Major Groove Binding Track Residues of the Connection Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Enhance cDNA Synthesis at High Temperatures

At high temperatures, RNA denaturation can improve the efficiency and specificity of reverse transcription. Refined structures and molecular models of HIV-1 reverse transcriptases (RTs) from phylogenetically distant clades (i.e., group M subtype B and group O) revealed a major interaction between the template-primer and the Arg³⁵⁸-Gly³⁵⁹-Ala³⁶⁰ triad in the large subunit of HIV-1_M/B RT. However, fewer contacts were predicted for the equivalent Lys³⁵⁸-Ala³⁵⁹-Ser³⁶⁰ triad of HIV-1_O RT and the nucleic acid. An engineered HIV-1_O K358R/A359G/S360A RT showed increased cDNA synthesis efficiency above 68 °C, as determined by qualitative and quantitative reverse transcription polymerase chain reactions. In comparison with wild-type HIV-1_O RT, the mutant enzyme showed higher thermal stability but retained wild-type RNase H activity. Mutations that increased the accuracy of HIV-1_M/B RTs were tested in combination with the K358R/A359G/S360A triple mutation. Some of them (e.g., F61A, K65R, K65R/V75I, and V148I) had a negative effect on reverse transcription efficiency above 65 °C. RTs with improved DNA binding affinities also showed higher cDNA synthesis efficiencies at elevated temperatures. Two of the most thermostable RTs (i.e., mutants T69SSG/K358R/A359G/S360A and K358R/A359G/S360A/E478Q) showed moderately increased fidelity in forward mutation assays. Our results demonstrate that the triad of Arg³⁵⁸, Gly³⁵⁹, and Ala³⁶⁰ in the major groove binding track of HIV-1 RT is a major target for RT stabilization, and most relevant for improving reverse transcription efficiency at high temperatures

Mobility of the Native <i>Bacillus subtilis</i> Conjugative Plasmid pLS20 Is Regulated by Intercellular Signaling

<div><p>Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native <i>Bacillus subtilis</i> plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default “OFF” state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed.</p></div

pLS20 gene 27c (<i>rco_LS20</i>) encodes a repressor of conjugation.

<p>pLS20 gene 27c (<i>rco_LS20</i>) encodes a repressor of conjugation.</p

Rap_LS20 stimulates conjugation.

<p>Conjugation kinetics of pLS20cat and pLS20rap were determined with and without ectopic expression of Rap_LS20 as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003892#s4" target="_blank">Materials and Methods</a>. PKS7 was used as recipient strain. GR23 (pLS20cat) and PKS79 (pLS20rap) were used as donor strains. GR23 contains an ectopic copy of <i>rap_LS20</i> under the control of the IPTG inducible P_spank promoter at the chromosomal <i>amy</i>E locus. t = 0 corresponds to the end of the exponential growth phase. Control experiments showed that overexpression of Rap_LS20 in strain GR20 did not significantly affect growth (not shown).</p

Conjugation kinetics of pLS20cat without and with replacing the recipient growth medium.

<p>Conjugation kinetics of pLS20cat was determined as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003892#s4" target="_blank">Materials and Methods</a> using strains PKS11 and PKS7 as donor and recipient strain, respectively. At each time point donor cells were mixed with recipient cells either directly (broken line) or after the recipient growth medium had been replaced with fresh LB medium (continuous line), and plated on selective agar plates after a 15 min mating period. t = 0 corresponds to the end of the exponential growth phase. Control experiments showed that the centrifugation step did not affect conjugation efficiency (not shown).</p

Genetic map of pLS20cat.

<p>(Putative) genes are numbered. Gene 1 corresponds to the homologue of gene 1 of the related <i>Bacillus pumilus</i> NRS576 plasmid p576 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003892#pgen.1003892-Singh2" target="_blank">[37]</a>. The positions and the lengths of the (putative) genes are indicated by arrows. Rightward and leftward oriented genes are indicated in purple and orange, respectively. Putative Rho-independent transcriptional terminators are indicated with green hairpin structures. The origin of replication region and the gene conferring resistance to chloramphenicol are labeled with green rectangles. The DNA region containing the chloramphenicol gene was cloned into the unique <i>Sal</i>I site located in pLS20 gene 13 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003892#pgen.1003892-Itaya1" target="_blank">[24]</a>. The sequences flanking the Cm resistance cassette coding for the N- and C-terminal regions of gene 13 are labeled 13-N and 13-C, respectively. The putative conjugation operon encompassing genes 28 to 74, is highlighted by a blue background. Genes showing significant homology with genes reported to be involved in conjugation in other systems are shown in black. Recently, the complete pLS20cat sequence has been deposited by Itaya,M., <i>et al</i>. (Mitsuhiro Itaya Keio University, Japan) in public database under accession numbers NC_015148.1 and AB615352.1. pLS20cat gene 25, according to our nomenclature, corresponds to gene 001 of the deposited sequence. Due to differences in annotation we prefer to maintain our nomenclature.</p

Model of regulatory circuitry of pLS20 conjugation genes.

<p>A. Repressed state due to Rco_LS20. Gene <i>rco_LS20</i> (red arrow, rco) encoding the master repressor of conjugation genes Rco_LS20 is divergently transcribed from the putative conjugation operon encompassing genes 28 to 74 (light blue arrows). Rco_LS20 inhibits expression of the conjugation genes by repressing a promoter, P_c, located upstream of gene 28, the first gene of the putative conjugation operon (our unpublished results). B. Activation of conjugation by Rap_LS20 anti-repressor. Gene _rapLS20 (green arrow, rap) encodes the anti-repressor of Rco_LS20 leading to de-repression of the conjugation genes. C. Repressed state due to inactivation of Rap_LS20 by signaling peptide Phr*_LS20. Gene <i>phr_LS20</i> (brown arrow, phr) encodes a pre-pro-protein of 44 residues. This protein is subject to an export-maturation-import route. The mature pentapeptide inhibits activity of the Rap_LS20 anti-repressor protein. For simplicity, import of the mature peptide has been shown into the cell producing the peptide. Grey cylinders labeled sec and imp, respectively, indicate the secretion and import routes. Extracellular processing of the secreted peptide is symbolized by the brown interrupted rectangle. QS, quorum sensing.</p

Conserved residues important for Rap proteins known to interact with Spo0F or ComA are not conserved in Rap_LS20.

<p>Alignment of the N-terminal regions of <i>Bacillus</i> Rap proteins. In addition to Rap_LS20 and Rap₅₇₆, the alignment includes Rap proteins that previously have been demonstrated to dephosphorylate Spo0F (RapP, RapA, RapB, RapE, RapI, RapJ RapH, Rap_XO1 ( = BXA0205), and Rap60 [Spo0F-phosphatase activity has not been demonstrated biochemically for Rap60]), and those shown to interact with ComA (RapF, RapC and RapH). Regions adapting an α-helical formation in RapH are indicated with green cylinders above the alignment. The highly conserved tryptophan residue present in all these Rap proteins is indicated in green. The catalytic Gln47 residue of RapH that is conserved in six of the seven other Spo0F-interacting Rap proteins as well as in RapI is highlighted in red. Alanine substitutions in Rap proteins that cause complete or significant loss of function/interaction with Spo0F and ComA are highlighted by blue boxes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003892#pgen.1003892-Parashar2" target="_blank">[44]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003892#pgen.1003892-Baker1" target="_blank">[45]</a>. RapH residue Leu55 is conserved in Rap_LS20 and Rap₅₇₆. It is worth mentioning that although the L55A mutant affected the function of RapH <i>in vivo</i>, no loss of RapH function was observed for this mutant <i>in vitro</i> <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003892#pgen.1003892-Parashar2" target="_blank">[44]</a>. Positions of the α-helices are indicated above the alignment.</p

Heat map representation of the expression levels of the pLS20cat genes at late exponential phase under various conditions analyzed by RNAseq.

<p>Left lane (“wt”) shows the expression level of pLS20cat genes in the wild type strain background at late exponential phase when conjugation efficiency is at its maximum. Expression levels are presented on a log₂ scale covering a range from 0 (white, lowest level) to 16 (blue, highest level). Middle (+Rco) and right (+Rap) lanes represent the effects of ectopic expression of Rco_LS20 (middle lane) or Rap_LS20 (right lane), respectively, on the expression of the pLS20cat genes. Differential expression levels are presented on a log₂ scale covering a range of −16 to 16 using shades of red and green for repression and overexpression, respectively. White reflects no change in expression. Gene numbers according to our nomenclature and those deposited in database under accession number NC_015148.1 (preceded by “J”) are given on the right). “c” corresponds to leftward oriented genes.</p

Phr*_LS20 pentapeptide inhibits conjugation in an <i>opp</i> dependent manner.

<p>A. Effects of synthetic Phr* peptide on conjugation in the wild type and an <i>opp</i> deficient background. Conjugation efficiencies of pLS20cat were determined at late exponential growth phase using as recipient strain PKS7, and as donor either strain PKS11 (wild type, black bars) or PKS98 (<i>oppA</i>, grey bars). Diluted overnight grown cultures of donor cells were split in two, and Phr*_LS20 pentapeptide was added to a final concentration of 6 µM to one of the cultures and equal volume of the peptide buffer to the other. B. Conjugation kinetics of pLS20cat and pLS20phr. Conjugation kinetics was determined as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003892#s4" target="_blank">Materials and Methods</a> using PKS7 as recipient strain and PKS14 (pLS20cat) or PKS117 (pLS20phr) as donor strains. t = 0 corresponds to the end of the exponential growth phase. Both donor strains contain an ectopic copy of <i>rco_LS20</i> under the IPTG inducible P_spank promoter at the chromosomal <i>amy</i>E locus. Overnight cultures of donor cells were grown in the presence of 1 mM IPTG and diluted in fresh pre-warmed LB medium without IPTG. C. Conjugation kinetics of pLS20cat after re-dilution of the donor cell culture. Conjugation kinetics using PKS7 and PKS11 as recipient and donor strains, respectively, was determined as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003892#s4" target="_blank">Materials and Methods</a> with the following modification. Overnight cultures were diluted, grown until late exponential growth phase (OD₆₀₀ = 0,8), and diluted again (to OD₆₀₀ = 0.05) before starting the experiment. B and C. t = 0 corresponds to the end of the exponential growth phase.</p