measurements. (E) Genomic DNA from the indicated strains was digested with SalI and the methylation-sensitive enzyme BsrFI, which does not cleave methylated RCCGGY motifs.

Fig 2. V. cholerae cytosine methylation and its associations with gene expression.

(A) The extent of VchM target site (RCCGGY) methylation on chromosomes I and II, as determined by bisulfite sequencing of DNA from exponential phase cultures, is plotted against the extent of methylation for these sites during infection. Three sites (#1-#3) were found to be persistently non-methylated; numbers designate the genomic position of each site on the forward (+) or reverse (-) strand. (B) Genes that were significantly (p-val <0.01) differentially expressed by wt and ∆vchM cells were correlated with the presence of intragenic RCCGGY motifs. Significance was assessed using a Fisher’s exact test by the comparing the fraction of differentially expressed genes containing RCCGGY motifs (e.g., 22 out of 63) to the fraction of all genes in V. cholerae containing RCCGGY motifs (1460 out of 3842 genes). Significant enrichment of motifs was found within only the genes that were upregulated in ∆vchM cells. DE = number of differentially expressed genes; 5mC = number of genes containing methylated RCCGGY motifs. (C) Differences in transcript abundance between exponential phase cultures of ∆vchM and WT cells are plotted relative to the number of RCCGGY motifs present within the coding region for each gene. The boxes represent the fold change of genes in the 25%-75% quartile with the median fold change shown as a line. The whiskers represent 1.5 fold of the interquartile range (the third quartile minus the first quartile) away from the box. (D) The numbers of genes that are differentially expressed (p-value <0.05) by ∆vchM strains of C6706 and O395 V. cholerae compared to their parental strains are shown for a variety of biological categories.

Fig 3. TIS-based identification of loci that interact genetically with vchM or rpoE.

(A) For two comparative analyses (∆vchM vs. wt and ∆vchM ∆rpoE vs. ∆vchM), the read count ratio of transposons inserted into each genetic locus was determined, and is plotted against the inverse of the associated p-value as determined by Mann-Whitney U analysis of read counts. Genes plotted above the horizontal line are considered significantly different in each analysis (p-value <0.001). (B) The raw number of reads originating from insertions on the forward (red) or reverse strand (green) in wt and ∆vchM insertion libraries are shown for selected loci from both libraries. All potential insertion sites (TA dinucleotides) are marked by black bars. (C) The predicted σ^E stress response pathway in V. cholerae.

Fig 4. Deletion of vchM suppresses the essentiality of rpoE but ∆vchM ∆rpoE mutants show impaired growth under stress conditions.

(A) Allelic exchange to replace rpoE with a kanamycin resistance cassette was performed in WT and ∆vchM cells. After patching colonies to determine if candidate colonies still retained the knockout vector, the false positive and rpoE deletion frequencies were calculated. (B) A representative western blot of lysates from cultures of WT, ∆vchM and ∆vchM ∆rpoE cells probed with polyclonal antisera against σ^E. n.s. = non-specific signal. (C) Culture density (OD 600) of the indicated strains was monitored for cells grown in LB and in LB supplemented with polymyxin B (poly B). (D) In vitro competition assays were performed between LacZ- V. cholerae and the indicated mutants. Cultures were grown in LB under aerobic or anaerobic conditions and supplemented with fumarate as noted. Competitive indices were calculated as in Fig 1. (E) Competitive infections were performed using differentially marked versions (LacZ^+/-) of the indicated strains. Competitive indices were calculated for bacteria in intestinal homogenates as in Fig 1A. The open circle represents the limit of detection where no ∆vchM ∆rpoE mutants were recovered.

Fig 5. Other cell envelope-related loci and VchM control of LPS modification contribute to basal levels of σ^E.

(A) Western blotting and relative abundance (compared to wt) of σ^E in mutants lacking genes that could not be disrupted in the ∆vchM ∆rpoE mutant. Mutants contain transposon insertions in the genes of interest, and are derived from a wt strain, not ∆vchM. RpoB was used as a loading control to normalize for total protein. Results are the average of three independent experiments. (B) Relative abundance of transcripts for LPS synthesis genes in mutants containing synonymous mutations that abolished RCCGGY motifs in these genes. Abundance is relative to transcript levels in wt cells, as determined by qRT-PCR in three independent biological replicates. (C) The relative abundance of σ^E in wt, ∆vchM and ∆vchM ∆rpoE cells and in a vc2437 insertion mutant (vc2437::pGP). Error bars represent data from two independent replicates normalized to wt basal σ^E levels. (D) Model for VchM-mediated modulation of the σ^E envelope stress response in V. cholerae. In wt cells, VchM methylation of some LPS modification genes restricts their expression, leading to aberrant LPS structure, generation of envelope stress and the induction of the σ^E response, which renders rpoE essential.