60 research outputs found

    5′RACE analysis of <i>mosA</i> transcription start site.

    No full text
    <p>A) Schematic of <i>mosA</i> transcription start site based on the 5′ RACE results shown in B). The +1 refers to the start of transcription as defined by the DNA sequence of the 5′RACE product obtained using primer A (small black arrow). The length of the PCR product is indicated. Black arrows represent ORFs as predicted using bioinformatics; the previously annotated <i>s052</i> start codon is indicated. B) Shows a 1% agarose gel of the product of 5′RACE reaction using primer A.</p

    Schematic of a positively selectable reporter of SXT loss.

    No full text
    <p>SXT containing <i>lacI<sup>Q</sup></i> was introduced into a <i>lacI E. coli</i> host containing Plac<i>-aad7</i> (a spectinomycin-resistance gene). If SXT (and <i>lacI<sup>Q</sup></i>) is lost, the cells become Spec<sup>R</sup>. The black diamond represents the site of insertion for a fragment containing <i>lacI<sup>Q</sup></i> (pFRTIq), thick black arrows represent ORFs, and the thin black line indicates the approximate location of the Δ9 deletion. Abbreviations: <i>tet</i> = tetracycline resistance gene, <i>kan</i> = kanamycin resistance gene, <i>int</i> = integrase, <i>prfC</i> = site of SXT insertion. Figure not to scale.</p

    Influence of Xis and SetCD on <i>mosA</i> expression.

    No full text
    <p>A) Schematic of chromosomal <i>mosA</i>:: <i>lacZ</i> transcription reporter within the <i>mosAT</i> locus. Thick black arrows represent ORFs as predicted by bioinformatics and the thin arrow represents the predicted location of the <i>mosA</i> promoter. B, D) β-galactosidase activities of the chromosomal <i>mosA::lacZ</i> fusion in CAG18439 containing wt SXT (B) or in CAG18439 containing Δint SXT (D) along with the indicating expression vectors. C) β-galactasidase activities from a plasmid-borne <i>mosA-lacZ</i> fusion (in pPmosA) in the indicated strains. All β-galactasidase measurements were conducted on 15 hr cultures and the results shown are the means and standard deviation from at least 9 independent cultures.</p

    Conjugation does not increase SXT loss.

    No full text
    1<p>SXTwt is strain RF146.</p>2<p>Loss was calculated as indicated in materials and methods.</p>3<p>Exconjugant formation was calculated as the number of exconjugant cfu/number of donor cfu. The data is from a representative experiment.</p

    Influence of genetic factors on the frequency of SXT loss.

    No full text
    <p>The frequency of SXT loss was calculated as the ratio of Spec<sup>R</sup>Cm<sup>S</sup> cfu / total cfu after 15 hour of growth. A) Factors that influence the frequency of SXT excision alter the frequency of SXT loss. The black diamonds indicate that the result is statistically different (p<0.05) than the result shown in the first column for the wild type reporter. The * signifies that the result was below the limit of detection which was ∼1×10<sup>−8</sup>. pXis and pXis-R harbor arabinose-inducible <i>xis</i> or its reverse complement respectively. B) SXT loss is not accompanied by heritable changes that influence maintenance of SXT. The ‘current SXT genotype’ refers to the ICE introduced into the host that lost the ICE designated as ‘previous SXT genotype’.</p

    Summarized deletions tested for SXT stability.

    No full text
    1<p>N, no elevation in the frequency of loss of the mutant SXT vs wt SXT; Y indicates at least an ∼5-fold increase in loss relative to wild type SXT.</p

    Strains and plasmids used in this study.

    No full text
    <p>Strains and plasmids used in this study.</p

    Genetic analysis of loss of <i>mosT</i> SXT.

    No full text
    <p>A) Schematic of the region deleted from Δ9 SXT. Black arrows represent ORFs, thin black arrows represent the deletions studied below. B) Frequency of loss of the indicated mutant SXT. C) Influence of <i>mosT</i> expression <i>in trans</i> on the stability of <i>mosT</i> SXT. All cultures were grown in the presence of 0.02% arabinose for 15 hr. Black diamond represents a statistically significant (p<.05) result compared to loss in the presence of pBAD33. D) Loss of <i>mosT</i> SXT is influenced by factors affecting SXT excision. All cultures were grown for 15 hr except as noted. Black diamonds represent a statistically significant (p<0.05) result compared to the <i>mosT</i> SXT grown for 15 hr. The * signifies that the result was below the limit of detection which was ∼1×10<sup>−8</sup>. <sup>a</sup>Loss was calculated following 3 hours of growth in an early log phase culture.</p

    <i>V</i>. <i>cholerae</i> endopeptidase are required to prevent lysis in response to inhibition of cell wall synthesis.

    No full text
    <p>(A) Wild type <i>V</i>. <i>cholerae</i> and a Δendo (Δ<i>shyABC</i> Δ<i>nlpC</i> Δ<i>tagE1</i> Δ<i>tagE2</i> P<sub>tac</sub>:<i>shyA</i>) mutant were grown in the presence or absence of 200 μM IPTG for 1.5 h (= T0) and then exposed to pen G. Cfu/ml at the indicated time points were normalized to cfu/ml at T0 and shown as fold change in viability. Data are mean of two biological replicates; error bars represent standard deviation. (B) Representative images of <i>V</i>. <i>cholerae</i> Δendo cells at different time points after exposure to penicillin G from the experiment shown in (A). (C) Time lapse images of Δendo cells initially grown for 1.5 h in either the presence or absence of IPTG (25 μM) and subsequently imaged on agarose pads containing 100 μg/ml pen G. The pad for ShyA+ cells also contained IPTG. The control panel depicts cells applied to an agarose pad containing neither antibiotic nor IPTG. Frames in the lower (control) panel are 10 min apart; in the other panels, frames are 5 min apart. Scale bar = 2 μm.</p

    Inhibition of cell wall synthesis in other bacteria does not always lead to cell lysis.

    No full text
    <p>(A) Influence of <i>shyA</i> or <i>yebA</i> overexpression on viability of <i>E</i>. <i>coli</i> EDL933 treated with meropenem. EDL933 carrying either pBAD33 (plasmid vector) or its derivatives encoding <i>yebA</i> or <i>shyA</i> was grown to exponential phase in the presence of arabinose, then meropenem (1 μg/ml) was added at (T0) and viable cell counts determined at the indicated times. Data are average of three independent experiments; error bars represent standard deviation. (B) Representative images from the 3 h time point of an experiment similar to the one depicted in (A), with or without the addition of 10 mM Mg<sup>2+</sup> in the growth medium. (C) Time lapse images of <i>P</i>. <i>aeruginosa</i>, <i>A</i>. <i>baumannii</i>, <i>V</i>. <i>cholerae</i> and EDL933 cells exposed to meropenem. Cells were grown in LB until OD<sub>600</sub> ~ 0.5 and then applied to an agarose pad (10% LB/PBS) containing meropenem (4 μg/ml) as well as MgSO<sub>4</sub> (10 mM)/CaCl<sub>2</sub> (1 mM) for <i>P</i>. <i>aeruginosa</i> and <i>A</i>. <i>baumannii</i> only. Frames are 9 min apart, scale bar = 5 μm. No AB = no antibiotic added.</p
    corecore