Regulation of the mobile genetic element ICEBs1 by a conserved repressor and anti-repressor

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010."February 2010." Cataloged from PDF version of thesis.Includes bibliographical references.The mobile genetic element ICEBs1 is an integrative and conjugative element (a conjugative transposon) found in the Bacillus subtilis chromosome. The SOS response and the RapI-Phrl sensory system activate ICEBsl gene expression, excision, and transfer by inactivating the ICEBsl repressor protein ImmR. Although ImmR is similar to many characterized phage repressors, we found that, unlike these repressors, inactivation of ImmR requires an ICEBslencoded anti-repressor ImmA (YdcM). Under ICEBsl-inducing conditions, ImmA cleaves ImmR at a specific site to induce the element. We found that changing the amount or the specific activity of ImmA can cause derepression of ICEBs1 without activation by RecA or RapI. We isolated and characterized mutations in immA (immAh) that cause derepression of ICEBsl gene expression in the absence of inducing signals. However, we also found that ImmA levels did not significantly change during activation by RapI, indicating that RapI-mediated induction is likely due to increased activity of ImmA. Therefore, we propose that RapI and RecA induce ICEBs1 by increasing its specific activity. Along with earlier observations, some ImmAh mutants highlighted the importance of ImmA's C-terminal sequence for regulation of ImmA protein levels. We demonstrated that GFP tagged with C-terminal residues of ImmA was less abundant in vivo than untagged GFP. We screened cells with mutations of ATP-dependent proteases for effects on ICEBsl expression, and found that ClpXP might play a role in regulating ImmA stability and ICEBs1 gene expression.(cont.) To learn more about the repressor, ImmR, we isolated and characterized mutants of immR (immR(ind-)) that attenuate induction of ICEBs1 gene expression under the normally inducing conditions of treatment with DNA damaging reagent and overproduction of RapI. All four identified immR(ind-) mutations fall within a stretch of 10 residues flanking the cleavage site, emphasizing the importance of this sequence for ImmR proteolysis and ICEBsl induction. To further characterize the C-terminal portion of ImmR, we demonstrated that it interacts with ImmA and with itself in yeast two-hybrid assays, indicating that this part of the protein likely functions in ImmR oligomerization and recognition of ImmR by ImmA. Homologs of ImmA and ImmR are found in many mobile genetic elements, so the mode of regulation by ImmA and ImmR may be conserved in various systems.by Baundauna Bose.Ph.D

Regulation of Horizontal Gene Transfer in Bacillus subtilis by Activation of a Conserved Site-Specific Protease▿

The mobile genetic element ICEBs1 is an integrative and conjugative element (a conjugative transposon) found in Bacillus subtilis. The RecA-dependent SOS response and the RapI-PhrI cell sensory system activate ICEBs1 gene expression by stimulating cleavage of ImmR, the ICEBs1 immunity repressor, by the protease ImmA. We found that increasing the amount of wild-type ImmA in vivo caused partial derepression of ICEBs1 gene expression. However, during RapI-mediated derepression of ICEBs1 gene expression, ImmA levels did not detectably increase, indicating that RapI likely activates the protease ImmA by increasing its specific activity. We also isolated and characterized mutations in immA (immAh) that cause partial derepression of ICEBs1 gene expression in the absence of inducing signals. We obtained two types of immAh mutations: one type caused increased amounts of the mutant proteins in vivo but no detectable effect on specific activity in vitro; the other type had no detectable effect on the amount of the mutant protein in vivo but caused increased specific activity of the protein (as measured in vitro). Together, these findings indicate that derepression of ICEBs1 gene expression is likely caused by an increase in the specific activity of ImmA. Homologs of ImmA and ImmR are found in many mobile genetic elements, so the mechanisms that regulate ImmA-mediated cleavage of ImmR may be widely conserved

Missense Mutations Allow a Sequence-Blind Mutant of SpoIIIE to Successfully Translocate Chromosomes during Sporulation.

SpoIIIE directionally pumps DNA across membranes during Bacillus subtilis sporulation and vegetative growth. The sequence-reading domain (γ domain) is required for directional DNA transport, and its deletion severely impairs sporulation. We selected suppressors of the spoIIIEΔγ sporulation defect. Unexpectedly, many suppressors were intragenic missense mutants, and some restore sporulation to near-wild-type levels. The mutant proteins are likely not more abundant, faster at translocating DNA, or sequence-sensitive, and rescue does not involve the SpoIIIE homolog SftA. Some mutants behave differently when co-expressed with spoIIIEΔγ, consistent with the idea that some, but not all, variants may form mixed oligomers. In full-length spoIIIE, these mutations do not affect sporulation, and yet the corresponding residues are rarely found in other SpoIIIE/FtsK family members. The suppressors do not rescue chromosome translocation defects during vegetative growth, indicating that the role of the γ domain cannot be fully replaced by these mutations. We present two models consistent with our findings: that the suppressors commit to transport in one arbitrarily-determined direction or delay spore development. It is surprising that missense mutations somehow rescue loss of an entire domain with a complex function, and this raises new questions about the mechanism by which SpoIIIE pumps DNA and the roles SpoIIIE plays in vivo

Intragenic <i>spoIIIEΔγ</i> suppressor mutations alter residues in the linker and motor domains.

<p>A. Positions of intragenic suppressor mutations are indicated on a schematic of the <i>spoIIIEΔγ</i> linear sequence. The γ domain is not shown, but is C-terminal to the β subdomain in full-length SpoIIIE. Numbers in parentheses indicate the number of times each mutation was isolated. Underlined mutations were identified only after the P492 codon was mutated from ccg to cct to lessen the chances of obtaining P492Q. In this study, SpoIIIE codons are numbered for the protein beginning with the sequence MSVAKKKRKS. This presumes an earlier translation start and thus the codons are numbered +2 relative to some annotations of SpoIIIE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148365#pone.0148365.ref050" target="_blank">50</a>]. (B-E). Positions of intragenic suppressor mutations are shown in a 3D-model of the SpoIIIE motor domain, obtained by threading the SpoIIIE sequence onto a FtsK crystal structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148365#pone.0148365.ref025" target="_blank">25</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148365#pone.0148365.ref044" target="_blank">44</a>]. B. Two of the six subunits of a SpoIIIE hexamer are shown. The Walker A and Walker B sites are shown in red and ADP is shown in brown. P319 (pink), A343 (orange), E347 (yellow), P492 (green), H493 (cyan), D586 (blue), and T617 (purple) are shown as space-filled residues. C. A343 (orange) and E347 (yellow) lie on the same face of a helix in the α domain. D. P319 (pink), P492 (green) and H493 (cyan) are near each other. E. D586 (blue) and T617 (purple) are near the Walker B motif (red) in the β domain.</p

Spore formation in various genetic backgrounds.

<p>Strains were induced to sporulate for 24–36 h in DSM medium, unsporulated cells were eliminated by heat-kill, and the number of spores was measured by plating for cfu. The average of at least 3 replicates is plotted. Error bars indicate one standard deviation in each direction. A. Intragenic mutants are partially or fully dominant to <i>spoIIIEΔγ</i>. Strains expressing both <i>spoIIIEΔγ</i> and an intragenic <i>spoIIIEΔγ</i> suppressor mutant exhibit sporulation efficiencies that are intermediate between those of strains with only the <i>spoIIIEΔγ</i> or the mutant allele, or that are similar to that of the mutant allele. All displayed strains express the indicated <i>spoIIIE</i> allele from the <i>spoIIIE</i> promoter at the ectopic locus <i>ycgO</i>. White bars indicate strains that also express <i>spoIIIEΔγ</i> from the <i>spoIIIE</i> promoter at the ectopic locus <i>yhdGH</i>. Asterisks mark pairs of sporulation efficiencies that were significantly different from each other by t-tests (p<0.05). Sporulation efficiencies are shown for <i>spoIIIEΔγ</i> (BOSE2042; BOSE2301) and 10 <i>spoIIIEΔγ</i> mutants: P260L (BOSE2286; BOSE2311), S264I (BOSE2540; BOSE3089), E312A (BOSE2121; BOSE2305), Y316D (BOSE2284; BOSE2309), P319S (BOSE2321; BOSE3083), A343V (BOSE2288; BOSE2313), E347G (BOSE2323; BOSE3085), P492Q (BOSE2120; BOSE2303), H493Y (BOSE2538; BOSE3087), and T617A (BOSE2123; BOSE2307). B. Missense mutations that suppress the <i>spoIIIEΔγ</i> phenotype do not affect the function of full-length SpoIIIE. Site-directed mutagenesis was used to introduce each indicated mutation into a <i>spoIIIE</i> allele that was then expressed under its native promoter at <i>ycgO</i> in <i>ΔspoIIIE</i> strains. Each mutant sporulated as well as cells with wild-type <i>spoIIIE</i> (bKM776). Seven mutants were tested: P260L (BOSE2298), E312A (BOSE2290), Y316D (BOSE2296), E347G (BOSE2325), P492Q (BOSE2294), D586N (BOSE3091), and T617A (BOSE2292). All eight sporulation efficiencies were not significantly different from each other by single factor ANOVA (p = 0.503).</p

Missense mutations in <i>spoIIIEΔγ</i> rescue sporulation and chromosome transport <i>in vivo</i>.

<p>A. Suppressor mutations rescue spore formation. Intragenic mutations identified by suppressor selection were remade in a <i>spoIIIEΔγ</i> allele by site-directed mutagenesis and expressed from the <i>spoIIIE</i> promoter at an ectopic locus (<i>ycgO</i>) in a <i>ΔspoIIIE</i> strain. Strains were induced to sporulate for 24–36 h in DSM medium, unsporulated cells were eliminated by heat-kill, and the number of spores was measured by plating for cfu. The number of heat-resistant spores per ml is indicated for strains harboring full-length (“f.l.”) <i>spoIIIE</i> (bKM776), <i>spoIIIEΔγ</i> (BOSE2042), and 11 <i>spoIIIEΔγ</i> mutants: P260L (BOSE2286), S264I (BOSE2540), E310K (BOSE2411), E312A (BOSE2121), Y316D (BOSE2284), P319S (BOSE2321), A343V (BOSE2288), E347G (BOSE2323), P492Q (BOSE2120), H493Y (BOSE2538), and T617A (BOSE2123). Each number is the average of at least 3 replicates. Error bars indicate one standard deviation in each direction. B. Suppressor mutations rescue chromosome transport <i>in vivo</i>. Sporulation was induced by resuspension and DNA transport was evaluated using a previously-established fluorescent microscopy assay [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148365#pone.0148365.ref012" target="_blank">12</a>]. <i>yfp</i> and <i>cfp</i> genes are expressed from a forespore-specific promoter (<i>PspoIIQ</i>). <i>yfp</i> is integrated near the origin (<i>yycR</i>), and its expression indicates that asymmetric septation is complete. <i>cfp</i> is integrated near the terminus (<i>pelB</i>), and its expression indicates that the terminus has been transported into the forespore. Percent of termini in forespores is the percent of YFP+ cells that are also CFP+. Data are shown for full-length (“f.l.”) <i>spoIIIE</i> (bBB128), <i>spoIIIEΔγ</i> (bBB412), and 5 <i>spoIIIEΔγ</i> mutants: P260L (BOSE2331), E312A (BOSE2201), A343V (BOSE2332), P492Q (BOSE2200), and T617A (BOSE2202). Each data point represents the average of ≥ 3 replicates, with ≥ 500 forespores scored for each.</p

Mutations that rescue the sporulation defect of <i>spoIIIEΔγ</i> strains do not rescue a vegetative translocation defect.

<p>Growth of strains in various concentrations of the replication-stress-inducing antibiotic novobiocin (nov) was evaluated. Cells were grown to mid-exponential phase in LB, and then diluted to OD₆₀₀ 0.0125 in LB containing 960 ng ml^-1 nov (A), 480 ng ml^-1 (B), or no nov (C). OD600 measurements are plotted versus hours after dilution. All strains harbor Δ<i>spoIIIE</i>::<i>neo</i>. As indicated, samples were from strains with no ectopic <i>spoIIIE</i> (bDR1066), <i>spoIIIE</i> (bKM776), <i>spoIIIEΔγ</i> (BOSE2042), or a <i>spoIIIEΔγ</i> mutant: P260L (BOSE2286), E312A (BOSE2121), Y316D (BOSE2284), A343V (BOSE2288), E347G (BOSE2323), P492Q (BOSE2120), T617A (BOSE2123). Representative data from one of at least two replicates are plotted.</p

SftA is not required for rescue of spore formation by <i>spoIIIEΔγ</i> intragenic suppressor mutations.

<p>Strains were induced to sporulate for 24–36 h in DSM medium, unsporulated cells were eliminated by heat-kill, and the number of spores was measured by plating for cfu. Sporulation levels are similar for <i>sftA+</i> and Δ<i>sftA</i> strains bearing <i>spoIIIEΔγ</i> mutant alleles: P260L (BOSE2286; BOSE2492), E312A (BOSE2121; BOSE2486), Y316D (BOSE2284; BOSE2490), P319S (BOSE2321; BOSE2496), A343V (BOSE2288; BOSE2494), E347G (BOSE2323; BOSE2498), P492Q (BOSE2120; BOSE2935), and T617A (BOSE2123; BOSE2488). Sporulation efficiencies for strains harboring full-length (“f.l.”) <i>spoIIIE</i> (bKM776) and <i>spoIIIEΔγ</i> (BOSE2042) are plotted for comparison. The average of at least 3 replicates is plotted. Error bars indicate one standard deviation in each direction.</p

Structural characterization of the late competence protein ComFB from Bacillus subtilis

Many bacteria take up DNA from their environment as part of the process of natural transformation. DNA uptake allows microorganisms to gain genetic diversity and can lead to the spread of antibiotic resistance or virulence genes within a microbial population. Development of genetic competence (Com) in Bacillus subtilis is a highly regulated process that culminates in expression of several late competence genes and formation of the DNA uptake apparatus. The late competence operon comF encodes a small protein of unknown function, ComFB. To gain insight into the function of ComFB, we determined its 3D structure via X-ray crystallography. ComFB is a dimer and each subunit consists of four α-helices connected by short loops and one extended β-strand-like stretch. Each subunit contains one zinc-binding site formed by four cysteines, which are unusually spaced in the primary sequence. Using structure- and bioinformatics-guided substitutions we analyzed the inter-subunit interface of the ComFB dimer. Based on these analyses, we conclude that ComFB is an obligate dimer. We also characterized ComFB in vivo and found that this protein is produced in competent cells and is localized to the cytosol. Consistent with previous reports, we showed that deletion of ComFB does not affect DNA uptake function. Combining our results, we conclude that ComFB is unlikely to be a part of the DNA uptake machinery under tested conditions and instead may have a regulatory function

Pervasive prophage recombination occurs during evolution of spore-forming <i>Bacilli</i>

Phages are the main source of within-species bacterial diversity and drivers of horizontal gene transfer, but we know little about the mechanisms that drive genetic diversity of these mobile genetic elements (MGEs). Recently, we showed that a sporulation selection regime promotes evolutionary changes within SPβ prophage of Bacillus subtilis, leading to direct antagonistic interactions within the population. Herein, we reveal that under a sporulation selection regime, SPβ recombines with low copy number phi3Ts phage DNA present within the B. subtilis population. Recombination results in a new prophage occupying a different integration site, as well as the spontaneous release of virulent phage hybrids. Analysis of Bacillus sp. strains suggests that SPβ and phi3T belong to a distinct cluster of unusually large phages inserted into sporulation-related genes that are equipped with a spore-related genetic arsenal. Comparison of Bacillus sp. genomes indicates that similar diversification of SPβ-like phages takes place in nature. Our work is a stepping stone toward empirical studies on phage evolution, and understanding the eco-evolutionary relationships between bacteria and their phages. By capturing the first steps of new phage evolution, we reveal striking relationship between survival strategy of bacteria and evolution of their phages