Burkholderia multivorans requires species‐specific GltJK for entry of a contact‐dependent growth inhibition system protein

Abstract

Interbacterial antagonism and communication are driving forces behind microbial community development. In many Gram‐negative bacteria, contact‐dependent growth inhibition (CDI) systems contribute to these microbial interactions. CDI systems deliver the toxic C‐terminus of a large surface exposed protein to the cytoplasm of neighboring bacteria upon cell−contact. Termed the BcpA‐CT, import of this toxic effector domain is mediated by specific, yet largely unknown receptors on the recipient cell outer and inner membranes. In this study, we demonstrated that cytoplasmic membrane proteins GltJK, components of a predicted ABC‐type transporter, are required for entry of CDI system protein BcpA‐2 into Burkholderia multivorans recipient cells. Consistent with current CDI models, gltJK were also required for recipient cell susceptibility to a distinct BcpA‐CT that shared sequences within the predicted “translocation domain” of BcpA‐2. Strikingly, this translocation domain showed low sequence identity to the analogous region of an Escherichia coli GltJK‐utilizing CDI system protein. Our results demonstrated that recipient bacteria expressing E. coli gltJK were resistant to BcpA‐2‐mediated interbacterial antagonism, suggesting that BcpA‐2 specifically recognizes Burkholderia GltJK. Using a series of chimeric proteins, the specificity determinant was mapped to Burkholderia‐specific sequences at the GltK C‐terminus, providing insight into BcpA transport across the recipient cell cytoplasmic membrane.

Recipient Cell Factors Influence Interbacterial Competition Mediated by Two Distinct Burkholderia dolosa Contact-Dependent Growth Inhibition Systems

“…Toward understanding the mechanism of Burkholderia CDI system effector import, we sought to identify recipient cell factors necessary for susceptibility to these two active CDI systems. A B. dolosa mutant lacking both CDI system-1 and CDI system-2 (Δ bcp-1 Δ bcp-2 ) was mutagenized using a miniTn5-based transposon and serially competed against B. dolosa donor cells that produced either CDI system-1 (Δ bcp-2 ) or CDI system-2 (Δ bcp-1 ), as previously described ( 15 ). Transposon insertion sites identified from resistant clones found one unique insertion site, in gene BDAG_00967, from individual colonies resistant to CDI system-2 (Table S5).…”

“…According to the current model, intoxication via the CDI system protein BcpA requires both extracellular and intracellular proteins to facilitate translocation into the recipient cell cytoplasm. Inner membrane proteins GltJK and Bth_II0599 have been found to facilitate translocation of BcpA-CT from B. multivorans and B. pseudomallei , respectively ( 15 , 16 ). This study has not identified candidate inner membrane receptors for B. dolosa BcpA-1 or BcpA-2, potentially due to a lack of saturation in the mutagenesis scheme or essentiality of the receptor encoding genes.…”

“…Random transposon mutagenesis of the AU0158 Δ bcp-1 Δ bcp-2 double mutant or Δ bcp-1 and Δ bcp-2 single mutants was conducted by delivering pUT-miniTn5-Kn ( 15 , 44 ) by conjugation. The mating was collected, serially diluted in PBS, and plated on LSLB with 250 μg/ml kanamycin to select for transposon insertion mutants.…”

“…For selection of CDI resistant mutants, sequential interbacterial competition assays were used ( 15 ). In brief, 25 μL of the transposon pool was inoculated into 25 ml LSLB with 250 μg/ml kanamycin and cultured overnight.…”

“…Little is known about CDI system toxin translocation in species outside gammaproteobacteria. Inner membrane proteins Bth_II0599 and GltJK have been shown to mediate import of specific Burkholderia pseudomallei and Burkholderia multivorans effectors, respectively ( 10 , 15 ). Alterations to Burkholderia thailandensis lipopolysaccharide (LPS) were also shown to disrupt entry of a B. pseudomallei BcpA-CT toxin ( 16 ).…”