LctD is required for growth using L-lactate.

<p>Growth curves of wild-type <i>A. actinomycetemcomitans</i> and the <i>A. actinomycetemcomitans lctD</i> insertion mutant in chemically defined medium (CDM) containing 20 mM glucose or 20 mM L-lactate as the sole source of energy. Glucose-grown <i>A. actinomycetemcomitans</i> (♦), L-lactate-grown <i>A. actinomycetemcomitans</i> (▴), glucose-grown <i>A. actinomycetemcomitans lctD⁻</i> (▪), L-lactate-grown <i>A. actinomycetemcomitans lctD⁻</i> (•).</p

LctD-his₆ catalyzes oxidation of L-lactate, but not D-lactate, to pyruvate.

<p>HPLC chromatogram of LctD-his₆ in vitro enzyme reactions with (A) L-lactate as the substrate at time 0, (B) L-lactate as the substrate after 15 min, and (C) D-lactate as the substrate after 15 min. Pyruvate and D- and L-lactate were detected using a refractive index detector, and data are displayed as peak height (microvolts). Using commercially available standards, it was determined that pyruvate is detected at approximately 11.6 to 12.1 min and lactate (D and L) is detected at approximately 14.6 to 15.2 min. Representative data are shown for experiments that were performed in duplicate or triplicate.</p

Pyruvate is a poor inhibitor of LctD-his₆.

<p>The ability of pyruvate and oxalate to inhibit LctD-his₆ enzymatic activity was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007864#s4" target="_blank">Materials and Methods</a>. 50% inhibition of LctD-his₆ activity was observed with 10–50 mM pyruvate and 2.1 mM oxalate.</p

PTS substrate exclusion model.

<p>Lactate enters the cell through the lactate permease (LctP) and is converted to pyruvate by L-lactate dehydrogenase (LctD). Intracellular levels of pyruvate increase and prevent autophosphorylation of protein EI, thus inhibiting PTS-mediated carbohydrate transport. PEP is phosphoenolpyruvate.</p

Kinetic analysis of LctD-his₆.

<p>LctD-his₆ was incubated with L-lactate and enzymatic activity assessed by monitoring reduction of MTT as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007864#pone.0007864-Futai1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007864#pone.0007864-Futai2" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007864#pone.0007864-Kohn1" target="_blank">[17]</a>. (A) Enzymatic activity was assessed over time for multiple LctD-his₆ concentrations (4, 5, 8, 10, and 12 nM) in the presence of saturating substrate concentrations. For K_m calculations, 8 nM LctD-his₆ was used. Representative data are shown. (B) The LctD-his₆ K_m for lactate was calculated as 152±23 µM (average±standard deviation) by averaging values from quadruplicate experiments. Representative data are shown.</p

Purification of LctD-his₆.

<p>(A) SDS-PAGE analysis of LctD-his₆. LctD-his₆ was purified using a HisTrap nickel column and examined by SDS-PAGE and Coomassie staining. Lane designations above the gel are: (La), molecular weight ladder; (Ly), cell lysate; (W1), buffer A flow-through; (W2), buffer B with 0.15 M imidazole flow-through; (W3), buffer B with 0.5 M imidazole flow-through; (LctD), phosphate buffer-exchanged LctD-his₆. Numbers to the left of the SDS-PAGE gel represent size standards in kilodaltons. Phosphate buffer-exchanged LctD-his₆ was used for enzymatic activity studies. (B) Western blot analysis of purified LctD-his₆. Purified LctD-his₆ was separated on a 10% SDS-PAGE gel, transferred to nitrocellulose membrane, and detected using an anti-his₆ antibody and chemiluminescence. Image represents an overlay of a white light image and a chemiluminescent image.</p

Communal feeding illustrated.

<p>Three dragons are feeding on a wild pig. The large prey size of Komodo dragons with overlapping lizard home range generally precludes a single lizard from consuming its large prey alone. Multiple or shared feeding facilitates spread of infectious bacteria between lizard mouths. (Photo by Achmad Ariefiandy.)</p

Communal feeding by dragons.

<p>Incidence of single versus multiple Komodo dragons feeding on large prey (Timor deer, wild pigs, water buffalo, and a Hawksbill sea turtle). Based on 20 independent observations of Komodo dragon feedings noted during fieldwork activities between 2002–2009.</p

Iron and Fur regulate Dispersin B.

<p>(A) Structure of the <i>dspB</i> promoter. Gray, OxyR box; orange, Fur box; underlined, -35 and -10 regions; +1, transcriptional start site; bold, start codon. (B) <i>dspB</i> transcription in colony biofilms was measured using a <i>dspB</i> promoter-<i>lacZ</i> transcriptional fusion. Left panel: blue, <i>A</i>. <i>actinomycetemcomitans</i> strain 624; red, <i>A</i>. <i>actinomycetemcomitans</i> strain VT1169. Right panel: blue, <i>A</i>. <i>actinomycetemcomitans</i> strain 624 wild type (wt); red, <i>A</i>. <i>actinomycetemcomitans</i> strain 624 <i>Δfur</i> (<i>Δfur</i>). Chelator is 250 μM 2,2’-dipyridyl, and Fe is 250 μM FeSO₄. Y axis is fold change (FC) in <i>dspB</i> expression relative to no chelator (-chelator) and no FeSO₄ (-Fe) addition. Error bars represent standard deviation (n = 3). (C) <i>dspB</i> mRNA levels in colony biofilms was measured using reverse transcriptase PCR in iron-replete (+Fe) and iron-restricted (-Fe) conditions. <i>clpX</i> serves as a control that is not regulated by iron or Fur. Wild type (wt), <i>Δfur</i> (<i>Δfur</i>), <i>Δfur</i> + <i>fur-vsv-g</i> (<i>Δfur</i> genetically complemented with VSV-G tagged Fur). (D) Biofilm dispersal assay. A second, higher ring biofilm (indicated by arrow) indicates dispersal. The purple stain is crystal violet. Chelator is 250 μM 2,2’-dipyridyl; -oxygen is anaerobic growth; +oxygen is aerobic growth.</p

Pursuit of an escaped prey by a second dragon.

<p>An injured Timor deer that has escaped an initial lizard has fled to the ocean and is being stalked by a different dragon. This second lizard succeeded in killing the deer. The Komodo dragon pictured is wearing a GPS collar used for tracking the animal. Occasional prey escape is essential to the operation of the lizard-lizard epidemic model. (Photo by Achmad Ariefiandy.)</p