Name: a non-coding small rna micc contributes to virulence in outer membrane proteins in salmonella enteritidis
Uploaded: 2021-01-27T14:00:00
Description: Watch this Scientific Journal Video about A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis at JoVE.com

This study was supported by grants from the Chinese National Science Foundation (Nos. 31972651 and 31101826), Jiangsu High Education Science Foundation (No.14KJB230002), State Key Laboratory of Veterinary Biotechnology (No.SKLVBF201509), Nature Science Foundation Grant of Yangzhou (No.YZ2014019), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

All the experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council. The animal care and use committee of Yangzhou University approved all experiments and procedures applied on the animals (SYXK2016-0020).
1. Bacterial strains, plasmids, and culture conditions
<ol>
	<li>Use the bacteria and plasmids listed in Table 1.</li>
	<li>Culture bacteria in LB broth or on LB agar plates at 37 &#176;C, in the prese...

<ol>
	<li>J&#248;rgensen, M. G., Pettersen, J. S., Kallipolitis, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=sRNA-mediated+control+in+bacteria:+An+increasing+diversity+of+regulatory+mechanisms.">sRNA-mediated control in bacteria: An increasing diversity of regulatory mechanisms.</a> Biochimica et Biophysica Acta-Gene Regulatory Mechanisms. 1863 (5), 194504 (2020).</li><li>Wagner, E. G. H., Romby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+RNAs+in+bacteria+and+archaea:+who+they+are,+what+they+do,+and+how+they+do+it.">Small RNAs in bacteria and archaea: who they are, what they do, and how they do it.</a> Advances In Genetics. 90, 133-208 (2015).</li><li>Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rough+guide+to+the+non-coding+RNA+world+of+Salmonella.">A rough guide to the non-coding RNA world of Salmonella.</a> Molecular Microbiology. 71 (1), 1-11 (2009).</li><li>Dutta, T., Srivastava, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+RNA-mediated+regulation+in+bacteria:+A+growing+palette+of+diverse+mechanisms.">Small RNA-mediated regulation in bacteria: A growing palette of diverse mechanisms.</a> Gene. 656, 60-72 (2018).</li><li>Waters, L. S., Storz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+RNAs+in+bacteria.">Regulatory RNAs in bacteria.</a> Cell. 136 (4), 615-628 (2009).</li><li>Chen, S., Zhang, A., Blyn, L. B., Storz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicC,+a+second+small-RNA+regulator+of+Omp+protein+expression+in+Escherichia+coli.">MicC, a second small-RNA regulator of Omp protein expression in Escherichia coli.</a> Journal of Bacteriology. 186 (20), 6689-6697 (2004).</li><li>Pfeiffer, V., Papenfort, K., Lucchini, S., Hinton, J. C., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coding+sequence+targeting+by+MicC+RNA+reveals+bacterial+mRNA+silencing+downstream+of+translational+initiation.">Coding sequence targeting by MicC RNA reveals bacterial mRNA silencing downstream of translational initiation.</a> Nature Structural &amp; Molecular Biology. 16 (8), 840-846 (2009).</li><li>Dam, S., Pag&#232;s, J. M., Masi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+Regulation+of+the+Small+RNA+MicC+and+the+Quiescent+Porin+OmpN+in+Response+to+Antibiotic+Stress+in+Escherichia+coli.">Dual Regulation of the Small RNA MicC and the Quiescent Porin OmpN in Response to Antibiotic Stress in Escherichia coli.</a> Antibiotics (Basel). 6 (4), 33 (2017).</li><li>Hao, 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=Porin+Deficiency+in+Carbapenem-Resistant+Enterobacter+aerogenes+Strains.">Porin Deficiency in Carbapenem-Resistant Enterobacter aerogenes Strains.</a> Microbial Drug Resistance. 24 (9), 1277-1283 (2018).</li><li>Wroblewska, Z., Olejniczak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hfq+assists+small+RNAs+in+binding+to+the+coding+sequence+of+ompD+mRNA+and+in+rearranging+its+structure.">Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure.</a> RNA. 22 (7), 979-994 (2016).</li><li>Santiviago, C. A., Toro, C. S., Hidalgo, A. A., Youderian, P., Mora, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+regulation+of+the+Salmonella+enterica+serovar+typhimurium+major+porin,+OmpD.">Global regulation of the Salmonella enterica serovar typhimurium major porin, OmpD.</a> Journal of Bacteriology. 185 (19), 5901-5905 (2003).</li><li>Hara-Kaonga, B., Pistole, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OmpD+but+not+OmpC+is+involved+in+adherence+of+Salmonella+enterica+serovar+typhimurium+to+human+cells.">OmpD but not OmpC is involved in adherence of Salmonella enterica serovar typhimurium to human cells.</a> Canadian Journal of Microbiology. 50 (9), 719-727 (2004).</li><li>Datsenko, K. A., Wanner, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+inactivation+of+chromosomal+genes+in+Escherichia+coli+K-12+using+PCR+products.">One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (12), 6640-6645 (2000).</li><li>Meng, X., 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=The+RNA+chaperone+Hfq+regulates+expression+of+fimbrial-related+genes+and+virulence+of+Salmonella+enterica+serovar+Enteritidis.">The RNA chaperone Hfq regulates expression of fimbrial-related genes and virulence of Salmonella enterica serovar Enteritidis.</a> FEMS Microbiology Letters. 346 (2), 90-96 (2013).</li><li>Livak, K. J., Schmittgen, T. D. <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+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Vander Velden, A. W., B&#228;umler, A. J., Tsolis, R. M., Heffron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+fimbrial+adhesins+are+required+for+full+virulence+of+Salmonella+typhimurium+in+mice.">Multiple fimbrial adhesins are required for full virulence of Salmonella typhimurium in mice.</a> Infection and Immunity. 66 (6), 2803-2808 (1998).</li><li>Prescott, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+enterica+serovar+enteritidis+in+humans+and+animals:+Epidemiology,+pathogenesis,+and+control.">Salmonella enterica serovar enteritidis in humans and animals: Epidemiology, pathogenesis, and control.</a> Canadian Veterinary Journal La Revue Veterinaire Canadienne. 40 (10), 736 (1999).</li><li>Balasubramanian, R., 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=The+global+burden+and+epidemiology+of+invasive+non-typhoidal.">The global burden and epidemiology of invasive non-typhoidal.</a> Hum Vaccin Immunother. 15 (6), 1421-1426 (2019).</li><li>De Buck, J., Van Immerseel, F., Haesebrouck, F., Ducatelle, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization+of+the+chicken+reproductive+tract+and+egg+contamination+by+Salmonella.">Colonization of the chicken reproductive tract and egg contamination by Salmonella.</a> Journal of General and Applied Microbiology. 97 (2), 233-245 (2004).</li><li>Padalon-Brauch, G., 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=Small+RNAs+encoded+within+genetic+islands+of+Salmonella+typhimurium+show+host-induced+expression+and+role+in+virulence.">Small RNAs encoded within genetic islands of Salmonella typhimurium show host-induced expression and role in virulence.</a> Nucleic Acids Research. 36 (6), 1913-1927 (2008).</li><li>Santiviago, C. 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=Analysis+of+pools+of+targeted+Salmonella+deletion+mutants+identifies+novel+genes+affecting+fitness+during+competitive+infection+in+mice.">Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice.</a> PLoS Pathogens. 5 (7), 1000477 (2009).</li><li>Gong, H., 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=A+Salmonella+small+non-coding+RNA+facilitates+bacterial+invasion+and+intracellular+replication+by+modulating+the+expression+of+virulence+factors.">A Salmonella small non-coding RNA facilitates bacterial invasion and intracellular replication by modulating the expression of virulence factors.</a> PLoS Pathogens. 7 (9), 1002120 (2011).</li><li>H&#233;brard, 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=sRNAs+and+the+virulence+of+Salmonella+enterica+serovar+Typhimurium.">sRNAs and the virulence of Salmonella enterica serovar Typhimurium.</a> RNA Biology. 9 (4), 437-445 (2012).</li><li>Vogel, J., Papenfort, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+non-coding+RNAs+and+the+bacterial+outer+membrane.">Small non-coding RNAs and the bacterial outer membrane.</a> Current Opinion in Microbiology. 9 (6), 605-611 (2006).</li><li>Papenfort, K., 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=SigmaE-dependent+small+RNAs+of+Salmonella+respond+to+membrane+stress+by+accelerating+global+omp+mRNA+decay.">SigmaE-dependent small RNAs of Salmonella respond to membrane stress by accelerating global omp mRNA decay.</a> Molecular Microbiology. 62 (6), 1674-1688 (2006).</li><li>Udekwu, K. I., 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=Hfq-dependent+regulation+of+OmpA+synthesis+is+mediated+by+an+antisense+RNA.">Hfq-dependent regulation of OmpA synthesis is mediated by an antisense RNA.</a> Genes and Development. 19 (19), 2355-2366 (2005).</li><li>Papenfort, K., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+target+regulation+by+small+noncoding+RNAs+rewires+gene+expression+at+the+post-transcriptional+level.">Multiple target regulation by small noncoding RNAs rewires gene expression at the post-transcriptional level.</a> Research in Microbiology. 160 (4), 278-287 (2009).</li><li>Lease, R. A., Cusick, M. E., Belfort, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riboregulation+in+Escherichia+coli:+DsrA+RNA+acts+by+RNA:RNA+interactions+at+multiple+loci.">Riboregulation in Escherichia coli: DsrA RNA acts by RNA:RNA interactions at multiple loci.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (21), 12456-12461 (1998).</li><li>Sharma, C. M., Darfeuille, F., Plantinga, T. H., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+RNA+regulates+multiple+ABC+transporter+mRNAs+by+targeting+C/A-rich+elements+inside+and+upstream+of+ribosome-binding+sites.">A small RNA regulates multiple ABC transporter mRNAs by targeting C/A-rich elements inside and upstream of ribosome-binding sites.</a> Genes and Development. 21 (21), 2804-2817 (2007).</li><li>Boisset, S., 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=Staphylococcus+aureus+RNAIII+coordinately+represses+the+synthesis+of+virulence+factors+and+the+transcription+regulator+Rot+by+an+antisense+mechanism.">Staphylococcus aureus RNAIII coordinately represses the synthesis of virulence factors and the transcription regulator Rot by an antisense mechanism.</a> Genes and Development. 21 (11), 1353-1366 (2007).</li><li>Mass&#233;, E., Vanderpool, C. K., Gottesman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+RyhB+small+RNA+on+global+iron+use+in+Escherichia+coli.">Effect of RyhB small RNA on global iron use in Escherichia coli.</a> Journal of Bacteriology. 187 (20), 6962-6971 (2005).</li><li>Papenfort, K., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+RNA+in+bacterial+pathogens.">Regulatory RNA in bacterial pathogens.</a> Cell Host &amp; Microbe. 8 (1), 116-127 (2010).</li></ol>

S. Enteritidis is an important facultative intracellular pathogen that can infect young chickens and produces symptoms from enteritis to systemic infection and death17,18. In addition, S. Enteritidis causes latent infections in adult chickens and chronic carriers contaminate poultry products, resulting in food-borne infections in humans19. The pathogenic mechanism of S. Enteritidis remains to be further probed. T...

Non-coding small RNAs (sRNAs) are 40-400 nucleotides in length, which generally do not encode proteins but could be transcribed independently in bacterial chromosomes1,2,3. Most sRNAs are encoded in the intergenic regions (IGRs) between gene-coding regions and interact with target mRNAs through base-pairing actions, and regulate target genes expression at the post-transcriptional level4,5. They play important regulation roles in substance metabolism, outer membrane protein synthesis, quorum sensing and virulence gene expression5.
MicC is a 109-nucleotide small RNA transcript present in Escherichia coli and Salmonella enterica serovar Typhimurium, which could regulate multiple outer membrane protein expression such as OmpC, OmpD, OmpN, Omp35 and Omp366,7,8,9. MicC regulates the expression of OmpC by inhibiting ribosome binding to the ompC mRNA leader in vitro and it requires the Hfq RNA chaperone for its function in Escherichia coli6. In Salmonella Typhimurium, MicC silences ompD mRNA via a &#8804;12-bp RNA duplex within the coding sequence (codons 23-26) and then destabilizes endonucleolytic mRNA7. This regulation process is assisted by chaperone protein Hfq10. The OmpC is an abundant outer membrane protein that was thought to be important in environments where nutrient and toxin concentrations were&#160;high, such as in the intestine6. The OmpD porin is the most abundant outer membrane protein in Salmonella Typhimurium and represents about 1% of total cell protein11. OmpD is involved in adherence to human macrophages and intestinal epithelial cells12. MicC also represses the expression of both OmpC and OmpD porins. It is thought that MicC may regulate virulence. To explore new target genes regulated by MicC and study the virulence regulation function of micC, we cloned the micC gene in the Salmonella Enteritidis (SE) strain 50336, and then constructed the mutant 50336&#916;micC and the complemented mutant 50336&#916;micC/pmicC. Novel target genes were screened by qRT-PCR. The virulence of 50336&#916;micC was detected by mice and chicken infections.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>dextrose</td>
			<td>Sangon Biotech</td>
			<td>A610219</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>DNA purification kit</td>
			<td>TIANGEN</td>
			<td>DP214</td>
			<td>for DNA purification</td>
		</tr>
		<tr>
			<td>Ex Taq</td>
			<td>TaKaRa</td>
			<td>RR01A</td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10017608</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>20032116</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>L-Arabinose</td>
			<td>Sangon Biotech</td>
			<td>A610071</td>
			<td>&#955;-Red recombination</td>
		</tr>
		<tr>
			<td>Mini Plasmid Kit</td>
			<td>TIANGEN</td>
			<td>DP106</td>
			<td>plasmid extraction</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10019308</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10002917</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>PrimeScriptRRT reagent Kit with gDNA Eraser&#160;</td>
			<td>TaKaRa</td>
			<td>RR047</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>SYBRR Premix Ex Taq II</td>
			<td>TaKaRa</td>
			<td>RR820</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202</td>
			<td>Ligation</td>
		</tr>
		<tr>
			<td>TRIzol&#160;</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Oxoid</td>
			<td>LP0021</td>
			<td>for broth preparation</td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Eppendorf</td>
			<td>5418</td>
			<td>centrifugation</td>
		</tr>
		<tr>
			<td>Electrophoresis apparatus</td>
			<td>Bio-Rad</td>
			<td>164-5050</td>
			<td>Electrophoresis</td>
		</tr>
		<tr>
			<td>&#160;Electroporation System</td>
			<td>Bio-Rad</td>
			<td>165-2100</td>
			<td>for bacterial transformation</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>BioTek</td>
			<td>Epoch</td>
			<td>Absorbance detection</td>
		</tr>
		<tr>
			<td>Real-Time PCR system</td>
			<td>Applied Biosystems</td>
			<td>7500 system</td>
			<td>qRT-PCR</td>
		</tr>
	</tbody>
</table>

a non-coding small rna micc contributes to virulence in outer membrane proteins in salmonella enteritidis

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia coli and Salmonella Typhimurium, could repress the expression of outer membrane proteins. To further investigate the regulation function of micC in Salmonella Enteritidis, we cloned the micC gene in the Salmonella Enteritidis strain 50336, and then constructed the mutant 50336&#916;micC by the &#955; Red-based recombination system and the complemented mutant 50336&#916;micC/pmicC carrying recombinant plasmid pBR322 expressing micC. qRT-PCR results demonstrated that transcription of ompD in 50336&#916;micC was 1.3-fold higher than that in the wild type strain, while the transcription of ompA and ompC in 50336&#916;micC were 2.2-fold and 3-fold higher than those in the wild type strain. These indicated that micC represses the expression of ompA and ompC. In the following study, the pathogenicity of 50336&#916;micC was detected by both infecting 6-week-old Balb/c mice and 1-day-old chickens. The result showed that the LD50 of the wild type strain 50336, the mutants 50336&#916;micC and 50336&#916;micC/pmicC for 6-week-old Balb/c mice were 12.59 CFU, 5.01 CFU, and 19.95 CFU, respectively. The LD50 of the strains for 1-day-old chickens were 1.13 x 109 CFU, 1.55 x 108 CFU, and 2.54 x 108 CFU, respectively. It indicated that deletion of micC enhanced virulence of S. Enteritidis in mice and chickens by regulating expression of outer membrane proteins.

Construction of the mutant 50336&#916;micC and complemented strain 50336&#916;micC /pmicC 
The micC gene clone result indicated that this gene was composed of 109 bp showing 100% identity with that of S. Typhimurium. Based on the sequence data, the deletion mutant 50336&#916;micC and the complemented mutant 50336&#916;micC/pmicC w...

Watch this Scientific Journal Video about A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis at JoVE.com

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

An &#955;-Red-mediated recombination system was used to create a deletion mutant of a small non-coding RNA micC.

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia ...

This protocol can be used for inactivating chromosomal genes to isolate mutants for gene function study and bacteria. It makes the homologous recombination process simple, fast, and highly efficient. Using this technique, the virulence related genes of the pathogen can be deleted to obtain the attenuated mutant which can be used as a candidate for the attenuated vaccine.Begin by amplifying the chloramphenicol cassette containing hemology fragments of the micC gene. Design forward and reverse primers to amplify the chloramphenicol cassette from plasmid PKD3, including 50 base pair homology extensions from the five prime and three prime ends of the micC gene. Perform PCR as described in the text manuscript using the PKD3 plasmid as the template to amplify the chloramphenicol cassette.Determine the size of the PCR product with agarose gel electrophoresis. Then purify the product with a DNA gel recovery kit and determine the concentration of DNA by spectrophotometry. Carry out a second PCR reaction to eliminate the interference of further recombination by the PKD3 plasmid.Dilute the first PCR product at a ratio of 1 to 200 and use it as a template for the secondary PCR. To construct the first recombinant strain 50336 delta micC dot cat, mix 100 microliters of SE 50336 competent cells with five microliters of PKD 46 plasmid and incubate on ice for 30 minutes. Heat shock the mixture at 42 degrees Celsius for 90 seconds, and rapidly transfer it back on ice for two minutes to transform the plasmid into the cells.Screen positive colonies by culturing the cells overnight at 30 degrees Celsius on an ampicillin resistant plate. The next day, add 30 milli more L arabinose to the SE 50336 PKD 46 liquid culture and induce recombinase expression with a one hour incubation at 30 degrees Celsius while shaking. Mix 100 nanograms of the purified PCR product and 40 microliters of SE 50336 PKD 46 competent cells in an electric shock cup.Then carry out electric shock transformation. After electro transformation, transfer the mixture to one milliliter of SOC medium and a shaking culture incubator at 150 RPM and 30 degrees Celsius for one hour. Spread the mixture on a chloramphenicol resistant LB plate and culture at 37 degrees Celsius overnight to screen for positive colonies.Culture the positive colonies at 42 degrees Celsius for two hours. Then screen the colony for sensitivity to ampicillin and resistance to chloramphenicol at 37 degrees Celsius overnight to obtain the first recombinant strain without PKD 46. After extracting the 50336 Delta micC dot cat genomic DNA using PCR and gel electrophoresis, electroplate 100 nanograms of plasmid PCP 20 into 40 microliters of 50336 Delta micC dot cat competent cells.Screen positive transformants on both ampicillin and chloramphenicol resistant plates at 30 degrees Celsius. Transfer the positive transformants into non-resistant LB liquid medium and culture them overnight at 42 degrees Celsius. Then isolate single colonies on an LB plate at 37 degrees Celsius.Select the colony that is sensitive to both ampicillin and chloramphenicol. This mutant is the micC deletion mutant SE 50336 Delta micC. Verify 50336 Delta micC by performing PCR as described in the text manuscript.This protocol was used to construct the deletion mutant 50336 Delta micC in the complimented mutant 50336 Delta micC, pmicC. The first recombinant 50336 Delta micC dot cat was validated by PCR with an expected band size of about 1200 base pairs of PCR products with the chloramphenicol insertion compared to 279 base pairs in the wild type strain. In the second recombination, the chloramphenicol cassette was eliminated by PCP 20.The PCR results combined with sequencing confirmed that the isogenic micC mutant was constructed successfully. The expression of ompA, ompC, and ompD genes in strains 50336, 50336 Delta micC, and 50336 delta micC pmicC were analyzed by realtime quantitative PCR. Transcription of ompA and ompC and 50336 delta micC increased about 2.2 fold and threefold compared to the wild type strain.While transcription of ompD only increased slightly. LD 50 assays showed that after infecting six to eight week old BALB/c mice with each of the three strains, most mice infected by 50336 Delta micC displayed lassitude, inappetence, or diarrhea 48 hours post-infection and died after 96 hours. The mice infected by wild type strain and 50336 delta micC pmicC displayed the same symptoms 72 hours post infection and died after 120 hours.The LD 50s of the three strains indicated that the virulence of the mutant 50336 Delta micC enhanced 2.5 fold as compared to the wild type. This technique helps researchers study the function of bacterial genes and obtain the desired engineered strain by deleting target genes.

Research

JoVE Journal

Immunology and Infection

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