Intercellular communication and conjugation are mediated by ESX secretion systems in mycobacteria

Abstract

Communal bacterial processes require intercellular communication mediated by secretion systems to coordinate appropriate molecular responses. Intercellular communication has not been described previously in mycobacteria. Here we show that the ESX secretion-system family member ESX-4 is essential for conjugal recipient activity in Mycobacterium smegmatis Transcription of esx4 genes in the recipient requires coculture with a donor strain and a functional ESX-1 apparatus in the recipient. Conversely, mutation of the donor ESX-1 apparatus amplifies the esx4 transcriptional response in the recipient. The effect of ESX-1 on esx4 transcription correlates with conjugal DNA transfer efficiencies. Our data show that intercellular communication via ESX-1 controls the expression of its evolutionary progenitor, ESX-4, to promote conjugation between mycobacteria.

Fig. 1.. Recipient ESX-4 is required for mycobacterial conjugation.

(A) DNA transfer efficiencies (transconjugants per donor cell) show that DCT requires an intact esx4 locus in the recipient strain. Transconjugants were not recovered in matings that lacked a recipient ESX-4 component. Mating-pair genotypes are indicated for donor and recipient, above and below the line, respectively. (B) Schematic showing the conserved gene content and order of esx4 loci. ESX nomenclature of the encoded proteins is shown above the arrows (ecc designates an ESX essential core protein). The gene specific to esx4 is indicated by its M. smegmatis gene name, Msmeg_1537. Recipient locus mutations are summarized by a transposon insertion (tn), a deletion (the absence of an arrow), or a complementing plasmid (green arrow with oval). M. smegmatis (Ms) and M. tuberculosis (Rv) gene numbers and amino acid identities (aa%) and the putative function of the encoded proteins are shown below each gene.

Fig. 2.. Coculture with donor induces recipient esx4 transcript levels and requires recipient ESX-1 activity.

(A) Experimental design for SNP-guided RNA-seq of DCT. The ESX-1_mut strains have targeted deletions of eccC_b1, encoding the ATPase required for ESX-1 secretion activity and required in the recipient for conjugation. The four strains were grown individually and in mixed cultures of WT × WT, WT × ESX-1_mut, or ESX-1_mut × WT. Conditions, processing, and analysis are as indicated at right. (B) Heat-map cells of WXG100 genes from esx4 (esxUT), esx1 (esxBA), and esx3 (esxHG), with changes upon coculture shown as log₂ insets. For each mating, the donor and recipient genotypes are shown, and the conjugal mating proficiencies are indicated. A housekeeping gene (rpoB) is included as an independent expression control.

Fig. 3.. ESX-1 and contact dependence of conjugal communication.

(A) DCT mating efficiencies for conjugal pairs used for RT-PCR. Strains are identified as WT or ESX-1_mut (ΔeccC_b1) donors (above the line) or recipients (below). The dashed line at bottom right indicates separation of conjugal strains by a porous membrane. (B) qRT-PCR analysis for esxU from cocultures of ESX-1 mutants or physically separated mycobacteria. esxU signals were normalized to rpoB expression. Error bars indicate SD (n = 3). (C) RT-PCR and restriction fragment length polymorphism analysis of BstUI fragments identifies recipient origin of the elevated esxUT transcripts. Arrows below the genetic map indicate the primers used for amplification, and the sites of BstUI cleavage are indicated by triangles. Monoculture genomic DNA (gDNA) controls show the expected parental patterns upon digestion (+) with BstUI. Digestion of coculture-derived cDNA shows a recipient pattern. bp, base pairs.