Name: conjugative mating assays for sequence-specific analysis of transfer proteins involved in bacterial conjugation
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Description: Watch this Scientific Journal Video about Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation at JoVE.com

This research was supported by a Discovery Grant from the Natural Sciences &#38; Engineering Council of Canada (NSERC).

1. Generation of DNA Constructs
<ol>
	<li>Designing Oligomers for Homologous Recombination of the Target Gene
	<ol>
		<li>Design a single 55-72 bp forward oligomer as follows: (a) pick a 19-32 bp long nucleotide sequence that is homologous to a DNA sequence in the region 10-100 bp upstream of the 5&#39; start site of the chloramphenicol acetyltransferase gene in the commercial pBAD33 plasmid,32 and (b) select a 36-54 bp long nucleotide sequence homologous to a region 10-150 bp downstream of ...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+diversity+of+prokaryotic+type+IV+secretion+systems.">Biological diversity of prokaryotic type IV secretion systems.</a> Microbiol Mol Biol Rev. 73 (4), 775-808 (2009).</li><li>Lawley, T. D., Klimke, W. A., Gubbins, M. J., Frost, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F+factor+conjugation+is+a+true+type+IV+secretion+system.">F factor conjugation is a true type IV secretion system.</a> FEMS Microbiol Lett. 224 (1), 1-15 (2003).</li><li>Lederberg, J., Tatum, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Recombination+in+Escherichia+Coli.">Gene Recombination in Escherichia Coli.</a> Nature. 158 (4016), 558-558 (1946).</li><li>Smillie, C., Garcillan-Barcia, M. P., Francia, M. V., Rocha, E. P. C., de la Cruz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobility+of+Plasmids.">Mobility of Plasmids.</a> Microbiol Mol Biol Rev. 74 (3), 434-452 (2010).</li><li>Willetts, N., Skurray, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Conjugation+System+of+F-Like+Plasmids.">The Conjugation System of F-Like Plasmids.</a> Annu Rev Genet. 14 (1), 41-76 (1980).</li><li>Frost, L. S., Ippen-Ihler, K., Skurray, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+sequence+and+gene+products+of+the+transfer+region+of+the+F+sex+factor.">Analysis of the sequence and gene products of the transfer region of the F sex factor.</a> Microbiol Rev. 58 (2), 162-210 (1994).</li><li>Audette, G. F., Manchak, J., Beatty, P., Klimke, W. A., Frost, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+exclusion+in+F-like+plasmids+requires+intact+TraG+in+the+donor+that+recognizes+its+cognate+TraS+in+the+recipient.">Entry exclusion in F-like plasmids requires intact TraG in the donor that recognizes its cognate TraS in the recipient.</a> Microbiology. 153, 442-451 (2007).</li><li>Christie, P. J., Atmakuri, K., Krishnamoorthy, V., Jakubowski, S., Cascales, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+Architecture,+and+Function+of+Bacterial+Type+Iv+Secretion+Systems.">Biogenesis, Architecture, and Function of Bacterial Type Iv Secretion Systems.</a> Annu Rev Microbiol. 59 (1), 451-485 (2005).</li><li>Christie, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+IV+secretion:+the+Agrobacterium+VirB/D4+and+related+conjugation+systems.">Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems.</a> Biochim Biophys Acta - Mol Cell Res. 1694 (1-3), 219-234 (2004).</li><li>Bhatty, M., Laverde Gomez, J. a., Christie, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expanding+bacterial+type+IV+secretion+lexicon.">The expanding bacterial type IV secretion lexicon.</a> Res Microbiol. 164 (6), 620-639 (2013).</li><li>Christie, P. J., Cascales, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+dynamic+properties+of+bacterial+Type+IV+secretion+systems+(Review).">Structural and dynamic properties of bacterial Type IV secretion systems (Review).</a> Mol Membr Biol. 22 (1-2), 51-61 (2005).</li><li>Christie, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+IV+secretion:+Intercellular+transfer+of+macromolecules+by+systems+ancestrally+related+to+conjugation+machines.">Type IV secretion: Intercellular transfer of macromolecules by systems ancestrally related to conjugation machines.</a> Mol Microbiol. 40 (2), 294-305 (2001).</li><li>Christie, P. J., Whitaker, N., Gonz&#225;lez-Rivera, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+and+structure+of+the+bacterial+type+IV+secretion+systems.">Mechanism and structure of the bacterial type IV secretion systems.</a> Biochim Biophys Acta. 1843 (8), 1578-1591 (2014).</li><li>Cascales, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+type+VI+secretion+toolkit.">The type VI secretion toolkit.</a> EMBO Rep. 9, 735 (2008).</li><li>Silverman, J. M., Brunet, Y. R., Cascales, E., Mougous, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Regulation+of+the+Type+VI+Secretion+System.">Structure and Regulation of the Type VI Secretion System.</a> Annu Rev Microbiol. 66 (1), 453-472 (2012).</li><li>Chandran, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+outer+membrane+complex+of+a+type+IV+secretion+system.">Structure of the outer membrane complex of a type IV secretion system.</a> Nature. 462 (7276), 1011-1015 (2009).</li><li>Rivera-Calzada, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+bacterial+type+IV+secretion+core+complex+at+subnanometre+resolution.">Structure of a bacterial type IV secretion core complex at subnanometre resolution.</a> EMBO J. 32 (8), 1195-1204 (2013).</li><li>Waksman, G., Fronzes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+architecture+of+bacterial+type+IV+secretion+systems.">Molecular architecture of bacterial type IV secretion systems.</a> Trends Biochem Sci. 35, 691 (2010).</li><li>Fronzes, R., Christie, P. J., Waksman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+biology+of+type+IV+secretion+systems.">The structural biology of type IV secretion systems.</a> Nat Rev Microbiol. 7 (10), 703-714 (2009).</li><li>Kaplan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+a+cell-embedded+megadalton+protein+complex+by+DNP-supported+solid-state+NMR.">Probing a cell-embedded megadalton protein complex by DNP-supported solid-state NMR.</a> Nat Methods. 12 (7), 5-9 (2015).</li><li>Guyer, M. S., Reed, R. R., Steitz, J. A., Low, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+sex-factor-affinity+site+in+E.+coli+as+gamma+delta.">Identification of a sex-factor-affinity site in E. coli as gamma delta.</a> Cold Spring Harb Symp Quant Biol. 45, 135-140 (1981).</li><li>Anthony, K. G., Sherburne, C., Sherburne, R., Frost, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+pilus+in+recipient+cell+recognition+during+bacterial+conjugation+mediated+by+F-like+plasmids.">The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F-like plasmids.</a> Mol Microbiol. 13 (6), 939-953 (1994).</li><li>Jobling, M. G., Holmes, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+vectors+with+the+pl5a+replicon,+kanamycin+resistance,+inducible+lacZ&#945;+and+pUC18+or+pUC19+multiple+cloning+sites.">Construction of vectors with the pl5a replicon, kanamycin resistance, inducible lacZ&#945; and pUC18 or pUC19 multiple cloning sites.</a> Nucleic Acids Res. 18 (17), 5315 (1990).</li><li>Guzman, L. M., Belin, D., Carson, M. J., Beckwith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+regulation,+modulation,+and+high-level+expression+by+vectors+containing+the+arabinose+P(BAD)+promoter.">Tight regulation, modulation, and high-level expression by vectors containing the arabinose P(BAD) promoter.</a> J Bacteriol. 177 (14), 4121-4130 (1995).</li><li>Yu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+recombination+system+for+chromosome+engineering+in+Escherichia+coli.">An efficient recombination system for chromosome engineering in Escherichia coli.</a> Proc Natl Acad Sci U S A. 97 (11), 5978-5983 (2009).</li><li>Lawley, T. D., Gilmour, M. W., Gunton, J. E., Standeven, L. J., Taylor, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+Mutational+Analysis+of+Conjugative+Transfer+Region+1+(Tra1)+from+the+IncHI1+Plasmid+R27.">Functional and Mutational Analysis of Conjugative Transfer Region 1 (Tra1) from the IncHI1 Plasmid R27.</a> J Bacteriol. 184 (8), 2173-2180 (2002).</li><li>Elton, T. C., Holland, S. J., Frost, L. S., Hazes, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F-Like+Type+IV+Secretion+Systems+Encode+Proteins+with+Thioredoxin+Folds+That+Are+Putative+DsbC+Homologues.">F-Like Type IV Secretion Systems Encode Proteins with Thioredoxin Folds That Are Putative DsbC Homologues.</a> J Bacteriol. 187 (24), 8267-8277 (2005).</li><li>Hazes, B., Frost, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+systems+biology+approach+to+study+type+II/IV+secretion+systems.">Towards a systems biology approach to study type II/IV secretion systems.</a> Biochim Biophys Acta. 1778, 1839-1850 (2008).</li><li>Lento, C., Ferraro, M., Wilson, D., Audette, G. F., Tsolis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDX-MS+and+deletion+analysis+of+the+type+4+secretion+system+protein+TraF+from+the+Escherichia+coli+F+plasmid.">HDX-MS and deletion analysis of the type 4 secretion system protein TraF from the Escherichia coli F plasmid.</a> FEBS Lett. 590 (3), 376-386 (2016).</li><li>Audette, G. F., Van Schaik, E. J., Hazes, B., Irvin, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-binding+protein+nanotubes:+Learning+from+nature's+nanotech+examples.">DNA-binding protein nanotubes: Learning from nature's nanotech examples.</a> Nano Lett. 4, 1897-1902 (2004).</li><li>Harris, R. L., Silverman, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tra+proteins+characteristic+of+F-like+type+IV+secretion+systems+constitute+an+interaction+group+by+yeast+two-hybrid+analysis.">Tra proteins characteristic of F-like type IV secretion systems constitute an interaction group by yeast two-hybrid analysis.</a> J Bacteriol. 186 (16), 5480-5485 (2004).</li><li>Moore, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+F-Plasmid+Conjugative+Transfer+Gene+traU.">Characterization of the F-Plasmid Conjugative Transfer Gene traU.</a> J Bacteriol. 172 (8), 4263-4270 (1990).</li><li>Anthony, K. G., Sherburne, C., Sherburne, R., Frost, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+pilus+in+recipient+cell+recognition+during+bacterial+conjugation+mediated+by+F-like+plasmids.">The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F-like plasmids.</a> Mol Microbiol. 13 (6), 939-953 (1994).</li><li>Klimke, W. A., Frost, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+the+role+of+the+transfer+gene,+traN,+of+the+F+and+R100-1+plasmids+in+mating+pair+stabilization+during+conjugation.">Genetic analysis of the role of the transfer gene, traN, of the F and R100-1 plasmids in mating pair stabilization during conjugation.</a> J Bacteriol. 180 (16), 4036-4043 (1998).</li><li>Jiang, W., Marraffini, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas:+New+Tools+for+Genetic+Manipulations+from+Bacterial+Immunity+Systems.">CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems.</a> Annu Rev Microbiol. 69 (1), 209-228 (2015).</li><li>Dahlberg, C., Bergstrom, M., Andreasen, M., Christensen, B. B., Molin, S., Hermansson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecies+bacterial+conjugation+by+plasmids+from+marine+environments+visualized+by+gfp+expression.">Interspecies bacterial conjugation by plasmids from marine environments visualized by gfp expression.</a> Mol Biol Evol. 15 (4), 385-390 (1998).</li></ol>

Bacterial conjugation process provides a means by which bacteria can spread genes providing an evolutionary advantage for growth in challenging environments, such as the spread of antibiotic resistance markers. Because many of the conjugative plasmids are so large,12 functional studies on the proteins involved in assembly of the transfer apparatus through mutation of target genes on the conjugative plasmid itself are unwieldy. The protocols detailed herein provide a means by which one can more readily assess t...

The transfer of genes and proteins at the micro-organismal level plays a central role in bacterial survival and evolution as well as infection processes. The exchange of DNA between bacteria or between a bacterium and a cell can be achieved through transformation, conjugation or vector transduction.1,2 Conjugation is unique in comparison to transformation and transduction in that during conjugation between gram-negative bacteria such as Escherichia coli, the transfer of DNA occurs in a donor-controlled fashion whereby a complex macromolecular system connects donor and recipient cells. Conjugation is also the most direct way in which bacterial cells interact with host cells to inject genes, proteins or chemicals in to host systems.3 Quite often, the transfer of such agents has remarkable effects on the host, ranging from pathogenesis and carcinogenesis to host evolution and adaptation. It has been shown that conjugative recombination increases the rate of adaptation 3-fold in bacteria with high mutation rates under conditions of environmental stress.4 Moreover, conjugation is by far the most common route through which antibiotic resistance genes in bacterial strains are spread.5,6
Microorganisms have evolved specialized secretion systems to support the transfer of macromolecules across cellular membranes; there are currently 9 types of secretion systems (TSSs) in gram negative bacteria that have been described: T1SS, T2SS, T3SS, T4SS, T5SS, T6SS, T7SS, as well as the Sec (secretion) and Tat (two-arginine translocation) pathways.7,8 Each type of secretion system is further divided into different subtypes, a necessity due to diversity of proteins and the distinctiveness of pathways involved, in different bacterial strains. For example, in the type IV secretion system (T4SS), the Ti and Cag systems facilitate effector transport whereas the F-plasmid, R27 and pKM101 T4SSs facilitate transfer of a conjugative plasmid.7,9,10 A detailed understanding of the mechanisms by which organisms assemble their respective secretion systems from their component proteins and share cellular contents with a recipient or their surrounding environment is an important factor in development of targeted strategies to combat pathogenic microorganisms and processes of cellular infection.
Following the initial identification bacterial conjugation in E. coli by Lederberg &#38; Tatum,11 a large number of mobile and conjugative plasmids have been identified and characterized.12 Such mobile plasmids show considerable range is size (from 1 to over 200 kilobases (kb)), however all mobile plasmids contain a relaxase, which recognizes the origin of transfer (oriT) thereby enabling transmission of the plasmid. Conjugative plasmids further encode genes for assembly of a functional T4SS as well as a type IV coupling protein.12 For example the 100 kb F plasmid of E. coli encodes all the conjugative genes within a 33.3 kB transfer (tra) region.13 The genes in the tra region of the F plasmid encode all proteins that facilitate pilus formation, mating pair formation (Mpf), DNA transfer and exclusion functions during conjugative plasmid transfer.10,14,15 A significant body of knowledge is available for conjugative T4SSs, however detailed structural studies of the conjugative proteins and complexes are only more recently becoming available.16-28
In order to assemble a comprehensive view of the conjugative process, a coupling of detailed structural studies to mutational analyses of conjugative transfer proteins is required. This can be achieved through conjugative mating assays. For the F plasmid, each protein encoded within the tra region plays a role in the F-mediated conjugation; therefore, the knockout/deletion of a transfer gene will abolish the conjugative capacity of the cell (Figure 1). While smaller mobile plasmids are more conducive to standard deletion procedures, for larger conjugative plasmids such as F, gene knockouts are more readily achieved via homologous recombination where the target gene is replaced with one conveying a distinct antibiotic resistance gene. In the current protocol, we employ homologous recombination to replace a transfer gene of interest with chloramphenicol acetyltransferase (CAT) in the 55 kb F plasmid derivative pOX38-Tc;29,30 the resultant knockout plasmid, pOX38-Tc &#916;gene::Cm, facilitates resistance to the presence of chloramphenicol (Cm) in the growth media. Donor cells harboring pOX38-Tc &#916;gene::Cm are unable to affect conjugative DNA transfer/mating as observed through the use of a mating assay; the mating efficiency of a pOX38-Tc &#916;gene::Cm donor cell and a normal recipient will decrease or, more often, be abolished. Conjugative transfer of the pOX38-Tc &#916;gene::Cm plasmid can be restored via a small recovery plasmid harboring the targeted transfer gene. This recovery plasmid can be one that provides constitutive expression, such as plasmid pK184 (pK184-gene),31 or one that provides inducible expression so long as that plasmid properly targets the gene to the correct location within the cell (cytoplasm or periplasm). Consequently, in mating assays between this new donor (harboring pOX38-Tc &#916;gene::Cm + pK184-gene plasmids) and a recipient cell, the mating efficiency is expected to restore to nearly that of a normal donor-recipient mating assay. This system enables one to probe the function of the knocked out gene through the generation of a series of pK184-gene constructs (deletions or point mutations) and testing each construct&#39;s ability to restore the mating capacity of the pOX38-Tc &#916;gene::Cm harboring donor cells.

<table border="0" cellpadding="0" cellspacing="0" width="762">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">GeneJet Plasmid Mini-Prep Kit</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">K0503</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">GeneJet Gel Extraction Kit</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">K0692</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">GeneJet PCR Purification Kit</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">K0702</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Q5 Site-Directed Mutagenesis Kit</td>
			<td>New England Biolabs</td>
			<td>E0554S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Broad Range DNA Ladder</td>
			<td>New England Biolabs</td>
			<td>N0303A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Petri Dishes</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">FB0875713</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Electroporator</td>
			<td style="width:165px;">Eppendorf</td>
			<td style="width:131px;">4309000027</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Electroporation cuvettes</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">FB101</td>
			<td style="width:212px;">Cuvettes have a 1 mm gap.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Enzymes</td>
			<td style="width:165px;"></td>
			<td style="width:131px;"></td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">AvaI</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0152S</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">EcoRI</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0101S</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">HindIII</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0104L</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">NdeI</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0111S</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Phusion DNA Polymerase</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">M0530L</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">T4 DNA Ligase</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">M0202S</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DpnI</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0176S</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antibiotics</td>
			<td style="width:165px;"></td>
			<td style="width:131px;"></td>
			<td style="width:212px;">Final Concentrations</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Chloramphenicol (Cm)</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">BP904-100</td>
			<td style="width:212px;">20 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Kanamycin (Km)</td>
			<td style="width:165px;">BioBasic Inc.</td>
			<td style="width:131px;">DB0286</td>
			<td style="width:212px;">50 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Nalidixic acid (Nal)</td>
			<td style="width:165px;">Sigma-Aldrich</td>
			<td style="width:131px;">N8878-25G</td>
			<td style="width:212px;">10 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Rifampicin (Rif)</td>
			<td style="width:165px;">Calbiochem</td>
			<td style="width:131px;">557303</td>
			<td style="width:212px;">20 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tetracycline (Tc)</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">BP912-100</td>
			<td style="width:212px;">10 &#181;g/mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Streptomycin (Sm)</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">BP910-50</td>
			<td style="width:212px;">50 &#181;g/mL</td>
		</tr>
	</tbody>
</table>

conjugative mating assays for sequence-specific analysis of transfer proteins involved in bacterial conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

The process of F plasmid-driven bacterial conjugation is a coordinated process that involves transfer proteins within the tra region of the F-plasmid that assembles a T4SS to facilitate pilus synthesis and conjugative DNA transfer. The protein TraF (GenBank accession # BAA97961; UniProt ID P14497) is required for conjugative F-pilus formation.10,14,35-37 The protein contains a C-terminal thioredoxin-like domain, though it does not have the catalyt...

Watch this Scientific Journal Video about Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation at JoVE.com

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

The overall goal of this mating assay is to assess the effects of mutations within the conjugative transfer gene on its ability to affect plasmid transfer within the context of a functional type four secretion complex. This method allows for asking protein specific questions, such as identifying regions of protein-protein interactions that occur between transfer proteins as they assemble a functional type four secretion system in bacterial conjugation. In this technique, genes on large conjugative plasmids are knocked out by homologous recombination.Gene function is assessed by providing the target gene to the knock-outs on a smaller plasmid. Generally, individuals new to this method will struggle because organization and set-up are key to successfully conduct this procedure. Proper experimental preparation is necessary to get interpretable results.Begin this protocol with preparation of the digested pBAD33 plasmid as described in the text protocol. Run each digest on an agarose gel. Using a UV cabinet and a sterile razor, cut the 2.8 kilo baseband that cotresponds to the cath sequence out of the gel.Work quickly to minimize the UV exposure of the DNA. Extract the DNA from the cut out gel slice using a gel extraction kit as per the manufacturer's protocol. To amplify the extracted cat cassette by polymerase chain reaction or PCR.Prepare the reaction mixture in a sterile PCR tube. Use primers that contain overhangs that are homologous to the target gene sequence for homologous recombination. Set-up a negative control that bears the same components, except replace the template DNA with double distilled water.Also, set-up a positive control using template DNA and primers that have been proven to work in a PCR reaction. Mix all the reaction contents gently by pipetting. Then, amplify the extracted cat cassette using the PCR parameters listed in the text protocol.Use only five microliters of each reaction to confirm the correct size of amplification via agarose gel electrophoresis. Purify the PCR amplicon using a PCR purification kit with the manufacturer's protocol. Add 300 nanograms of the amplified cat cassette into 50 microliters of electrocompetent DY330R cells, harboring the pOX38-Tc plasmid on ice.Gently pipette up and down to mix. Repeat this step using unmodified pBAD33 plasmid as a positive control. Then, transfer the cells to a pre-cooled one millimeter electroporation cuvette.Electroporate the cells at 1.8 kilovolts with a time constant of 5.5 milliseconds using an electroporator. Immediately, after applying the pulse, dilute the cells with one milliliter of SOC media and transfer to a fresh microfuge tube. Incubate the cells at 32 degrees Celsius for two hours.Next, aliquot 100 microliters of each sample unto agar plates containing 10 micrograms per milliliter tetracycline and 20 micrograms per milliliter chloramphenicol. Spread the aliquot over the plate using a sterile spreader. Incubate the plate upside down at 32 degrees celsius overnight.The next day, select for the successful recombinants. The cat cassette introduced into the cell will undergo homologous recombination with the gene of interest and create the pOX38-Tc chloramphenicol knock-out clone. Separate the electroporate 300 nanograms of pK184 gene or pK184 gene mutant plasmid into 50 microliters of electrocompotent DY330R-cells harboring the pOX38-Tc chloramphenicol knock-out plasmid as before.To generate the XK1200 donor cells perform conjugative mating followed by electroporation to generate XK1200 cells harporing both the chloramphenicol knock-out and the PK184 plasmid as described in the text protocol. Preparing overnight culture of these XK1200 donor cells in 20 milliliters of sterile LB with chloramphenicol and kanamycin. Also prepare MC4100 recipients cells in 50 milliliters of LB with 50 micrograms per milliliter of streptomycin using cells from a glycerol stock or from a single colony on ang agar plate.Grow the cultures at 37 degrees celsius, shaking at 200 rpm. Add glucose to a final concentration of 100 millimolar to all donor cells. The next day, make 1 in 70 dilutions from each overnight culture separately in two milliliters of sterile LB with the same antibiotics.Grow the cells to mid log phase at 37 degrees celsius with shaking at 200 rpm. Centrifuge the cell culture to pellet the cells and discard the supernatant. Then, wash the cell pellet once with cold sterile LB to remove the antibiotics.After centrifuging a second time as before we suspend the cells in two milliliters of cold sterile LB.In duplicate, aliquot 100 microliters of donor cells and 100 microliters of recipient cells to 800 microliters of sterile LB media for one milliliter total volume. Allow the cells to mate at 37 degrees celsius for one hour without shaking. After an hour, vortex the cells for 30 seconds to disrupt the mating pairs.Then, place the cells on ice for 10 minutes to prevent further mating. Using the mid log cultures and fresh sterile LB, prepare six serial dilutions of the donor and recipient cells from 1 and 100 to 1 and 10 million. On each of two halves of an agar plate containing naladixic acid, chloramphenicol, and kanamycin, spot 10 microliter aliquots of each dilution of XK1200 donor cells.Repeat spotting for the dilutions of the recipient MC4100 cells on agar plates containing streptomycin. Then, incubate the plates overnight at 37 degrees celsius. Next, using the vortex mixture and fresh sterile LB, prepare six dilutions of the transconjugants.Select for the transconjugant MC4100 cells that harbor the pOX38-Tc chloramphenicol knock-out bu spotting ten microliter aliquots of each dilution on each half of the agar plates containing streptomycin and chloramphenicol. Repeat for both duplicate mixtures. Then, incubate the plates overnight at 37 degrees celsius.Calculate the mating efficiency by counting the number of colonies from the same dilution spotting for each donor, recipient and transconjugants cells on their respective plates. Also, count the recipient colonies to test for any bias resulting from a larger number of transconjugants than recipients at that given dilution. Calculate the mating efficiency as the number of transconjugant colonies divided by the number of donor colonies, multiplied by 100 to obtain the the efficiency value per 100 donor cells.When the type four secretion system gene-tra-F is knocked out of the pOX38-Tc plasmid, it results in a loss of conjugative transfer of the pOX38-Tc traF chloramphenicol knock out plasmid from donor to recipient cells. However, when the traF gene is provided in trans by the PK184 traF plasmid an the donor cells, a recovery of conjugative transfer of the pOX38-Tc traF chloramphenicol knock-out plasmid is observed. When provided with dilution mutants of traF and the PK184 plasmid in the donor cells, loss of conjugative transfer of the pOX38-Tc traF chloramphenicol knock-out plasmid can be observed.Loss of conjugative transfer indicates the region of the protein important for protein-protein interactions within the conjugative type four secretion system. This region can then be probed in more detail by point mutations that affect the efficiency for transfer. Once mastered, this technique can be performed in a single work day with expected growth times.This protocol can be done on cells other than the ones demonstrated here. The critical aspect, at this point, is that, donor and recipient cells don't harbor the plasmid of interest. So that when you add the knock-out plasmid, you don't get conflicting results.While attempting this procedure it is important to be very organized as there are different growth conditions and antibiotics for donor, recipient, and transconjugate cells. Following this procedure, other methods such as crystallography, NMR, or mass spectrometry can be performed in order to obtain a detailed structural and functional picture of the protein of interest.

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Research

JoVE Journal

Genetics

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little is known about the molecular control that governs their development. This is in part due to physical, resource and time limitations often associated with the study of secondary growth processes. A number of in vitro techniques have been used involving either plant part or whole plant system in both woody and non-woody plant species. However, questions about their applicability for the study of secondary stem growth processes, the recalcitrance of certain species and labor intensity are often prohibitive for medium to high throughput applications. Also, when looking at secondary stem development and wood formation the specific traits under investigation might only become measurable late in a tree&#39;s lifecycle after several years of growth. In addressing these challenges alternative in vivo protocols have been developed, named Induced Somatic Sector Analysis, which involve the creation of transgenic somatic tissue sectors directly in the plant&#39;s secondary stem. The aim of this protocol is to provide an efficient, easy and relatively fast means to create transgenic secondary plant tissue for gene and promoter functional characterization that can be utilized in a range of tree species. Results presented here show that transgenic secondary stem sectors can be created in all live tissues and cell types in secondary stems of a variety of tree species and that wood morphological traits as well as promoter expression patterns in secondary stems can be readily assessed facilitating medium to high throughput functional characterization.

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. Here, we present a method to selectively up-regulate protein expression of desired genes in cultured cells by means of synthetic antisense non-coding RNAs known as SINEUPs. This positive control of gene expression is at the post-transcriptional level and exerted by an inverted short interspersed nuclear element (SINE) repeat at the 3&#697; end of SINEUPs that comprises its effector domain (ED). SINEUPs can specifically bind to any protein-coding mRNA of choice through its binding domain (BD), a region designed to complement the sequence within the 5&#697; untranslated region (5&#697; UTR) and around the start codon of the mRNA. Target-specific SINEUPs designed in this manner are transfected to cultured cells, and protein and RNA are extracted for downstream analyses, generally 24-48 h post-transfection. SINEUP-induced protein up-regulation is detected by Western-blot analysis and RNA expression is measured using real-time quantitative reverse transcription PCR. We have observed that BD design is critical for achieving optimum SINEUP activity and that testing different BD sizes and positions with respect to the start codon of the target mRNA is recommended. Therefore, we describe here a semi-automated high-throughput imaging method based on fluorescence detection that can be implemented to target mRNA fused with green fluorescent protein (GFP). SINEUPs specifically enhance translation within normal physiological range of the cell, without altering the target transcript level. This method has been successfully employed against a range of endogenous and exogenous targets, in a wide variety of human, mouse, and insect cell lines along with in vivo systems. Moreover, SINEUPs have been reported to increase antibody production and work as an RNA therapeutic against haploinsufficient genes. The versatile and modular nature of SINEUPs makes them a suitable tool for gene-specific translational control.

SINEUPs are synthetic antisense non-coding RNAs, which contain a binding domain (BD) and an effector domain (ED) and up-regulate translation of target mRNA. Here, we describe detection methods for SINEUPs in cultured cell lines, analysis of their translation-promoting activity by Western-blot and a semi-automated high throughput imaging system.

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. ...

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast permitting in vivo phenotypic analysis. This method does not rely on maize transformation but exploits microbial genetics and secretory capabilities of pathogens. Herein, it allows inspection of in vivo delivered secreted proteins with high spatiotemporal resolution at different kinds of infection sites and tissues. The Trojan horse strategy can be utilized to transiently complement maize loss-of-function phenotypes, to functionally characterize protein domains, to analyze off-target protein effects, or to study onside protein overdosage, making it a powerful tool for protein studies in the maize crop system. This work contains a precise protocol on how to generate a Trojan horse strain followed by standardized infection protocols to apply this method to three different maize tissue types.

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

This work describes the cloning of an Ustilago maydis Trojan horse strain for the in situ delivery of secreted maize proteins into three different types of maize tissues.

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast ...

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to agriculture. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed a method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 in tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Here, we present a versatile method for tomato root transformation followed by inoculation with Ralstonia solanacearum to perform straightforward genetic analysis for the study of bacterial wilt disease.

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to ...

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...