The Burkholderia bcpAIOB genes define unique classes of two-partner secretion and contact dependent growth inhibition systems

Abstract

Microbes have evolved many strategies to adapt to changes in environmental conditions and population structures, including cooperation and competition. One apparently competitive mechanism is contact dependent growth inhibition (CDI). Identified in Escherichia coli, CDI is mediated by Two-Partner Secretion (TPS) pathway proteins, CdiA and CdiB. Upon cell contact, the toxic C-terminus of the TpsA family member CdiA, called the CdiA-CT, inhibits the growth of CDI(-) bacteria. CDI(+) bacteria are protected from autoinhibition by an immunity protein, CdiI. Bioinformatic analyses indicate that CDI systems are widespread amongst α, β, and γ proteobacteria and that the CdiA-CTs and CdiI proteins are highly variable. CdiI proteins protect against CDI in an allele-specific manner. Here we identify predicted CDI system-encoding loci in species of Burkholderia, Ralstonia and Cupriavidus, named bcpAIOB, that are distinguished from previously-described CDI systems by gene order and the presence of a small ORF, bcpO, located 5' to the gene encoding the TpsB family member. A requirement for bcpO in function of BcpA (the TpsA family member) was demonstrated, indicating that bcpAIOB define a novel class of TPS system. Using fluorescence microscopy and flow cytometry, we show that these genes are expressed in a probabilistic manner during culture of Burkholderia thailandensis in liquid medium. The bcpAIOB genes and extracellular DNA were required for autoaggregation and adherence to an abiotic surface, suggesting that CDI is required for biofilm formation, an activity not previously attributed to CDI. By contrast to what has been observed in E. coli, the B. thailandensis bcpAIOB genes only mediated interbacterial competition on a solid surface. Competition occurred in a defined spatiotemporal manner and was abrogated by allele-specific immunity. Our data indicate that the bcpAIOB genes encode distinct classes of CDI and TPS systems that appear to function in sociomicrobiological community development.

Figure 1. Organization and diversity of Burkholderia-type CDI-encoding loci.

A) Schematic of Burkholderia- and E. coli-type CDI-encoding loci. Burkholderia-type CDI loci are encoded by genes bcpAIOB and differ from E. coli-type CDI loci in gene order and content; Burkholderia-type CDI loci contain an additional ORF, bcpO, not present in E. coli-type CDI loci. B) Schematic of the amino acid sequence of a single representative allele for each of the B. thailandensis and B. pseudomallei CDI-encoding loci. The alleles have strong sequence similarity (∼89% similar) amongst the N-terminal encoded ∼2750 aa of BcpA, BcpB, and some BcpO proteins, but fall into three phylogenetic groups (indicated by shades of gray). The diversity of the CDI-encoding proteins is located within the ∼350 aa C-terminal to the Nx(E/Q)LYN motif in BcpA, BcpI, and some BcpO proteins, with no more than ∼10% similarity between any two alleles (indicated by different colors). For some BcpO proteins, the diversity is restricted to the predicted signal sequence peptide (alleles 1–5, 7, and 8) whereas the functional domain of the protein is highly conserved.

Figure 2. Examination of the bcpAIOB locus in B. thailandensis.

A) Top panel, analysis of the operon structure of bcpAIOB. Primer sets 1, 2, and 3 flanking the intergenic region of bcpA and bcpI, bcpI and bcpO, and bcpO and bcpB, respectively, (as indicated in 2C) were used for RT-PCR. Middle and bottom panels, analysis of the approximate start site of transcription of bcpA. RT-PCR was performed using primers annealing 50, 70, 120, 150, 200, 250, and 300 nt 5′ to bcpA with a reverse primer internal to bcpA. All products were visualized by ethidium bromide. B) Expression of bcpA. P_S12-lacZ and P_neg-lacZ (dark gray bars) and P_bcpA-lacZ (light gray bars) reporter strains were cultured under various conditions and assayed for β-galactosidase activity. Error bars represent the mean ± 1 SEM. C) Schematic of B. thailandensis E264 strain constructs, including WT, ΔbcpO, ΔbcpB, and ΔbcpAIOB, used throughout the study.

Figure 3. Analysis of BcpA-HA.

Immunoblot of whole cell lysates of WT (E264BcpA-HA::pECG22), ΔbcpO (E264BcpA-HAΔbcpO::pECG22), ΔbcpB (E264BcpA-HAΔbcpB::pECG22), ΔbcpO::bcpO (E264BcpA-HAΔbcpO::bcpO::pECG22), ΔbcpB::bcpB (E264BcpA-HAΔbcpB::bcpB::pECG22), no tag (E264::pECG22) strains producing BcpA-HA were separated by SDS-PAGE and stained with anti-HA antibody.

Figure 4. Per cell analysis of the probabilistic expression of bcpA.

A) P_bcpA-lacZ reporter strain plated on M63 minimal medium agar supplemented with X-gal. B) Confocal microscopy of gfp reporter strains, P_bcpA-gfp, P_S12-gfp, and E264Km^R (vector), cultured overnight in liquid medium. C) Representative flow cytometry analysis of gfp reporter strains, P_bcpA-gfp, P_S12-gfp, and E264Km^R (vector), cultured overnight in liquid medium. The dot plot (top left panel) of side scatter intensity (SS INT) vs. forward scatter intensity (FS INT) shows the events corresponding to B. thailandensis that were gated on (“Gate”) for subsequent analysis. Histograms show the number of GFP-positive events (Count) vs. relative fluorescence intensity (FL1 INT) for each reporter strain, and the percent of GFP-positive bacteria detected in each culture is indicated.

Figure 5. bcpAIOB-dependent autoaggregation in M63 minimal medium.

A) Indicated strains were cultured in M63 minimal medium for ∼48 hours with aeration. Four or 10 U of DNaseI was added to wild type E264 cultures upon inoculation. B) Wild type and P_S12-bcpAIOB strains were cultured in M63 minimal medium; photos were taken over the course of 48 hours.

Figure 6. Intracellular toxicity of BcpA-CT proteins and protection by BcpI proteins.

B. thailandensis producing BcpA-CT peptides with and without BcpI proteins from B. pseudomallei; specific combinations are indicated above each graph. Production of A) BcpA-CT_K96243 (left panel) and BcpA-CT_1106A-2 (right panel), B) BcpA-CT_K96243 and BcpI_K96243 (left panel) and BcpA-CT_1106A-2 and BcpI_1106A-2 (right panel), and C) BcpA-CT_K96243 and BcpI_1106A-2 (left panel) and BcpA-CT_1106A-2 and BcpI_K96243 (right panel) were repressed with 0.2% glucose (red bars) or induced with 0.2% rhamnose (blue bars). Error bars represent the mean ± 1 SEM.

Figure 7. bcpAIOB-dependent competition does not occur in liquid medium.

A) Wild type E264Cm^R (WT) co-cultured with ΔbcpAIOB (Δ). B) Wild type E264Cm^R (WT) co-cultured with ΔbcpAIOB::bcpI _E264 (Δ::bcpI). C) Wild type E264::bcpI _E264 (WT::bcpI) co-cultured with ΔbcpAIOB (Δ). D) Constitutive E264P_S12-bcpAIOB (P_S12) co-cultured with ΔbcpAIOB (Δ). For all assays, strains were cultured separately overnight, diluted to OD₆₀₀ 0.2, mixed at a 1∶1 ratio, and co-cultured for 24 hours. Aliquots were taken at 0 hours and 24 hours, serially diluted, and plated with antibiotic selection to calculate the cfu/ml of each strain. Error bars represent the mean ± 1 SEM.

Figure 8. bcpAIOB-mediated contact dependent growth inhibition on solid medium.

A) Colony biofilms of indicated strains and competitions were observed for four days by microscopy. B) Samples from the center of each colony biofilm competition were taken on each day and plated with antibiotic selection to determine the competitive index (C.I.) of wild type E264Cm^R (WT) bacteria compared to ΔbcpAIOB (left panel), ΔbcpAIOB::bcpI _E264 (middle panel), and ΔbcpAIOB::bcpI _K96243 (right panel). C) Samples from a single location along the leading edge of each colony biofilm (as indicated by the red asterisks(*)) were plated as in B. In cases where only wild type bacteria were recovered, the actual C.I. is greater than or equal to the value stated.

Figure 9. Analysis of 24–hour time course of CDI-mediated competition.

A) Wild type E264Cm^R (WT) bacteria and ΔbcpAIOB mutant bacteria were co-incubated on solid medium. Samples were taken from the center (top panel) and edge (bottom panel) at the indicated times. The C.I. was determined as in Figure 8B. Red data points indicate only wild type bacteria were recovered, and the actual C.I. is therefore greater than or equal to the represented value. B) Microscopy of E264P_S12-rfp and E264ΔbcpAIOBP_S12-gfp mixed at a 1∶1 ratio in the center (left column) and edge (right column) of colony biofilms at the indicated times.

Figure 10. P_S12-bcpAIOB-mediated contact dependent growth inhibition.

E264P_S12-bcpAIOB (P_S12) co-cultured with ΔbcpAIOB (Δ) mutant bacteria at a 1∶1,000 ratio (left column) or a 1∶1 ratio (right column) on solid medium. The C.I. was determined for the center (top panel) and edge (bottom panel) of the colony biofilm as described in Figure 8B. Red data points indicate only wild type bacteria were recovered, and the actual C.I. is therefore greater than or equal to the represented value.

Figure 11. ΔbcpO-mediated contact dependent growth inhibition.

Cultures of bacteria were mixed at a 1∶1 ratio and colony biofilms were plated on solid LSLB agar. Samples were taken from the center (top panels) and leading edge (bottom panels) of the colony biofilms at day 1 and plated with antibiotic selection to determine the C.I. of ΔbcpO compared to ΔbcpAIOB (column I), ΔbcpO::bcpO _E264 compared to ΔbcpAIOB (column II), ΔbcpO compared to ΔbcpAIOB::bcpI _E264 (column III), and ΔbcpO compared to ΔbcpAIOB::bcpI _K96243 (column IV). Red data points indicate only wild type bacteria were recovered, and the actual C.I. is therefore greater than or equal to the represented value.