PMC

(A–C) B. thai and P. putida growth following inoculation of competitive cultures (A, B) or mono-cultures (C) onto LB 3% w/v agar. (D, E) B. thai and P. putida growth following inoculation of competitive cultures into LB broth. (F) Quantification of dead cells 7.5 hours after initiating competition between P. putida and the indicated B. thai strain on LB 3% w/v agar (n≥7,000). Error bars are ± SD.

Fluorescence confocal microscopy images of B. thai (green) and P. putida (cyan) biofilm formation in flow chambers. (A) Representative images of monotypic B. thai biofilms of the indicated strains immediately following seeding (Day 0) and after four days of maturation. (B) Representative images of mixed biofilms seeded with a 1∶1 mixture of P. putida with the indicated B. thai strains.

(A) Growth competition assays between the indicated GFP-labeled B. thai strains and the TDCs. Standard light photographs and fluorescent images of the competition assays are shown. (B) Fluorescence images of GFP-labeled B. thai wild-type and ΔT6SS-1 grown in the presence of the TDCs with (no contact, NC) or without (contact, C) an intervening filter. (C) Fluorescence images of growth competition assays between GFP-labeled B. thai ΔclpV-1 or complemented ΔclpV-1 with the TDCs. (D) Quantification of c.f.u before (initial) and after (final) growth competition assays between the indicated organisms. The c.f.u. ratio of the B. thai strain versus competitor bacteria is plotted. Error bars represent ± SD.

Mice were exposed to 10⁵ c.f.u./lung aerosolized B. thai wild-type or ΔtssK-5 bacteria and c.f.u. were monitored in the (A) lung after 4, 24, and 48 h (n = 6 per time point), and in the (B) liver and spleen after 24 and 48 h (n = 6 per time point). (C) C57BL/6 wild-type (n = 6) and MyD88^−/− mice (n = 7) were infected with the ΔtssK-5 strain and survival was monitored for 14 days. Error bars in (A) and (B) are ± SD.

C57BL/6 wild-type mice were infected by the aerosol-route with 10⁵ c.f.u./lung of B. thai strains and monitored for survival for 10–14 days post infection (p.i.). Survival of mice after exposure to B. thai (A) wild-type and strains harboring gene deletions in individual T6SS gene clusters (n = 5 per group), (B) wild-type and a strain bearing an in-frame tssK-5 deletion (ΔtssK-5) or its complemented derivative (ΔtssK-5-comp; n = 7, 7 and 8, respectively), (C) or a strain with inactivating mutations in T6SS-5 or in four T6SSs (ΔT6SS-1,2,4,6; n = 6 and 8, respectively).

(A) Overview of the B. thai T6SS gene clusters. Burkholderia T6SS-3 is absent from B.thai. Genes were identified according to the nomenclature proposed by Shalom and colleagues : tss, type six secretion conserved genes; tag, type six secretion-associated genes variably present in T6SSs. Genes are colored according to function and conservation (dark grey, tss genes; light grey, tag genes; color, experimentally characterized tss or tag genes; white, genes so far not linked to T6S). Brackets demarcate genes that were deleted in order to generate B. thai strains ΔT6SS-1, -2, -4 -5 and -6 and their assorted combinations. Locus tag numbers are based on B. thai E264 genome annotations. (B) Neighbor-joining tree based on 334 T6S-associated VipA orthologs. The locations of VipA proteins from T6SSs discussed in the text are indicated. Each line represents one or more orthologous T6SSs from a single species. Lines are colored based on bacterial taxonomy of the corresponding organism. Indicated bootstrap values correspond to 100 replicates. This phylogeny is available in expanded format in . A key for the coloring scheme is also present in .

(A) In vitro growth of B. thai wild-type and a strain bearing gene deletions in all five T6SSs (ΔT6S). The data presented are an average of three replicates. (error bars smaller than symbols). (B) B. thai wild-type and ΔT6S swimming motility in semi-solid LB agar (scale bar = 1.0 cm). (C) Fluorescence images of growth competition assays between GFP-labeled B. thai wild-type and ΔT6S strains against the indicated unlabeled competitor species. Competition assay outcomes could be divided into T6S-independent (AR, Agrobacterium rhizogenes; ATu, A. tumefaciens; AV, A. vitis; PD, Paracoccus denitrificans; RS, Rhodobacter sphaeroides; ATe, Acidovorax temperans; BT, B. thailandensis; BV, B. vietnamiensis; AC, Acinetobacter calcoaceticus; AH, Aeromonas hydrophila; PAt, Pectobacterium atrosepticum; FN, Francisella novicida; PAe, Pseudomonas aeruginosa; SM, Serratia marcescens; VC, Vibrio cholerae; VP, Vibrio parahaemolyticus; VV, V. vulnificus; XC, Xanthomonas campestris; XN, Xenorhabdus nematophilus; YP, Yersinia pestis LCR^–; BC, Bacillus cereus; BS, B. subtilis; ML, Micrococcus luteus; SA, Staphylococcus aureus; SP, Streptococcus pyogenes), those with modest T6S-effects (BA, B. ambifaria; EC, E. coli; KP, Klebsiella pneumoniae; ST, Salmonella typhimurium) and those in which B. thai proliferation was strongly T6S-dependent (dashed boxes – PP, P. putida E0044; PF, P. fluorescens ATCC27663; SP, S . proteamaculans 568). This latter group of organisms is referred to as the T6S-dependent competitors (TDCs).