Kinetics of transcript levels of <i>bont/A, botR/A, ntnh</i>, and <i>ha34</i> genes in Hall <i>botR/A</i> isogenic antisense strains.

<p>Transcript levels were quantified by qRT-PCR at 4, 6, 8, 10, 12, 18, 24, 30 and 48 h of culture and normalized versus housekeeping rpoB gene in Hall/pAT19 (empty vector), Hall/306 (antisense <i>botR/A</i> mRNA), and Hall/309 (<i>botR/A</i> overexpression). Data are mean values +/− SD from 9 independent experiments, *P<0.05 and **P<0.005.</p

BoNT/A production tested by mouse bioassay in 12 h culture supernatants of the control strain Hall/pAT19 and isogenic antisense strains.

<p>BoNT/A production tested by mouse bioassay in 12 h culture supernatants of the control strain Hall/pAT19 and isogenic antisense strains.</p

NTNH and HA34 production in Hall <i>botR/A</i> and 3 TCS isogenic antisense strains.

<p>Production of NTNH (A) and HA34 (B) was assayed by western blotting in 12 h culture supernatants of Hall/pAT19, Hall/306, Hall/309, Hall/707, Hall714, and Hall/1146. Western blots were quantified by ImageJ (B and D) with normalization of NTNH and HA34 production in Hall/pAT19 to 1. Data are mean values +/− SD from 3 independent experiments, *P<0.05 and **P<0.005.</p

BoNT/A production in isogenic antisense strains targeting the SHK gene from three TCSs.

<p>(A) Growth kinetics of Hall/707K, Hall/714K, and Hall/1146K were similar to that of the control Hall/pAT19 strain as monitored by OD₆₀₀ measurement. (B) BoNT/A production assayed by ELISA was significantly reduced in the culture supernatants of Hall/707K, Hall/714K, and to a higher extent of Hall/1146K. Data are mean values +/− SD from 3 independent experiments, *P<0.05 and **P<0.005.</p

Primers used to amplify internal fragments of the target genes by quantitative reverse transcriptase PCR.

<p>PCR efficiencies correspond to the optimized PCR parameters indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041848#s4" target="_blank">Materials and Methods</a> (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041848#pone.0041848.s001" target="_blank">Table S1</a>).</p><p>F, forward; R, reverse.</p

Kinetics of transcript levels of <i>bont/A, botR/A, ntnh</i>, and <i>ha34</i> genes in Hall/707, Hall/714, and Hall/1146 versus Hall/pAT19 and Hall/306.

<p>Transcript levels were quantified by qRT-PCR and normalized versus <i>rpoB</i> gene. Data are mean values +/− SD, from 9 independent experiments where *P<0.05 and **P<0.005.</p

Growth kinetics, BoNT/A, NTNH, and HA34 production in Hall <i>botR/A</i> isogenic antisense strains.

<p>(A) Growth curves of Hall/pAT19 (empty vector), Hall/306 (antisense <i>botR/A</i> mRNA), and Hall/309 (<i>botR/A</i> overexpression) were determined by measurement of optical density at 600 nm (OD₆₀₀). (B) BoNT/A was assayed in the culture supernatants by ELISA and expressed as OD at 490 nm. Production of NTNH (C) and HA34 (E) were determined in the supernatant of 12 h cultures by Western blotting with specific antibodies and quantification with ImageJ (D, F) (means +/− SD; n = 3), *P<0.05 and **P<0.005. Note that western blotting with anti-NTNH antibodies show native (130 kDa) and partially processed NTNH in its 115 kDa fragment.</p

Ultrastructural morphology of Hall/pAT19, Hall/1447, and Hall/1148.

<p>Bacteria from a 5 h culture in TGY were treated by high pressure and freeze substitution and then observed by electron microscopy. Hall/pAT19 (A) showed a well delineated wall, whereas in Hall/1147 (B) and Hall/1138 (C) the bacterial wall is transparent, almost invisible. In addition, Hall/1148 (C) showed numerous transparent inclusions. All scale bars represent 200 nm.</p

Growth kinetics and BoNT/A production in representative Hall isogenic antisense strains.

<p>(A) Hall/707, Hall/714, and Hall/1146 showed a similar growth curve monitored at OD₆₀₀ than the Hall/pAT19 strain. (B) Hall/1147 and Hall/1148 grew more rapidly than the control Hall/pAT19 strain, whereas Hall/1001 displayed a slower growth curve. Hall/652, Hall716, and Hall/723 show a similar kinetics compare to Hall/pAT19. (C) Monitoring of growth kinetics of Hall/pAT19, Hall/707 and Hall/1147 by bacterial counting on TGY agarose and expressed as unit forming colony (UFC)/ml. (D) BoNT/A production assayed by ELISA was significantly reduced in the culture supernatants of Hall/707, Hall/714, Hall/1146, Hall/1147, and Hall/748, versus the control Hall/pAT19 strain. BoNT/A production was significantly reduced in Hall/306 at 24 h culture. Data are mean values +/− SD from 3 independent experiments, *P<0.05.</p

Two-component system and orphan response regulator genes in <i>C. botulinum</i> strain Hall, homology with regulatory genes in other bacterial species, Hall isogenic antisense strains, and primers used for the construction of recombinant plasmids generating antisense mRNA.

a<p>In bold the accession number of the gene which has been targeted in the isogenic antisense strains and in bracket the associated gene of the corresponding TCS.</p>b<p>Protein identity >60% (<i>C. sporogenes</i> is omitted due to its high similarity to <i>C. botulinum</i>).</p