Interbacterial signaling via Burkholderia contact-dependent growth inhibition system proteins

Abstract

In prokaryotes and eukaryotes, cell-cell communication and recognition of self are critical to coordinate multicellular functions. Although kin and kind discrimination are increasingly appreciated to shape naturally occurring microbe populations, the underlying mechanisms that govern these interbacterial interactions are insufficiently understood. Here, we identify a mechanism of interbacterial signal transduction that is mediated by contact-dependent growth inhibition (CDI) system proteins. CDI systems have been characterized by their ability to deliver a polymorphic protein toxin into the cytoplasm of a neighboring bacterium, resulting in growth inhibition or death unless the recipient bacterium produces a corresponding immunity protein. Using the model organism Burkholderia thailandensis, we show that delivery of a catalytically active CDI system toxin to immune (self) bacteria results in gene expression and phenotypic changes within the recipient cells. Termed contact-dependent signaling (CDS), this response promotes biofilm formation and other community-associated behaviors. Engineered strains that are isogenic with B. thailandensis, except the DNA region encoding the toxin and immunity proteins, did not display CDS, whereas a strain of Burkholderia dolosa producing a nearly identical toxin-immunity pair induced signaling in B. thailandensis Our data indicate that bcpAIOB loci confer dual benefits; they direct antagonism toward non-self bacteria and promote cooperation between self bacteria, with self being defined by the bcpAIOB allele and not by genealogic relatedness.

Keywords: biofilm; contact-dependent competition; signal transduction; sociomicrobiology; two-partner secretion.

Fig. 1.

Community-associated behaviors require BcpA catalytic activity independently of interbacterial growth inhibition. (A) Proportion of CR-binding colonies of wild-type bacteria, ΔbcpAIOB mutant, B. thailandensis producing catalytically inactive BcpA (BtEKA), wild-type bacteria constitutively expressing bcpI (WT+bcpI), and B. thailandensis constitutively expressing WT bcpAIOB (P_S12-WT) or bcpAIOB in which bcpA contains toxin-inactivating mutations (P_S12-BtEKA). Lines show the mean of at least three independent experiments per strain (n = 3–6). (B) Aggregation in minimal medium (Upper) and pigment production by colony biofilms (Lower) of the strains in A. *P < 0.05; ns, not significant.

Fig. 2.

Catalytically active BcpA affects expression of genes associated with community behaviors. (A) Genes identified by RNA-seq as significantly differentially expressed (P < 0.05) by BtEKA mutants compared with wild-type bacteria (blue) and P_S12-WT mutants compared with the wild-type strain (yellow). (B) Heat map of RPKMs (reads per kilobase of transcript per million reads mapped) for the 35 genes that were oppositely expressed in P_S12-WT and BtEKA mutants compared with wild-type B. thailandensis (WT). Shading indicates low (black) and high (green) RPKM values for each of triplicate samples of each strain. Genes are arranged in chromosomal order, and putative operons/multigene loci are annotated to the right. Arrowhead shows position of csuD. (C) β-Galactosidase activity (in Miller units) for wild-type, BtEKA, and P_S12-WT bacteria carrying lacZ reporters within the coding regions of Bth_I0535 (Left, dark gray bars) or csuD (Right, light gray bars). Bars represent the mean of three independent experiments, and error bars show the SD. *P < 0.05 compared with wild-type cultures. (D) Proportion of CR-binding colonies of BtEKA, P_S12-WT, and WT and ΔBth_I0534 mutant bacteria with (+) and without (−) P_S12-Bth_I0534 at a chromosomal attTn7 site. Bars show the mean of three independent experiments (n = 3–6 per strain). *P < 0.05. (E) Z-stack images of 48-h biofilms formed by GFP-marked bacterial strains shown in D. (Scale bar: 20 μm.)

Fig. 3.

Catalytically active BcpA acquired from neighboring bacteria affects gene expression in recipients. (A) Expression of Bth_I0535 (Top) and csuD (Middle) in ΔbcpA recipient bacteria containing lacZ reporters for these genes after incubation with no BcpA donor (first spot) or coincubation (at a 1:1 ratio) with B. thailandensis constitutively producing wild-type (P_S12-WT, middle spot) or inactive BcpA (P_S12-BtEKA, last spot) on X-gal agar. Donor (Bottom) and recipient (first column) cells incubated alone are also shown. (B) Diagram of the modified P_bcp-flp reporter cassette. The putative bcpA promoter (black arrow) drives expression of flp recombinase. Gray arrows indicate constitutive promoters. Flp recombinase target (FRT) sites (black boxes) flank nptII (conferring kanamycin resistance) and gfp genes. A cat gene (conferring chloramphenicol resistance, Cm^R) is located outside the FRT sites. Tn7L and Tn7R (hatched boxes) sequences target the cassette to an attTn7 site on one of the two B. thailandensis chromosomes. (C) Proportion of wild-type bacteria carrying the P_bcp-flp reporter (recipients) that expressed bcpAIOB when incubated with the indicated (Cm^S) mutants (donors) at a 1:100 ratio (recipient:donor) on M63 agar. The fraction of the reporter recipient population having expressed bcpAIOB is shown as the percentage of Cm^R colonies having lost GFP fluorescence (% GFP⁻ recipient cfus). Reporter recipient bacteria were recovered from both the center (Left) and edge (Right) of the coculture colony spot. *P < 0.05.

Fig. 4.

B. thailandensis responds to self but not non-self BcpA. (A) Aggregation of wild-type B. thailandensis, BtEKA mutants, and chimeric strains Bt-Bp1106a-1 and Bt-Bp1106a-2 (gray bars, “native promoter”) or strains in which the bcpAIOB locus is driven by the constitutive promoter, P_S12 (black bars, “P_S12”). The ratio of the OD₆₀₀ of a settled culture to the OD₆₀₀ of the same culture after vortexing is shown. Dashed line shows an OD₆₀₀ ratio of 1, which indicates no aggregation. Bars represent the mean of four independent experiments, and error bars denote the SD. *P < 0.05 compared with WT or P_S12-WT aggregation. (B) β-Galactosidase activity (in Miller units) for WT and BtEKA bacteria, and chimeric strains Bt-Bp1106a-1 and Bt-Bp1106a-2 carrying lacZ reporters within the coding region of Bth_I0535. Bars represent the mean of three independent experiments, and error bars show the SD. *P < 0.05 compared with WT cultures. (C) Expression of Bth_I0535::lacZ (Top) and csuD::lacZ (Middle) reporters in B. thailandensis ΔbcpA recipient bacteria after coincubation (at a 1:5 ratio) with B. dolosa wild-type or ΔbcpA-1, ΔbcpA-2, or ΔbcpA-1ΔbcpA-2 mutant bacteria on X-gal agar. Donor (Bottom) and recipient (first column) cells incubated alone are also shown.