Mice infected with the <i>bbfA</i> mutant demonstrated a delay to death in comparison to those challenged with the parental strain.

<p>Groups of six BALB/c mice were challenged via the intra-peritoneal route and the median time to death was determined by the Mantel-Cox log-rank test at 35 days post-infection. Mice inoculated with the <i>bbfA</i> mutant had a mean survival time of 3.5 days versus 2 days for those dosed with the wild-type (<i>p</i> = 0.03).</p

Strains, plasmids and cell lines used in this study.

<p>Strains, plasmids and cell lines used in this study.</p

The <i>bpss1439</i> mutant displayed reduced biofilm formation which was complemented by <i>in trans bpss1439</i> expression.

<p>a. The wild-type and <i>bpss1439</i> mutant were grown in LB under static conditions at 37°C for 48 hours. Biofilms were stained with 1% w/v crystal violet, solubilised and quantified using an optimal density reading at 595 nm. Results are plotted with standard deviation error bars from triplicate experiments each consisting of eight experimental replicates; the <i>p</i> value was calculated using a paired Student's T test. b. The trans-complemented <i>bpss1439</i> mutant (pME-<i>1439</i>), as well as the wild-type and <i>bpss1439</i> pME strain, were also assessed for biofilm production as described previously.</p

Comparison of the <i>bbfA</i> mutant with other reported biofilm reduced strains.

†<p>EPS =  exopolysaccharide.</p

The arrangement of the <i>bpss1439</i> operon.

<p>The <i>bpss1439</i> gene is located in an operon with two downstream genes, <i>bpss1442</i> and <i>bpss1443</i>; both which encode hypothetical proteins of no significant database hits. The closest orthologue to <i>bpss1439</i> is the upstream gene (<i>bpss1434</i>) encoding another unstudied predicted TAA.</p

The <i>bbfA</i> mutant demonstrated reduced adhesion and microcolony formation.

<p>A. The wild-type, <i>bbfA</i> mutant and trans-complemented bbfA mutant (pME-<i>1439</i>), along with the control wild-type and <i>bbfA</i> mutant strains harbouring pME, were grown in LB under static conditions at 37°C for 48 hours. Biofilms were stained for exopolysaccharide using the periodic acid-Schiff protocol and examined by light microscopy. B. The wild-type and <i>bbfA</i> mutant biofilms were also fixed with 2.5% v/v glutaldehyde and examined by scanning electron microscopy.</p

Autotransporters of <i>B</i>. <i>pseudomallei</i> and their published functions.

<p>‡ nomenclature used in this manuscript</p><p>^ name based on phenotypic characterisation</p><p>Autotransporters of <i>B</i>. <i>pseudomallei</i> and their published functions.</p

Net intracellular replication of the <i>B</i>. <i>pseudomallei</i> AT mutants in J774.2 macrophage-like cells.

<p>Viable intracellular bacteria were enumerated (output divided by input CFU) for the duplicate experimental samples of each of the AT mutants and the data was normalised against the wild-type parent strain. Data from five experiments was expressed as the mean and standard error of the mean; statistical significance was analysed using Student’s <i>t</i> test.</p

Complementation of the <i>bpaC</i> mutant serum sensitivity and intracellular survival phenotypes.

<p>a. Complementation of the <i>bpaC</i> mutant with a full-length copy of the <i>bpaC</i> gene expressed <i>in trans</i> by the inducible pME6032 vector (pME-<i>bpaC</i>) abrogated the serum sensitive phenotype seen for the <i>bpaC</i> mutant harbouring empty pME6032 vector (pME). Graphs show mean results with error bars displaying standard error of the mean; statistical significance was calculated using Student’s <i>t</i> test. b. Complementation of the <i>bpaC</i> mutant with a full-length copy of the <i>bpaC</i> gene expressed <i>in trans</i> by the inducible pME6032 vector (pME-<i>bpaC</i>) partially complemented the defect in net intracellular replication seen for the <i>bpaC</i> mutant harbouring pME6032 vector with a full-length copy of the <i>bbfA</i> gene (pME-<i>bbfA</i>). Graphs show normalised mean results with error bars displaying standard error of the mean; statistical significance was calculated using Student’s <i>t</i> test.</p

Virulence of <i>B</i>. <i>pseudomallei</i> autotransporter mutants in a murine model of melioidosis.

<p>N/D = not done</p><p>Virulence of <i>B</i>. <i>pseudomallei</i> autotransporter mutants in a murine model of melioidosis.</p