Supplementary MaterialsAdditional file 1 Table S1. capsulatus /em with a 31% identity and a 73% similarity. DctP, along with DctQ and DctM, constitutes a tripartite ATP-independent periplasmic transporter (TRAP-T) system involved in succinate utilization, and DctP plays a role as an extracytoplasmic solute receptor in this transporter [34]. STM3170 and STM3171, which Velcade supplier are located immediately downstream from STM3169, have a 66% and an 80% similarity with DctQ and DctM, respectively. These suggest that the TRAP-T in em S /em . Typhimurium is composed of em stm3169 /em , em stm3170 /em , and em stm3171 /em genes. In addition, two hypothetical operons, em yiaOMN /em and em stm4052-4054 /em , are annotated as TRAP-T in the em S /em . Typhimurium strain LT2 [31]. Recently, it has been reported that the TRAP-T (SiaPQM) in em Haemophilus influenzae /em is essential for LPS sialylation and virulence [35]. Further research is necessary to determine the role of these transporters Velcade supplier in em S /em . Typhimurium virulence. Conclusions We constructed an agarose 2-DE reference map of amino-acid starved em S /em . Typhimurium and identified a novel virulence-associated factor, STM3169, regulated by ppGpp by applying the map to comparative proteomics. em stm3169 /em is also regulated by an SPI-2 two-component regulator, SsrB. Recently, it has been reported that the lack of ppGpp synthesis in em Salmonella /em strains attenuates virulence and induces immune responses in mice [36]. Thus, further analysis of proteins regulated by ppGpp may lead to the development of new vaccines. Methods Bacterial strains, primers, and culture conditions The bacterial strains and plasmids used in this study are listed in Table ?Table2.2. The oligonucleotide primers used are listed in Table ?Table3.3. Bacteria were grown in Luria-Bertani (LB) medium or on LB agar under conditions suitable for selection for resistance to ampicillin (100 g/mL), chloramphenicol (25 g/mL), nalidixic acid (50 g/mL), or spectinomycin (50 g/mL), as appropriate. To induce the bacterial stringent response, serine hydroxamate (Sigma; 0.005%), an inhibitor of serine tRNA synthetase, was added to a 12 h culture in LB broth, and the bacteria were further incubated for 1 h [26]. Magnesium minimal medium (MgM, pH 5.8) was used to induce SPI-2 gene Velcade supplier expression [6]. Table 2 Bacterial strains and plasmids used. thead th align=”left” rowspan=”1″ colspan=”1″ Strains /th th align=”left” rowspan=”1″ colspan=”1″ Relevant characteristics /th th align=”left” rowspan=”1″ colspan=”1″ Source/Ref. /th /thead Bacterial strains br / em S /em . Typhimurium14028wild-typeATCCSH100Spontaneous nalidixic acid resistant derivative of wild-type 14028[44]TM157SH100 em relA /em :: em cat /em em spoT /em :: em kan /em this studyYY2SH100 em relA /em :: em cat /em em spoT /em :: em kan /em em ssrB /em :: em tet /em this studyTH973SH100 em stm3169 /em :: em kan /em this studyTH1162SH100 em stm3169 /em :: em lacZ /em this studyTH1164TM157 em stm3169 /em :: em lacZ /em this studyYY3TH1164 em ssrB /em :: em tet /em this studyTM129SH100 em ssaG /em :: em lacZ /em this studyYY1SH100 em ssrB /em :: em tet /em this studySH113SH100 em ssaV /em :: em cat /em [11]TM548SH100 em sseF /em :: em kan /em this study em E. coli /em DH5K-12 em recA1 endA1 gyrA96 thi-1 hsdR17 supE44 /em ( em lacXYA-argR /em ) em U169 deoR /em ( em 80 dlac /em ( em lacZ /em ) em M15 /em )InvirtogenSM10 em pir /em em thi-1 thr leu tonA lacY supE recA /em ::RP4-2-Tc::Mu em pir /em [45]PlasmidspGEM-TeasyTA cloning vectorPromegapMW118pSC101-based low copy number plasmidNippon GenepACYC184p15A-based low copy number plasmid, em tet /em templateNew England BiolabspFLAG-CTCFLAG tag expression vectorSigmapLD- em lacZ /em Integrational plasmid with promoterless em lacZ /em gene[39]pBAD-HisAExpression vector for His6 fusion proteinInvitrogenpMW-Stm3169 em stm3169 /em gene in pMW118this studypLD-stm3169Z em stm3169 /em :: em lacZ /em operon fusion in pLD- em lacZ /em this studypLD-ssaGZ em ssaG /em :: em lacZ /em operon fusion in pLD- em lacZ /em this studypRelApBAD-HisA expressing em relA /em genethis studypSsrBpFLAG-CTC expressing em ssrB /em genethis studypKD46Red recombinase expression plasmid[41]pKD4 em CTSD kan /em cassette used for gene deletion[41] Open in a separate window Table 3 Primers thead th align=”left” rowspan=”1″ colspan=”1″ Name /th th align=”left” rowspan=”1″ colspan=”1″ Nucleotide sequence (5′ to 3′) em a /em /th /thead Construction of the deletion mutantsrelA-FWCGCCATCCCGCAATGGTTTACATAArelA-RVTCATTGTTCTGGCCATAACAGCspoT-FWCTTGAAAACCATCATTCGCGCTGAACGspoT-RVTCTGCGGTACGAATGATTGCAGAAACGstm3169-red-FWACGTTCATTCACAACATCAGCGGTATTACTGGCCGGCTGTGTGTAGGCTGGAGCTGCTTCstm3169-red-RWACATATTCTCGATGTATTCCAGATCCTTCGCTGACTGAGCCATATGAATATCCTCCTTAGsseF-red-FWAACAGAACGAAATATGAAAATTCATATTCCGTCAGCGGCAGTGTAGGCTGGAGCTGCTTCsseF-red-RWTGTCCATTAATGCAGGTGTAGTAGCAGATTGACAGAGCGCCATATGAATATCCTCCTTAGpAC-tet-FWTTGGTAGCTCAGAGAACCTTCGAAAAACCGpAC-tet-RVTCGCTCGCGTATCGGTGATTCATTCGCTAConstruction of plasmids for the complementationsrelA-FW2AGGCTCGAGGTCGCGGTAAGAAGTrelA-RV2ACAAGCTTACTGTCTGGGGTTTACssrB-FWGGGCTCGAGGAATATAAGATCTTATTAGTAssrB-RVCCCGGATCCATACTCTATTAACCTCATTCTstm3169-FWCCGCTCGAGAACACACGTTCATTCACAACATCAGstm3169-RVGGAAGATCTATTCTCGATGTATTCCAGATCCTTCConstrucion of the em lacZ /em fusionsssaG-Pro-FWAAAGTCGACCAAATGCTCAGGTAGGAGGGCssaG-Pro-RVAAAGGATCCATCATCGATTCTGGGTTGAGCstm3169-Pro-FWACGCGTCGACGACGATTTAGCCGGTATGAAAATCAstm3169-Pro-RVCGGGATCCTTACATATTCTCGATGTATTCCAGAComfirmation of gene expression by qRT-PCRgyrA-FWAAGAGCTCCTATCTGGATTATGCgyrA-RVTATTTACCGATTACGTCACCAACrelA-FWATTGTGCCATTCACCTATCAGTTrelA-RVGATATTTTTGTCACGATCCTGCTinvF-FWATCGCTGCTGAATAGTGTAGAAGinvF-RVCATTTGTCTGCCAATTGAATAATstm0209-FWCCTGAACGTAGAAAATTACGAGAstm0209-RVGTCAGGTTTTTCACCATGTTACTstm0435-FWGTCAATCAGTTGCTCGATATTCTGstm0435-RVTTTAATCAGCTTGACGATTTTCTTCstm0748-FWTGAACCTGTACGTTATGGATCTCstm0748-RVCGCCGTTAATGTTCATTTTATACstm0781-FWGAAGGCAAGATCACCGTATTTstm0781-RVCTGATCAGCAGAGATGAAGAGATstm1478-FWACAAAAGTTGAGGAGCTGAATAAAGstm1478-RVGCCACTGACGCGTAATATGATAAstm1720-FWTTGGTTGTAAAATTGAAGACAAAGGstm1720-RVGTCCCCTTCAGGATAAAGGTGTAATstm1746-FWCGAATTATTCCAGAAACTGAAGAAAstm1746-RVATCGCCCTGATTTTTAACCTTATTAstm2638-FWTATTCTGACGGTCTGTTTAGCTTTTstm2638-RVGTACTGCCCTGAATTTGATACTGTCstm3169-FWGTTACCAGAATAATGTCGCAGCTATstm3169-RVAATCATCCACATAAAAAGAATCTGGstm3318-FWCAAACTCAGCCTTAATCTTATGCstm3318-RVACTTTATCGGCGTTGATCTTAATstm4319-FWATTAGTATTATCCGAGGCCAGACstm4319-RVCAGTCTTGCAAACTCTACTGCTCstm4403-FWATTGATATTCACAGCAACAAACCstm4403-RVAGGTCAGGTTTTTAATACGTTCCstm4405-FWCGAACTGACGTTGAATAAAGATGAGstm4405-RVAATTGTGGTCGTCTGGTATCTGT Open in a separate window aUnderlined part indicates P1 or P2 site for pKD4, or restriction sites. Construction of mutants Nonpolar mutants of em relA /em and em spoT /em were constructed by allele exchange using the temperature- and sucrose-sensitive suicide vector pCACTUS [37]. The em relA /em and em spoT /em genes were amplified by PCR with the following primers: (1) relA-FW and relA-RV for em relA /em and (2) spoT-FW and spoT-RV for em spoT. S /em . Typhimurium strain SH100 genomic DNA was used as the template. The PCR products were cloned into TA cloning vector pGEM-T Easy (Promega) generating plasmid pGEM- em relA /em and pGEM- em spoT /em , respectively. A disruption mutation of em relA /em was created by the insertion of the HincII-digested promoterless em cat /em gene into a unique NruI site in the coding sequence of em relA /em on pGEM- em relA /em . The resulting plasmid pGEM- em relA /em :: em Velcade supplier cat /em was digested with BglII and then self-ligated, yielding plasmid pGEM- em relA /em :: em Velcade supplier cat /em . In contrast, the em spoT /em gene was disrupted by the insertion of a SmaI-digested Kmr-encoding gene ( em kan /em ) cassette from pUC18K [38] into NruI sites in the coding sequence of em spoT /em on pGEM- em spoT /em , thus generating pGEM- em spoT /em :: em kan /em . The disrupted gene.