Consequences of a mobile genetic element integrated at secondary locations

Thesis (Ph. D.)--Massachusetts Institute of Technology, Microbiology Graduate Program, 2013.Cataloged from PDF version of thesis.Includes bibliographical references (pages 91-96).Integrative and conjugative elements (ICEs) are widespread mobile genetic elements that are integrated in bacterial chromosomes, but can excise and transfer to a recipient through conjugation. ICEs are important agents of evolution, contributing to the acquisition of new traits, including antibiotic resistance. Many ICEs are site-specific in that they integrate preferentially into a primary attachment site in the bacterial chromosome. Site-specific ICEs can integrate into secondary locations, but little is known about the consequences of integration. Using ICEBs1, a site-specific ICE from Bacillus subtilis, I found that integration into secondary attachment sites is detrimental to both ICEBs1 and the host cell. Integration at secondary locations is detrimental to ICEBsJ. Once integrated in the chromosome, excision of ICEBs1 from all secondary attachment sites analyzed was either reduced (4 sites) or undetectable (3 sites) compared to ICEBs1 excision from the primary site. Additionally, from two of the four secondary sites that exhibited reduced but detectable excision, the excised, circular form of ICEBs1 was present at lower levels than expected, indicating that circular ICEBs1 may be unstable. Defects in excision and stability of ICEBs] severely limit its ability to spread to other cells. Integration at secondary locations is detrimental to the host cell. Induction of ICEBs1 gene expression in secondary integration sites resulted in a defect in cell proliferation and/or viability, as well as induction of the SOS response. These effects are likely due to DNA damage resulting from plasmid-like, rolling-circle replication of the excision-defective ICEBs1 in the chromosome. Consistent with this model, deletion of ICEBs] replication genes (nicK and helP) alleviated the proliferation and viability defects. Implications for the evolution of ICEs. These previously unrecognized detrimental effects may provide selective pressure against propagation of ICEBs1 in secondary attachment sites. Such detrimental effects could explain the maintenance and prevalence of site-specific integration among ICEs.by Kayla L. Menard.Ph.D

Impact of Toxoplasma gondii Infection on Host Non-coding RNA Responses

As an intracellular microbe, Toxoplasma gondii must establish a highly intimate relationship with its host to ensure success as a parasite. Many studies over the last decade-and-a-half have highlighted how the host reshapes its immunoproteome to survive infection, and conversely how the parasite regulates host responses to ensure persistence. The role of host non-protein-coding RNA during infection is a vast and largely unexplored area of emerging interest. The potential importance of this facet of the host-parasite interaction is underscored by current estimates that as much as 80% of the host genome is transcribed into non-translated RNA. Here, we review the current state of knowledge with respect to two major classes of non-coding RNA, microRNA (miRNA) and long non-coding RNA (lncRNA), in the host response to T. gondii infection. These two classes of regulatory RNA are known to have profound and widespread effects on cell function. However, their impact on infection and immunity is not well-understood, particularly for the response to T. gondii. Nevertheless, numerous miRNAs have been identified that are upregulated by Toxoplasma, and emerging evidence suggests a functional role during infection. While the field of lncRNA is in its infancy, it is already clear that Toxoplasma is also a strong trigger for this class of regulatory RNA. Non-coding RNA responses induced by T. gondii are likely to be major determinants of the host's ability to resist infection and the parasite's ability to establish long-term latency

Selective Pressures to Maintain Attachment Site Specificity of Integrative and Conjugative Elements

Integrative and conjugative elements (ICEs) are widespread mobile genetic elements that are usually found integrated in bacterial chromosomes. They are important agents of evolution and contribute to the acquisition of new traits, including antibiotic resistances. ICEs can excise from the chromosome and transfer to recipients by conjugation. Many ICEs are site-specific in that they integrate preferentially into a primary attachment site in the bacterial genome. Site-specific ICEs can also integrate into secondary locations, particularly if the primary site is absent. However, little is known about the consequences of integration of ICEs into alternative attachment sites or what drives the apparent maintenance and prevalence of the many ICEs that use a single attachment site. Using ICEBs1, a site-specific ICE from Bacillus subtilis that integrates into a tRNA gene, we found that integration into secondary sites was detrimental to both ICEBs1 and the host cell. Excision of ICEBs1 from secondary sites was impaired either partially or completely, limiting the spread of ICEBs1. Furthermore, induction of ICEBs1 gene expression caused a substantial drop in proliferation and cell viability within three hours. This drop was dependent on rolling circle replication of ICEBs1 that was unable to excise from the chromosome. Together, these detrimental effects provide selective pressure against the survival and dissemination of ICEs that have integrated into alternative sites and may explain the maintenance of site-specific integration for many ICEs.United States. Public Health Service (Grant GM050895

Cartoon of repeated rolling-circle replication from the ICE<i>Bs1 oriT</i> that is stuck in the chromosome.

<p>Rolling circle replication is induced in ICE<i>Bs1</i> insertions that are unable to excise from the chromosome. During this replication, the ICE<i>Bs1</i> relaxase NicK (black circles) nicks a site in <i>oriT</i>, the origin of transfer (gray bar) that also functions as an origin of replication <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003623#pgen.1003623-Lee3" target="_blank">[15]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003623#pgen.1003623-Lee4" target="_blank">[23]</a>. NicK presumably becomes covalently attached to the 5′ end of the nicked DNA. Replication extends (dotted line with arrow) from the free 3′-end, and regenerates a functional <i>oriT</i> that is a substrate for another molecule of NicK. The only other ICE<i>Bs1</i> product needed for ICE<i>Bs1</i> replication is the helicase processivity factor HelP <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003623#pgen.1003623-Thomas1" target="_blank">[19]</a>. The rest of the replication machinery (not shown) is composed of host-encoded proteins.</p

Primers used.

1<p>sequences are indicated 5′ to 3′.</p>2<p>the relevant location of each primer is indicated, along with how the primer was used. Primers to chromosomal regions are usually near the site of integration of ICE<i>Bs1</i>. The position, 5′ or 3′, in the indicated gene is relative to the direction of transcription of that gene, 5′ indicating extension in the same and 3′ indicating extension in the opposite direction as transcription. Left and right ends of ICE<i>Bs1</i> are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003623#pgen-1003623-g001" target="_blank">Figure 1</a>.</p

Induction of the SOS response.

<p>The ß-galactosidase specific activities from the SOS transcriptional reporter fusion <i>dinC</i>-<i>lacZ</i> in strains with ICE<i>Bs1</i> in the indicated secondary attachment sites are presented. Strains were grown as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003623#pgen-1003623-g004" target="_blank">Figure 4</a> and samples for ß-galactosidase assays were taken 3 hours after induction of ICE<i>Bs1</i> gene expression. Data presented are the averages of two biological replicates (four for Δ<i>attR</i> strain KM392)). For all of the strains with insertions in secondary attachment sites, the values from the biological replicates were within 20% of the average. Strains used include: wt, <i>attB</i>::ICE<i>Bs1</i> (KM390); <i>ykrP</i>::ICE<i>Bs1</i> (KM402); <i>mmsA</i>::ICE<i>Bs1</i> (KM394); <i>attB</i>::ICE<i>Bs1</i> Δ<i>attR</i>::<i>tet</i> (KM392); <i>srfAA</i>::ICE<i>Bs1</i> (KM400); <i>yvbT</i>::ICE<i>Bs1</i> (KM396); <i>yrkM</i>::ICE<i>Bs1</i> (KM404).</p

Excision of ICE<i>Bs1</i> from secondary attachment sites.

<p><b>A–B.</b> Excision frequencies and relative amounts of the excision products (circular ICE<i>Bs1</i> and empty chromosomal site) were determined as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003623#s4" target="_blank">Materials and Methods</a>. Cells were grown in defined minimal medium with arabinose as carbon source. Products from excision were determined two hours after addition of xylose to induce expression of Pxyl-<i>rapI</i> to cause induction of ICE<i>Bs1</i> gene expression. Primers for qPCR were unique to each attachment site. Strains used include: wt, that is, ICE<i>Bs1</i> inserted in <i>attB</i> (CAL874); <i>ΔattR</i>, ICE<i>Bs1</i> integrated in <i>attB</i>, but with the right attachment site deleted and ICE<i>Bs1</i> unable to excise (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003623#pgen-1003623-g001" target="_blank">Figure 1</a>) (CAL872); <i>mmsA</i>::ICE<i>Bs1</i> (KM70); <i>yrkM</i>::ICE<i>Bs1</i> (KM72); <i>srfAA</i>::ICE<i>Bs1</i> (KM141); <i>yycJ</i>::ICE<i>Bs1</i> (KM132); <i>ykrP</i>::ICE<i>Bs1</i> (KM77); <i>spoVD</i>::ICE<i>Bs1</i> (KM130); <i>yvbT</i>::ICE<i>Bs1</i> (KM94). Each strain was assayed at least three times (biological replicates) and qPCR was done in triplicate on each sample. Error bars represent standard deviation. <b>A.</b> Frequency of excision of ICE<i>Bs1</i> from the indicated site of integration. The relative amount of the empty chromosomal attachment site was determined and normalized to the chromosomal gene <i>cotF</i>. Data were also normalized to a strain with no ICE<i>Bs1</i> (JMA222), which represents 100% excision. <b>B.</b> Relative amount of circular ICE<i>Bs1</i> compared to the amount of empty chromosomal attachment site for the indicated insertions. The relative amount of the ICE<i>Bs1</i> circle, normalized to <i>cotF</i>, was divided by the relative amount of the empty attachment site, also normalized to <i>cotF</i>. These ratios were then normalized to those for wild type. <b>C.</b> Cartoon of integration of ICE<i>Bs1</i> into its primary bacterial attachment site <i>attB</i>. <i>attB</i> is identical to the attachment site on ICE<i>Bs1</i>, <i>att</i>ICE<i>Bs1</i>. They consist of a 17 bp region with 5 bp inverted repeats (gray boxes) on each side of a 7 bp spacer region (white box). During integration and excision, a recombination event occurs in the 7 bp spacer (crossover) region <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003623#pgen.1003623-Glaser1" target="_blank">[38]</a>. <b>D.</b> Cartoon of integration of ICE<i>Bs1</i> into secondary integration sites. A secondary integration site is indicated with a black box. When ICE<i>Bs1</i> integrates into a secondary site, the crossover regions in <i>att</i>ICE<i>Bs1</i> and that of the secondary site are not necessarily identical, potentially creating a mismatch. This mismatch, if not repaired, will be resolved by host replication, generating left and right ends with different crossover sequences. Excision would then create a circular ICE<i>Bs1</i> with a heteroduplex in the attachment site on ICE<i>Bs1</i>.</p

Map and DNA sequence of the primary and 15 secondary integration sites for ICE<i>Bs1</i>.

<p><b>A.</b> Approximate position of the primary and 15 secondary ICE<i>Bs1</i> integration sites on the <i>B. subtilis</i> chromosome. The circle represents the <i>B. subtilis</i> chromosome with the origin of replication (<i>oriC</i>) indicated by the black rectangle at the top. The slash marks represent the approximate location of the ICE<i>Bs1</i> insertion site. The name of the gene near which (<i>ygxA</i>) or into which (all other locations) ICE<i>Bs1</i> inserted is indicated on the outside of the circle. The arrows on the inside of the circle indicate the direction of ICE<i>Bs1</i> replication for each insertion. <i>trnS-leu2</i> (in bold) contains the primary ICE<i>Bs1</i> integration site <i>attB</i>. <b>B.</b> DNA sequence of the primary and 15 secondary integration sites. The gene name is indicated on the left, followed by the DNA sequence (chromosomal target). The primary attachment site (<i>attB</i>) is a 17 bp sequence with 5 bp inverted repeats (underlined) separated by a 7 bp spacer. Mismatches from <i>attB</i> are indicated in bold, capital letters. “mm” indicates the number of mismatches from the primary 17 bp <i>attB</i>. “occurrences” indicates the number of independent times an insertion in each site was identified. Percentages of the total (27) are indicated in parenthesis. The * next to <i>yqhG</i> indicates that two different ICE<i>Bs1</i> insertions were isolated in this gene, once in each orientation. <b>C.</b> Sequence logo of the ICE<i>Bs1</i> secondary attachment sites. Using Weblogo 3.3 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003623#pgen.1003623-Crooks1" target="_blank">[21]</a>, we generated a consensus motif of the 26 bases surrounding the insertion site of the 15 secondary insertion sites for ICE<i>Bs1</i>. For comparison, the primary attachment site for ICE<i>Bs1</i> is a 17 bp region with 5 bp inverted repeats and a 7 bp spacer region in the middle <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003623#pgen.1003623-Lee2" target="_blank">[10]</a>. The size of each nucleotide corresponds to the frequency with which that nucleotide was observed in that position in the secondary attachment sites.</p

Effects of induction of ICE<i>Bs1</i> gene expression on cell viability.

<p>The effects of induction of ICE<i>Bs1</i> gene expression on cell viability are shown for the indicated insertions and their derivatives. Cells were grown in defined minimal medium with arabinose to early exponential phase (OD600∼0.05) and xylose was added to induce expression of Pxyl-<i>rapI</i>, causing induction of ICE<i>Bs1</i> gene expression. The number of colony forming units was measured three hours after induction and compared to cells grown in the absence of xylose (uninduced). All experiments were done at least three times, except for the <i>helP</i> mutants (panel C), which were done twice with similar results. Data presented are averages of the replicates. Error bars represent the standard deviation of at least three replicates. <b>A.</b> Drop in viability of strains in which excision of ICE<i>Bs1</i> is defective. Strains used include: wt, that is, <i>attB</i>::ICE<i>Bs1</i> (CAL874); <i>attB</i>::ICE<i>Bs1</i> Δ<i>attR</i>::<i>tet</i> (CAL872); <i>mmsA</i>::ICE<i>Bs1</i> (KM70); <i>srfAA</i>::ICE<i>Bs1</i> (KM141); <i>yycJ</i>::ICE<i>Bs1</i> (KM132); <i>ykrP</i>::ICE<i>Bs1</i> (KM77); <i>yrkM</i>::ICE<i>Bs1</i> (KM72); <i>spoVD</i>::ICE<i>Bs1</i> (KM130); <i>yvbT</i>::ICE<i>Bs1</i> (KM94). <b>B.</b> Data are shown for two secondary insertion sites (<i>mmsA</i>::ICE<i>Bs1</i> and <i>yvbT</i>::ICE<i>Bs1</i>). Similar results were obtained with <i>ykrP</i>::ICE<i>Bs1</i> and <i>srfAA</i>::ICE<i>Bs1</i> (data not shown). Derivatives of each insertion that delete <i>nicK</i> and all downstream ICE<i>Bs1</i> genes (Δ<i>nicK-yddM</i>) or that leave <i>nicK</i> intact and delete just the downstream genes (Δ<i>ydcS-yddM</i>) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003623#pgen-1003623-g001" target="_blank">Figure 1</a>) were tested. Strains used include: <i>mmsA</i>::ICE<i>Bs1</i> (KM70); <i>mmsA</i>::{ICE<i>Bs1</i> Δ(<i>nicK-yddM</i>)::<i>cat</i>} (KM366); <i>mmsA</i>::{ICE<i>Bs1</i> Δ(<i>ydcS-yddM</i>)::<i>cat</i>} (KM358); <i>yvbT</i>::ICE<i>Bs1</i> (KM94); <i>yvbT</i>::{ICE<i>Bs1</i> Δ(<i>nicK-yddM</i>)::<i>cat</i>} (KM369); <i>yvbT</i>::{ICE<i>Bs1</i> Δ(<i>ydcS-yddM</i>)::<i>cat</i>} (KM362). Data for KM70 and KM94 are the same as those shown above in panel A and are shown here for comparison. <b>C.</b> The ICE<i>Bs1</i> helicase processivity protein encoded by <i>helP</i> is required for cell killing by ICE<i>Bs1</i>. Data are shown for two secondary integration sites (<i>ykrP</i> and <i>yvbT</i>) and the excision defective ICE<i>Bs1</i> Δ<i>attR</i>. The <i>helP</i> allele is a non-polar deletion <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003623#pgen.1003623-Thomas1" target="_blank">[19]</a>. Strains used include: <i>attB</i>::(ICE<i>Bs1</i> Δ<i>attR</i>::<i>tet</i>) (CAL872); <i>attB</i>::(ICE<i>Bs1</i> Δ<i>helP</i> Δ<i>attR</i>::<i>tet</i>) (KM437); <i>ykrP</i>::ICE<i>Bs1</i> (KM77); <i>ykrP</i>::(ICE<i>Bs1</i> Δ<i>helP</i>) (KM429); <i>yvbT</i>::ICE<i>Bs1</i> (KM94); <i>yvbT</i>::(ICE<i>Bs1</i> Δ<i>helP</i>) (KM459). Data for KM94, KM77, and CAL872 are the same as those shown above in panel A and are shown here for comparison.</p

Map of ICE<i>Bs1</i> and its derivatives.

<p><b>A.</b> The linear genetic map of ICE<i>Bs1</i> integrated in the chromosome. Open arrows indicate open reading frames and the direction of transcription. Gene names are indicated above or below the arrows. The origin of transfer (<i>oriT</i>) is indicated by a thick black line overlapping the 3′ end of <i>conQ</i> and the 5′ end of <i>nicK</i>. <i>oriT</i> functions as both the ICE<i>Bs1</i> origin of transfer and origin of replication <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003623#pgen.1003623-Lee3" target="_blank">[15]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003623#pgen.1003623-Lee4" target="_blank">[23]</a>. The thin black arrow indicates the direction of ICE<i>Bs1</i> rolling-circle replication. The small rectangles at the ends of ICE<i>Bs1</i> represent the 60 bp direct repeats that contain the site-specific recombination sites in the left and right attachment sites, <i>attL</i> and <i>attR</i>, that are required for excision of the element from the chromosome. <b>B–F.</b> Various deletions of ICE<i>Bs1</i> were used in this study. Thin horizontal lines represent regions of ICE<i>Bs1</i> that are present and gaps represent regions that are deleted. Antibiotic resistance cassettes that are inserted are not shown for simplicity. <b>B. </b><i>rapI</i> and <i>phrI</i> are deleted and a kanamycin resistance cassette inserted. <b>C.</b> The right attachment site (<i>attR</i>) is deleted and a tetracycline resistance cassette inserted. <b>D.</b> The genes from the 5′ end of <i>nicK</i> and into <i>yddM</i> are deleted and a chloramphenicol resistance cassette inserted. <b>E.</b> The genes from the 5′ end of <i>ydcS</i> and into <i>yddM</i> are deleted and a chloramphenicol resistance cassette inserted. <b>F.</b> The entire coding sequence of <i>helP</i> (previously known as <i>ydcP</i>) and 35 bp in the <i>helP</i>-<i>ydcQ</i> intergenic region is removed. There is no antibiotic resistance cassette in this construct.</p