Crosslinking of φBT1 integrase and Gp3.

<p>Enzymes were incubated in the absence or presence of dimethyl suberimidate (DMS) and analyzed by SDS-PAGE (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080434#s2" target="_blank">Materials and Methods</a>). The positions of integrase (Int) monomer and dimer, Gp3 monomer and dimer, as well as possible interaction complexes are illustrated. The bands indicated by hollow arrows could be higher order oligomers. M, protein molecular weight markers.</p

DataSheet_2_Identifying and ranking causal microbial biomarkers for colorectal cancer at different cancer subsites and stages: a Mendelian randomization study.docx

IntroductionThe gut microbiome is directly involved in colorectal carcinogenesis, but much of the epidemiological evidence for the effect of the gut microbiome on colorectal cancer (CRC) risk comes from observational studies, and it is unclear whether identified microbial alterations are the cause or consequence of CRC development.MethodsUnivariate Mendelian randomization (MR) analysis and multivariate MR analysis based on Bayesian model averaging were performed to comprehensively explore the microbial risk factors associated with CRC. The Network Module Structure Shift method was used to identify microbial biomarkers associated with CRC. Mediation analysis was used to explore the dietary habits-microbiota-CRC pathway.ResultsThe results of the four methods showed that 9 bacteria had a robust causal relationship with the development of CRC. Among them, Streptococcus thermophilus reduced the risk of CRC; Eubacterium ventriosum and Streptococcus were beneficial bacteria of malignant tumors of colon (CC); Erysipelotrichaceae was a protective factor for malignant tumors of rectal (CR); Bacteroides ovatus was a risk factor for benign tumors. Finally, the mediation analysis revealed 10 pathways by which dietary regulation bacteria affected the risk of CRC, including alcohol consumption increased the risk of CC by reducing the abundance of Eubacterium ventriosum (mediated proportion: 43.044%), and the mediated proportion of other pathways was 7.026%-34.22%.DiscussionThese findings will contribute to the understanding of the different carcinogenic mechanisms of intestinal flora in the colon and rectum and the risk of tumor transformation, thereby aiding CRC prevention, early screening, and the development of future strategies to reduce CRC risk.</p

DataSheet_1_Identifying and ranking causal microbial biomarkers for colorectal cancer at different cancer subsites and stages: a Mendelian randomization study.docx

IntroductionThe gut microbiome is directly involved in colorectal carcinogenesis, but much of the epidemiological evidence for the effect of the gut microbiome on colorectal cancer (CRC) risk comes from observational studies, and it is unclear whether identified microbial alterations are the cause or consequence of CRC development.MethodsUnivariate Mendelian randomization (MR) analysis and multivariate MR analysis based on Bayesian model averaging were performed to comprehensively explore the microbial risk factors associated with CRC. The Network Module Structure Shift method was used to identify microbial biomarkers associated with CRC. Mediation analysis was used to explore the dietary habits-microbiota-CRC pathway.ResultsThe results of the four methods showed that 9 bacteria had a robust causal relationship with the development of CRC. Among them, Streptococcus thermophilus reduced the risk of CRC; Eubacterium ventriosum and Streptococcus were beneficial bacteria of malignant tumors of colon (CC); Erysipelotrichaceae was a protective factor for malignant tumors of rectal (CR); Bacteroides ovatus was a risk factor for benign tumors. Finally, the mediation analysis revealed 10 pathways by which dietary regulation bacteria affected the risk of CRC, including alcohol consumption increased the risk of CC by reducing the abundance of Eubacterium ventriosum (mediated proportion: 43.044%), and the mediated proportion of other pathways was 7.026%-34.22%.DiscussionThese findings will contribute to the understanding of the different carcinogenic mechanisms of intestinal flora in the colon and rectum and the risk of tumor transformation, thereby aiding CRC prevention, early screening, and the development of future strategies to reduce CRC risk.</p

Plaque assay of mutated phage φC31.

<p>(A) Schematic representation of <i>gp3</i> and surrounding phage genes. Plasmid-phages φXD101(X02) and φXD101(X03) were derivatives of φXD101 by PCR-targeting technology. <i>gp3</i> of φXD101 was replaced by Chloramphenicol resistant gene (<i>Chl^R</i>) in φXD101(X02), and further replaced by <i>gp3</i>-φBT1 and <i>Chl^R</i> in φXD101(X03). The construction of φXD101(X02) and φXD101(X03) are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080434#s2" target="_blank">Materials and Methods</a>. Primers X09 and X12 were used for identification of the derivatives, and the results are shown in Figure S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080434#pone.0080434.s001" target="_blank">File S1</a>. (B) Plaque assays of phage φXD101, φXD101(X02) and φXD101(X03). The three plasmids were transformed into <i>Streptomyces coelicolor</i> strain J1929 by conjugation from <i>E.coli</i> strain ET12567/pUZ8002 and selected on Apramycin agar; the positive clones were isolated to burst phages. The phage suspension was then plated onto soft agar with spores of indicator strain to yield plaques. Plates 1, 2 and 3 were prepared using suspensions from J1929 harbouring φXD101, φXD101(X02) and φXD101(X03), respectively.</p

Control of Directionality in <i>Streptomyces</i> Phage φBT1 Integrase-Mediated Site-Specific Recombination

<div><p><i>Streptomyces</i> phage φBT1 integrates its genome into the <i>attB</i> site of the host chromosome with the <i>attP</i> site to generate <i>attL</i> and <i>attR</i>. The φBT1 integrase belongs to the large serine recombinase subfamily which directly binds to target sites to initiate double strand breakage and exchange. A recombination directionality factor (RDF) is commonly required for switching integration to excision. Here we report the characterization of the RDF protein for φBT1 recombination. The RDF, is a phage-encoded <i>gp3</i> gene product (28 KDa), which allows efficient active excision between <i>attL</i> and <i>attR</i>, and inhibits integration between <i>attB</i> and <i>attP</i>; Gp3 can also catalyze topological relaxation with the integrase of supercoiled plasmids containing a single excision site. Further study showed that Gp3 could form a dimer and interact with the integrase whether it bound to the substrate or not. The synapse formation of <i>attL</i> or <i>attR</i> alone with integrase and Gp3 showed that synapsis did not discriminate between the two sites, indicating that complementarity of central dinucleotides is the sole determinant of outcome in correct excision synapses. Furthermore, both <i>in vitro</i> and <i>in vivo</i> evidence support that the RDFs of φBT1 and φC31 were fully exchangeable, despite the low amino acid sequence identity of the two integrases.</p></div

Substrates with palindromic central dinucleotides participate in <i>attL/attL</i> and <i>attR/attR</i> excision.

<p>(<b>A</b>) Excision reactions were performed using <i>attL</i> and <i>attR</i> with palindromic central dinucleotides. Plasmid pZLLR03 was digested with EcoRI and AgeI to generate linearized <i>attL^GC</i> (4948 bp) and <i>attR^GC</i> (1798 bp). The product sizes were predicted as 4532 bp for <i>BOB</i> and 5364 bp for <i>P′O P′</i>, 1262 bp for <i>POP</i> and 2334 bp for <i>B′OB′</i>, 3433 bp for <i>BOB′</i>(<i>attB</i>) and 3313 bp for <i>POP′</i>(<i>attP</i>), 2897 bp for <i>BOP</i> and 3849 bp for <i>B′OP′</i>. The positions of the substrates and series of products are indicated. The concentrations of proteins were 270 nM for integrase and 350 nM for Gp3. The reactions were terminated by proteinase K after incubation at 30°C for eight hours. (<b>B</b>) Schematic diagrams of the excision reaction shown in (A). Both <i>attL</i> and <i>attR</i> may form parallel and anti-parallel alignment, however, only correct alignments of substrates could participate in correct excision synapses, followed by excision.</p

Gp3 acts as the RDF through interaction with integrase.

<p>Investigation of DNA binding properties of Gp3 with or without integrase (Int) using FAM-labeled <i>attB₂₁₂</i> (A), <i>attP₂₄₇</i> (B), <i>attL₃₀₆</i> (C) and <i>attR₁₅₃</i>(D). The Int concentration in the reactions was 135 nM, and concentrations of Gp3 were 175 nM and 1750 nM. The positions of free DNA, complex I, complex II and the synaptic complex are indicated. The bands indicated by hollow arrows in (B) and (C) could result from multiple Gp3 monomers interacting with the Int dimer at higher concentrations.</p

<i>In vitro</i> excision using Gp3 from φBT1 and φC31.

<p>Excision reactions were performed using <i>attL</i> and <i>attR</i> of φBT1 or φC31. <i>attL</i> and <i>attR</i> of φBT1 were obtained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080434#pone-0080434-g001" target="_blank">Figure 1</a>. For substrates of φC31, plasmid pZL5822 was digested with EcoRI to generate <i>attL</i>-φC31(1969 bp) and <i>attR</i>-φC31 (6270 bp); the product sizes were predicted as 3432 bp for <i>attB</i> φC31 and 4810 bp for <i>attP</i>-φC31. Pairs of excision substrates were incubated with their own integrase in the presence or absence of Gp3 from φBT1 or φC31. The concentrations of proteins used were 270 nM for integrase and 350 nM for Gp3. The reactions were terminated by proteinase K after incubation at 30°C for eight hours.</p

Quantification of <i>in vitro</i> excision efficiencies between two sites of <i>attB</i>, <i>attP</i>, <i>attL</i> or <i>attR</i>.

<p><i>In vivo</i> detection of <i>in vitro</i> recombination products by blue-white screen; the four recombination targets were inserted into <i>lacZα</i> such that LacZ activity was abolished by recombination. The four target plasmids were pZLB00 (<i>atttB</i>), pZP00 (<i>atttP</i>), pZLL00 (<i>atttL</i>) and pZLR00 (<i>atttR</i>); the plasmids containing partner DNA were pZL5812 (<i>attB</i>), pZLP00 (<i>attP</i>), pZL5816 (<i>attL</i>) and pZL5817 (<i>attR</i>). After <i>in vitro</i> recombination, the plasmids were transformed into <i>E.coli</i> DH10B and plated on IPTG/X-gal medium; the white clones were produced by recombination. The substrate ratio of each combination was 5∶1 (partner to target). The data shown are average values of three reactions. The substrates used in each reaction and the relative recombination efficiencies (%) are indicated.</p

Gp3 with integrase catalyze excision in phage φBT1 recombination.

<p>(<b>A and B</b>) <i>In vitro</i> excision and integration recombination using linearized DNA in the presence of Gp3 and integrase. For the substrates of excision, plasmid pZL5813 was digested with KpnI to generate <i>attL</i> (6247 bp) and <i>attR</i> (2477 bp); the product sizes were predicted as 3578 bp for <i>attB</i> and 5146 bp for <i>attP</i>. For integration, pZL5812 was digested with HindIII to generate <i>attB</i> (3578 bp), and pZL5811 was digested with EcoRI to generate <i>attP</i> (4434 bp); the product sizes were predicted as 1623 bp for <i>attL</i> and 6389 bp for <i>attR</i>. Substrates were incubated with or without 27 nM integrase and varying concentrations (nM) of Gp3 for two hours. (<b>C</b>) Schematic diagrams of substrates used and the expected products in the excision and integration reactions shown in (A). (<b>D</b>) Time course of <i>in vitro</i> excision, and the substrates used were as shown in (A). The concentrations of proteins were 270 nM for integrase and 350 nM for Gp3. The reaction times are indicated. (<b>E</b>) DNA topological relaxation assays of <i>attB</i> (plasmid pZLB00), <i>attP</i> (plasmid pZLP00), <i>attL</i> (plasmid pZL5816), and <i>attR</i> (plasmid pZL5817) were performed with or without integrase or Gp3. The bands between the supercoiled and relaxed circles formed a ladder of closed circular DNA species that are probably topoisomers with a declining degree of superhelicity. The positions of supercoiled substrate DNA (SC), relaxed circles (RC), open circles (OC) and dimeric supercoiled DNAs (SC dimer) are indicated.</p