Supplemental Materials - Genes & Development



Supplemental Data

Supplemental Materials and Methods

Analysis of Surface-Extracted Proteins

Surface-extracted fractions were prepared from L. monocytogenes cultures grown at RT for 20 hr without shaking as described (Gründling et al. 2004). A culture volume equivalent to 0.4 mL of an OD600=1.1 for wild-type, ∆degU, and ∆mogR and 0.8 mL for ∆mogR ∆degU was resolved on a 12% SDS-PAGE gel and stained with Coomassie.

Western Blot Analysis

For detection of FlaA in cellular fractions (Figure 1), L. monocytogenes were grown at RT without shaking for 20 hr and then pelleted at 16,000 x g for 1 min. Sample preparation and Western blot analysis were performed as described (Shen and Higgins 2006), except that an anti-FlaA antibody generated against L. monocytogenes His6-tagged FlaA purified from E. coli cell lysates was used (Cocalico).

For detection of (-O-linked GlcNAc modification of proteins, surface-extracted proteins were prepared from cultures of L. monocytogenes grown at RT for 20 hr as described (Gründling et al. 2004). For Western blot analysis, immunoblots were blocked in 2.5% BSA, then probed with an anti-(-O-linked GlcNAc specific antibody (mAb CTD110.6, Pierce). This antibody has been shown to be specific for (-O-linked GlcNAc and not (-O-linked GlcNAc residues (Comer et al. 2001). The primary antibody incubation was followed by an anti-mouse horseradish peroxidase-conjugated secondary antibody (BioRad), and detected by ECL using Western Lightning (Perkin Elmer).

For analysis of FlaA and Lmo0688 proteins in Figure 3 and Figure 6, 14 to 16 hr cultures grown at 37(C were diluted 1:12 in BHI and grown at either RT or 37(C without shaking for 6 hr. A culture volume equivalent of 1 mL of OD600=1.5 was pelleted and resuspended in 75 μL of TE/lysozyme (10 mM Tris-HCL [pH 8.0], 1 mM EDTA, 3 mg/mL lysozyme) and incubated at 37(C for 1 hr. Next, 75 (L of 2X final sample buffer was added, samples were boiled for 5 min at 95(C and then centrifuged for 5 min at 16, 000 x g. Ten microliters of the boiled sample was loaded onto a 6% SDS-PAGE gel for analysis of Lmo0688, and 2.5 (L of the sample was loaded onto a 12% SDS-PAGE gel for analysis of FlaA. Western blot analysis was performed as described (Shen and Higgins 2006) using either an anti-FlaA antibody or an anti-Lmo0688 antibody generated against His6-tagged Lmo0688 purified from E. coli lysates (Cocalico).

Northern Blot Analysis

Bacterial cultures were grown in BHI at RT either 20 hr for detection of flaA or 6 hr for detection of lmo0688. RNA was extracted from 10 mL of culture as previously described (Gründling et al. 2004). Primer pair #382 and #511 was used to amplify DNA probes for flaA and primer pair #530 and #531 was used to amplify DNA probes for lmo0688 from wild-type L. monocytogenes strain EGDe genomic DNA. The resulting DNA probes were gel-purified, eluted in TE, and approximately 100 ng of probe DNA was random-primed labeled with 50 (Ci [(32P]-dCTP using the Rediprime II random primer labeling kit (GE Healthcare) according to the manufacturer’s instructions. Unincorporated nucleotides were removed using G25 Sephadex TE columns (Roche). Fifteen micrograms of total RNA was mixed with 15 (L of loading buffer (12.9% 5X MOPS, 22.5% formaldehyde, 64.5% formamide), denatured for 5 min at 65(C, and immediately transferred to ice. 10X glycerol loading buffer (50% glycerol, 0.25% bromophenol blue, 0.25% xylene cyanol, 10 mM EDTA) was added, and samples were resolved on a 1.25% agarose gel containing 5% formaldehyde. RNA was transferred overnight in 20X SSC by capillary action to Hybond N+ membrane (GE Healthcare) and UV crosslinked to the membrane. Denatured radiolabeled DNA probe was added to the membrane in Church buffer (0.5 M sodium phosphate buffer, pH 7.2, 7% SDS, 1 mM EDTA, 1% BSA) and hybridized 14 to 16 hr at 65(C. The membrane was washed twice at 65(C for 20 min in 2X SSC, 0.5% SDS and once in 0.2X SSC, 0.5% SDS for 20 min. Autoradiography was used to visualize hybridized transcripts.

Purification of His6-Tagged Lmo0688

Fourteen to sixteen hour cultures of DH-E1392 and DH-E1393 were diluted 1:1000 in 2 L of 2YT media, grown 4 hr at 37˚C and then induced with 350 (M IPTG for 15 hr at 18˚C. Cultures were pelleted, resuspended in 30 mL of lysis buffer [500 mM NaCl, 100 mM Tris, pH 7.5, 15 mM imidazole, 10% glycerol supplemented with 2 mM (-mercaptoethanol, 1 mg/mL lysozyme, and Complete–EDTA protease inhibitor mixture (Roche)], and lysed by sonication. His6-tagged Lmo0688 and His6-tagged D83N D85N were purified from the cleared lysate by Ni-affinity chromatography using a 1 mL HiTrap FF column (GE Healthcare) on an Aktaprime FPLC (GE Healthcare). Protein concentration was determined by measuring the absorbance at 280 nm. His6-tagged Lmo0688 used in gel shift assays was diluted into 1X binding buffer (100 mM NaCl/10% glycerol/1 mM MgCl2/10 mM Tris pH 7.5/0.5 mM EDTA/0.5 mM DTT/12.5 (g/mL salmon sperm DNA/50 (g/mL BSA).

Purification of His6-Tagged MogR and His6-Tagged DegU

Ni2+-affinity purification of His6-tagged MogR and His6-tagged DegU was performed as previously described (Shen and Higgins 2006).

Gel Mobility Shift Analysis

Gel shift analysis was performed as previously described in the Materials and Methods with the exception that a cheY DNA probe was used (Shen and Higgins 2006).

Affinity Pull-Down Assays

Affinity pull-down assays were performed as described in Materials and Methods with the exception that 3 μg of His6-tagged MogR was added to each volume of lysate and Ni-NTA beads.

Strain Construction

Primer pair #279 and #280 was used with wild-type EGDe genomic DNA to amplify ~1.0 kB of the region upstream of lmo0688. Primers #281 and #282 and EGDe genomic DNA were used to amplify ~1 kB of sequence downstream of lmo0688. The 5’ and 3’ PCR products were gel purified using the QIAquick gel extraction kit (Qiagen) and used as templates for a splicing by overlap extension (SOE) PCR reaction (Horton et al. 1989). The flanking primers, #279 and #281, were used to amplify an ~2.0 kB PCR product containing an in-frame deletion of sequences encoding amino acids 11 to 627 of Lmo0688. The SOE PCR product was gel-purified, digested with XbaI and BamHI, ligated to plasmid pCON1 digested with the same restriction enzymes, and transformed into XL1-Blue to create strain DH-E1043. The resulting plasmid, pCON1/∆lmo0688 was introduced into wild-type, ∆mogR, and ∆mogR ∆flaA by electroporation, and allelic exchange was performed (Camilli et al. 1993) to generate strains ∆688 (DH-L1056), ∆mogR ∆688 (DH-L1371), ∆mogR ∆flaA ∆688 (DH-L1429) respectively.

To create a complementing construct of lmo0688, the lmo0688 coding sequence was cloned into pLOV (Higgins 2006). pLOV is a site-specific integration vector that allows over-expression of genes in L. monocytogenes based on the observation that fusion of coding sequences to the hly 5’ UTR can increase expression of genes in L. monocytogenes (Shen and Higgins 2005). The pLOV vector was created by amplifying a PCR fragment containing the hly 5’ UTR sequence lacking the hly ribosome binding site (RBS) using primer pair #168 and #204 and wild-type L. monocytogenes 10403S genomic DNA as a template. The resulting PCR product was gel purified, digested with EagI and PstI, ligated to pHPL3 (Gründling et al. 2004) digested with the same restriction enzymes, transformed into XL1-Blue to generate strain DH-E1225. The resulting plasmid was designated pLOV. lmo0688 carrying an optimized ermC RBS was amplified from EGDe genomic DNA using primers #513 and #313. The resulting PCR product was gel purified, digested with SalI and PstI, ligated into pLOV digested with the same restriction enzymes, and transformed into XL1-Blue to create strain DH-E1421. The resulting plasmid pLOV/c688 was sequenced and transformed into (688 by electroporation to create (688/c688 (DH-L1422). To create (degU/c688 (DH-L1423), the SM10 E. coli conjugation strain was transformed with pLOV/c688 yielding DH-E1428 and mated with (degU (DH-L1273).

Active site mutations in lmo0688 were introduced by allelic exchange into wild-type L. monocytogenes. Primer pair #279 and #515 was used with wild-type EGDe genomic DNA to amplify ~1.4 kB of the region upstream of the residues encoding the Lmo0688 active site. Primers #514 and #516 and EGDe genomic DNA were used to amplify ~1 kB of sequence downstream of the residues encoding the lmo0688 active site. The 5’ and 3’ PCR products were gel purified using the QIAquick gel extraction kit (Qiagen) and used as templates for a PCR SOE reaction. The flanking primers, #279 and #516, were used to amplify a ~2.4 kB PCR product containing point mutations G247A and G253A of lmo0688. The SOE PCR product was gel-purified, digested with XbaI and KpnI, ligated to plasmid pCON1 digested with the same restriction enzymes, and transformed into CLG190 to create strain DH-E1387. The resulting plasmid, pCON1/688*, was introduced into wild-type L. monocytogenes EGDe and allelic exchange was performed to generate strain 688* (DH-L1386).

To construct a pET vector for IPTG-inducible expression of flaA, primer pair #510 and #511 was used to amplify the flaA coding sequence lacking the stop codon using EGDe genomic DNA as the template. The resulting PCR product were digested with NdeI and SalI, ligated to pET22b digested with the same enzymes, and transformed into XL1-Blue to yield strain DH-E1388. The resulting plasmid, pET22b/FlaA, was transformed into BL21(DE3) (Novagen, EMD Biosciences) to yield strain DH-E1389.

To construct IPTG-inducible pET constructs for purification of His6-tagged Lmo0688 and His6-tagged D83N D85N, primer pair #519 and #520 was used to amplify the lmo0688 coding sequence lacking the stop codon using EGDe or 688* genomic DNA as the template. The resulting PCR products were digested with NcoI and SalI, then ligated into the pET28a expression vector digested with the same restriction enzymes. The ligations were transformed into XL1-Blue to create strains DH-E1391 and DH-E1394, respectively. The pET28a/lmo0688 and pET28a/D83N D85N plasmids were transformed into BL21(DE3) to create strains DH-E1392 and DH-E1393, respectively, for expression studies.

To construct an IPTG-inducible pET construct for purification of His6-tagged DegU, primer pair #535 and #486 was used to amplify the degU coding sequence including the stop codon using EGDe genomic DNA as the template. The resulting PCR product was digested with NdeI and SalI, then ligated into the pET28a expression vector digested with the same restriction enzymes. The ligation was transformed into XL1-Blue to create strain DH-E1445 harboring plasmid pET28a/degU, which was subsequently transformed into JM109(DE3) pLysS to create strain DH-E1446, which allows for tightly controlled, IPTG-inducible expression of N-terminally His6-tagged DegU.

To express mogR from an IPTG-inducible promoter in ∆mogR, the mogR coding sequence including the RBS was amplified from EGDe genomic DNA using primers #371 and #372. The resulting PCR product was digested with BamHI and SalI, ligated to pHLIV2 digested with the same enzymes, and transformed into XL1-Blue to create strain DH-E1152 harboring plasmid pHLIV2/mogR. The pHLIV2/mogR plasmid was electroporated into ∆mogR to generate imogR (DH-L1367). pHLIV2 is a site-specific integration vector derived from pPL2 (Lauer et al. 2002) that was modified to allow high-level, IPTG-inducible chromosomal expression of genes in L. monocytogenes. The HyperSPAC/lacOid promoter of pHLIV2 was generated by PCR using pLIV1 (Dancz et al. 2002) as a template and primer pair #324 and #325; this primer pair introduces a point mutation in the transcriptional start site of the SPAC/lacOid promoter that increases transcription initiating from the HyperSPAC/lacOid promoter. The resulting PCR product was digested with BstXI and EagI, ligated to pPL3 (Gründling et al. 2004) digested with the same enzymes, and transformed into XL1-Blue to yield strain DH-E1433 harboring plasmid pPL3+HyperOid. The lacI gene under the control of the constitutive p60 promoter (Kuhn and Goebel 1989) was cloned from pLIV1 using primer pair #508 and #509. The resulting PCR product was digested with SalI and KpnI, ligated to pPL3+HyperOid digested with the same enzymes, and transformed into XL1-Blue to yield strain DH-E1093 harboring plasmid pHLIV2.

To create the flaA::Tn917 strains constitutively expressing lmo0688, the L. monocytogenes phage P35 was used to transduce the flaA::Tn917 transposon insertion from DH-L975 as previously described (Gründling et al. 2004) into ∆688/c688 and ∆degU/c688 to generate strains DH-L1426 and DH-L1427.

Supplemental Tables

Table S1. Microarray analysis of genes down-regulated in Lmo0688-negative L. monocytogenes relative to wild-type during growth in BHI at RT

|Gene | *Fold | p-value |Sequence Description |

| |Change | | |

|lmo0036 |-2.6 |7.31E-06 |similar to ornithine carbamoyltransferase |

|lmo0037 |-2.6 |1.62E-14 |similar to amino acid transporter |

|lmo0398 |-5.3 |0.00004 |similar to phosphotransferase system enzyme IIA |

|lmo0399 |-5.6 |0.00022 |similar to fructose-specific phosphotransferase enzyme IIB |

|lmo0400 |-7.2 |0.00028 |similar to fructose-specific phosphotransferase enzyme IIC |

|lmo0464 |-6.0 |0.01924 |weakly similar to transposase |

|lmo0468 |-3.0 |0.01451 |unknown |

|lmo0585 |-4.9 |0.00588 |putative secreted protein |

|lmo0673 |-4.4 |6.95E-07 |unknown |

|lmo0675 |-3.3 |0.00102 |similar to flagellar switch protein FliN |

|lmo0676 |-2.9 |0.00005 |similar to flagellar biosynthetic protein FliP |

|lmo0677 |-4.0 |0.00568 |similar to flagellar biosynthetic protein FliQ |

|lmo0678 |-12.5 |1.71E-08 |similar to flagellar biosynthetic protein FliR |

|lmo0679 |-100.0 |6.44E-07 |similar to flagellar biosynthetic protein FlhB |

|lmo0680 |-100.0 |0 |similar to flagellar biosynthetic protein FlhA |

|lmo0681 |-4.5 |4.38E-09 |similar to flagellar biosynthetic protein FlhF |

|lmo0683 |-5.2 |0.01296 |similar to chemotactic methyltransferase CheR |

|lmo0684 |-5.6 |0.00006 |unknown |

|lmo0685 |-40.1 |0.00238 |similar to motility protein (flagellar motor rotation) MotA |

|lmo0686 |-7.7 |0.00003 |similar to motility protein (flagellar motor rotation) MotB |

|lmo0687 |-10.1 |1.42E-07 |unknown |

|lmo0689 |-97.1 |0 |similar to CheA activity-modulating chemotaxis protein CheV |

|flaA |-100.0 |0 |Flagellin protein |

|cheY |-9.2 |3.41E-10 |Chemotaxis response regulator CheY |

|cheA |-12.2 |5.20E-23 |two-component sensor histidine kinase CheA |

|lmo0693 |-57.4 |2.60E-18 |similar to flagellar motor switch protein FliY C-terminal part |

|lmo0694 |-10.2 |1.18E-09 |unknown |

|lmo0695 |-100.0 |2.14E-40 |unknown |

|lmo0696 |-12.2 |0.00229 |similar to flagellar hook assembly protein |

|lmo0697 |-87.0 |2.34E-36 |similar to flagellar hook protein FlgE |

|lmo0698 |-32.8 |9.09E-25 |weakly similar to flagellar switch protein |

|lmo0699 |-100.0 |0 |similar to flagellar switch protein FliM |

|lmo0700 |-10.6 |4.88E-41 |similar to flagellar motor switch protein FliY |

|lmo0701 |-100.0 |2.26E-25 |unknown |

|lmo0702 |-23.9 |0.00112 |unknown |

|lmo0703 |-35.9 |1.44E-08 |unknown |

|lmo0704 |-27.5 |1.04E-10 |unknown |

|lmo0705 |-99.5 |0 |similar to flagellar hook-associated protein FlgK |

|lmo0706 |-97.1 |0 |similar to flagellar hook-associated protein 3 FlgL |

|lmo0707 | -62.2 |1.59E-13 |similar to flagellar hook-associated protein 2 FliD |

|lmo0708 | -6.8 |4.72E-16 |similar to hypothetical flagellar protein |

|lmo0709 | -4.4 |1.56E-06 |unknown |

|lmo0710 |-27.5 |2.81E-06 |similar to flagellar basal-body rod protein FlgB |

|lmo0711 |-50.0 |2.12E-21 |similar to flagellar basal-body rod protein FlgC |

|lmo0714 |-100.0 |0.00011 |similar to flagellar motor switch protein FliG |

|lmo0715 |-37.7 |0.01219 |unknown |

|lmo0716 |-34.2 |6.42E-07 |similar to H+-transporting ATP synthase alpha chain FliI |

|lmo0724 |-2.6 |0.00667 |similar to B. subtilis YvpB protein |

|lmo0739 |-3.0 |1.29E-08 |similar to 6-phospho-beta-glucosidase |

|lmo0973 |-3.1 |0 |DltB protein for D-Ala esterification of lipoteichoic acid and teichoic acid |

|lmo0984 |-9.1 |0.00044 |weakly similar to two-component response regulator |

|lmo1099 |-100.0 |0.0028 |similar to a protein encoded by Tn916 |

|lmo1390 |-2.5 |0.00006 |similar to ABC transporter (permease proteins) |

|lmo1391 |-2.5 |0.00199 |similar to sugar ABC transporter, permease protein |

|lmo1699 |-71.4 |0.00002 |some similarities to methyl-accepting chemotaxis proteins |

|lmo1700 |-9.0 |0.01551 |unknown |

|lmo1733 |-2.9 |1.02E-10 |similar to glutamate synthase (small subunit) |

|lmo1864 |-4.4 |1.62E-08 |similar to hemolysin III proteins, putative integral membrane protein |

|lmo1961 |-2.9 |0.00002 |similar to oxidoreductases |

|lmo1998 |-40.5 |2.36E-06 |similar to opine catabolism protein |

|lmo1999 |-14.6 |0.00025 |weakly similar to glucosamine-fructose-6-phosphate aminotransferase |

|lmo2001 |-25.4 |0.00002 |similar to PTS mannose-specific enzyme IIC component |

|lmo2003 |-9.3 |0.00009 |similar to transcription regulator GntR family |

|lmo2004 |-5.0 |0.00063 |similar to transcription regulator GntR family |

|lmo2093 |-2.5 |3.30E-06 |unknown |

|lmo2159 |-2.8 |4.19E-16 |similar to oxidoreductase |

|lmo2160 |-3.1 |6.73E-11 |similar to unknown proteins |

|lmo2161 |-3.2 |5.23E-13 |unknown |

|lmo2163 |-2.6 |2.54E-07 |similar to oxidoreductase |

|lmo2175 |-3.0 |0.00036 |similar to dehydrogenase |

|lmo2469 |-2.6 |0.00923 |similar to amino acid transporter |

|lmo2762 |-3.6 |0.01217 |similar to PTS cellobiose-specific enzyme IIB |

|lmo2764 |-6.6 |2.48E-10 |similar to xylose operon regulatory protein and to glucose kinase |

|lmo2773 |-2.5 |1.70E-12 |similar to transcription antiterminator |

Gene corresponds to the gene name on the ListiList server .

*Only genes with an absolute fold-change greater than 2.5 (p < 0.02) are given. Rosetta Resolver software analysis sets the maximal fold-change possible as 100 (Hughes et al. 2000). The functions of the encoded proteins are indicated according to the EGD-e genome annotation where possible (Glaser et al. 2001). lmo0688 transcript levels could not be determined in Lmo0688-negative bacteria, since the lmo0688-specific oligonucleotide spotted on the microarray is within the region deleted in ∆688. Genes related to flagellar motility or in operons with flagellar genes are in bold.

Table S2. Listeria monocytogenes and Escherichia coli strains

Listeria monocytogenes

|Strain |Genotype and relevant features |Strain Designation |Reference |

|DH-L478 |Wild-type L. monocytogenes strain EGDe |wild-type |M. Loessner |

|DH-L975 |Tn917 insertion in flaA in EGDe |wt; flaA::Tn |(Gründling et al. 2004) |

|DH-L1056 |lmo0688 in-frame deletion in EGDe |(688 |This study |

|DH-L1156 |mogR in-frame deletion in EGDe |∆mogR |(Gründling et al. 2004) |

|DH-L1179 |Tn917 insertion in flaA in DH-L1156 |∆mogR; flaA::Tn |(Gründling et al. 2004) |

|DH-L1248 |flaA in-frame deletion in DH-L1156 |∆mogR ∆flaA |(Shen and Higgins 2006) |

|DH-L1274 |degU in-frame deletion in DH-L1156 |∆mogR ∆degU |(Shen and Higgins 2006) |

|DH-L1275 |degU in-frame deletion in DH-L975 |∆degU; flaA::Tn |(Shen and Higgins 2006) |

|DH-L1276 |degU in-frame deletion in DH-L1179 |∆mogR ∆degU; flaA::Tn |(Shen and Higgins 2006) |

|DH-L1273 |degU in-frame deletion in EGDe |(degU |(Shen and Higgins 2006) |

|DH-L1367 |IPTG-inducible mogR in DH-L1156 |imogR |This study |

|DH-L1371 |lmo0688 in-frame deletion in DH-L1156 |∆mogR ∆688 |This study |

|DH-L1386 |Point mutations in active site of lmo0688 |688* |This study |

|DH-L1422 |pLOV/c688 integrated into DH-L1056 |(688/c688 |This study |

|DH-L1423 |pLOV/c688 integrated into DH-L1273 |(degU/c688 |This study |

|DH-L1426 |Tn917 insertion in flaA in DH-L1422 |(688/c688; flaA::Tn |This study |

|DH-L1427 |Tn917 insertion in flaA in DH-L1423 |(degU/c688; flaA::Tn |This study |

|DH-L1429 |lmo0688 in-frame deletion in DH-L1248 |∆mogR ∆688 ∆flaA |This study |

Escherichia coli

|Strain |Genotype and relevant features |Reference |

|DH-E113 |BL21(DE3) |Novagen |

|DH-E121 |pET28a in BL21(DE3) |Novagen |

|DH-E122 |pET29b in BL21(DE3) |Novagen |

|DH-E123 |pCON1 in JM109 |(Higgins 2006) |

|DH-E182 |XL1-Blue {F’ proAB lacIq ∆(lacZ)M15 Tn10} |Stratagene |

| |recA1 endA1 gyrA96 thi-1 hsdR17 supE relA1 lac | |

|DH-E278 |JM109(DE3) pLysS |Novagen |

|DH-E375 |CLG190 (F' lac, pro, lacIq) ∆malF3, ∆phoA, phoR, ∆lacX74, ∆ara∆leu7697, araD139, galE, galK, |D. Boyd |

| |StrR, pcnB, zad::Tn10, recA | |

|DH-E898 |pPL3 in XL1-Blue |(Gründling et al. 2004) |

|DH-E899 |pHPL3 in XL1-Blue |(Gründling et al. 2004) |

|DH-E1043 |pCON1/∆lmo0688 in XL1-Blue |This study |

|DH-E1093 |pHLIV2 in XL1-Blue |This study |

|DH-E1152 |pHLIV2/mogR in XL1-Blue |This study |

|DH-E1225 |pLOV in XL1-Blue |(Higgins 2006) |

|DH-E1317 |pET22b in XL1-Blue |Novagen |

|DH-E1335 |pET29b derivative with mogR in BL21(DE3) |(Shen and Higgins 2006) |

|DH-E1387 |pCON1/688* CLG190 |This study |

|DH-E1388 |pET22b/FlaA in XL1-Blue |This study |

|DH-E1389 |pET22b/FlaA in BL21(DE3) |This study |

|DH-E1391 |pET28a/lmo0688 in XL1-Blue |This study |

|DH-E1392 |pET28a/lmo0688 in BL21(DE3) |This study |

|DH-E1393 |pET28a/D83N D85N in BL21(DE3) |This study |

|DH-E1394 |pET28a/D83N D85N in XL1-Blue |This study |

|DH-E1421 |pLOV/c688 in XL1-Blue |This study |

|DH-E1428 |pLOV/c688 in SM10 |This study |

|DH-E1433 |pPL3+HyperOid in XL1-Blue |This study |

|DH-E1445 |pET28a/degU in XL1-Blue |This study |

|DH-E1446 |pET28a/degU in JM109(DE3) pLysS |This study |

Table S3. Oligonucleotides used in this study

|Number |Sequence |Sitea |

|168 |AGATACCGGCCGATAAAGCAAGCATATAATATTGCGTT |EagI |

|204 |CAACAACTGCAGACATTTTTTAACCTAATAATGCC |PstI |

|279 |GCTCTAGAGATAGTTATATTGCAGCAGAGAATTTAGGAACG |XbaI |

|280 |AGTGTCTTGAAGTATCATACAAATCGAAATTAACGGCCGCAT | |

|281 |CGGGATCCGTTTGAACAGAAAAGTTGTCCAAACACTTC |BamHI |

|282 |ATTTGTATGATACTTCAAGACACTGTTACAAACAATCGATAG | |

|313 |ACGCGTCGACTTTCTTCTGTCATATTACTGGCCTCCTA |SalI |

|324 |CTGCAGAACCACCGCGGTGGATGCATCTAACAGCACAAGAGCGGAAAG |BstXI |

|325b |AGATACCGGCCGCACCTCCTTAAGCTTAATTGTGAGCGCTCACAATTACACACATTATGCCACACCTTG |EagI |

|371 |CGGGATCCCAATAATACTATAGGATAGAGAGGTATGTG |BamHI |

|372 |ACGCGTCGACGAATAGATTCACCATTTACGGTATAAATTTCG |SalI |

|382 |GATCACGTTGCAGACACTACTAAC | |

|486 |ACGCGTCGACGAGATTTCTTTAGCGAATG |SalI |

|508 |ACGCGTCGACTCGATCATCATAATTCTGTCTCATTATATAAC |SalI |

|509 |CTGCGGTACCCGGTGATCCTAACTCACATTAA |KpnI |

|510 |GGAATTCCATATGAAAGTAAATACTAATATCATTAGC |NdeI |

|511 |ACGCACGTCGACGCTGTTAATTAATTGAGTTAAC |SalI |

|513 |AAACTGCAGAGGAGGAAAAATATGCGGCCGTTAATTTCGATTTG |PstI |

|514b |ATGGATTTTAGCGATTAATGCAAATGAATGTTTGGAAGAGGA | |

|515b |TCCTCTTCCAAACATTCATTTGCATTAATCGCTAAAATCCAT | |

|516 |GGGGTACCAATCTTCACCTCTGTTGCTT |KpnI |

|519 |AACCATGGATGCGGCCGTTAATTTCGATTTG |NcoI |

|520 |GCACGTCGACTCGATTGTTTGTAACAGTGTC |SalI |

|530 |CATTATGGCTATATGTCGGAA | |

|531 |CTTCTAAGTAAATTGTTCCTAGC | |

|535 |GGAATTCCATATGGCACTCAAAATCATGAT |NdeI |

| | | |

a. The indicated restriction endonuclease site is underlined within the oligonucleotide sequence.

b. Deviations from the wild-type sequence are in bold within the oligonucleotide sequence.

Supplemental Figures

Figure S1. Loss of DegU expression in MogR-negative L. monocytogenes impairs FlaA glycosylation.

Analysis of FlaA and FlaA glycosylation levels in L. monocytogenes. Wild-type (wt), ΔmogR, ΔdegU, and ΔmogR ΔdegU L. monocytogenes strains were grown at room temperature (RT) for 20 hr. Left panel: total FlaA protein in cell wall-associated fractions was separated by SDS-PAGE and visualized by Coomassie stain. Right panel: glycosylation of FlaA was determined by Western blot analysis using a (-O-linked GlcNAc-specific antibody. Two-fold more protein sample was loaded for ∆mogR ∆degU.

Figure S2. Lmo0688 removes MogR bound to cheY promoter region DNA by protein-protein interaction.

(A) Gel shift analysis of MogR and Lmo0688 binding to cheY promoter region DNA. Radiolabeled cheY promoter region DNA spanning –108 to +74 relative to the transcriptional start site was incubated with a constant amount (40 nM) of purified His6-tagged MogR to which increasing concentrations of His6-tagged Lmo0688 (lanes 2-6) or 240 nM His6-tagged DegU was added (lane 7). Increasing concentrations of His6-tagged Lmo0688 alone was incubated with radiolabeled cheY promoter region DNA (lanes 8-11). The binding reactions were separated by non-denaturing PAGE and detected by autoradiography. Shifted (S), supershifted (SS) and super-supershifted (SSS) DNA complexes are indicated.

(B) Pull-down assay of Lmo0688 by Ni2+ affinity purification of His6-tagged MogR. Purified His6-tagged MogR was incubated with cell lysates prepared from L. monocytogenes strains (688/c688, wild-type (wt), and (688. His6-tagged MogR and interacting proteins were isolated using Ni-NTA agarose beads. Proteins isolated in the pull-down assay were separated on a 10% SDS-PAGE gel and analyzed by Western blot using either an Lmo0688- or MogR-specific antibody.

Supplemental References

Camilli, A., Tilney, L.G., and Portnoy, D.A. 1993. Dual roles of plcA in Listeria monocytogenes pathogenesis. Mol. Microbiol. 8: 143-157.

Comer, F.I., Vosseller, K., Wells, L., Accavitti, M.A., and Hart, G.W. 2001. Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine. Anal. Biochem. 293: 169-177.

Dancz, C.E., Haraga, A., Portnoy, D.A., and Higgins, D.E. 2002. Inducible control of virulence gene expression in Listeria monocytogenes: temporal requirement of listeriolysin O during intracellular infection. J. Bacteriol. 184: 5935-5945.

Glaser, P., Frangeul, L., Buchrieser, C., Rusniok, C., Amend, A., Baquero, F., Berche, P., Bloecker, H., Brandt, P., Chakraborty, T., Charbit, A., Chetouani, F., Couve, E., de Daruvar, A., Dehoux, P., Domann, E., Dominguez-Bernal, G., Duchaud, E., Durant, L., Dussurget, O., Entian, K.D., Fsihi, H., Garcia-del Portillo, F., Garrido, P., Gautier, L., Goebel, W., Gomez-Lopez, N., Hain, T., Hauf, J., Jackson, D., Jones, L.M., Kaerst, U., Kreft, J., Kuhn, M., Kunst, F., Kurapkat, G., Madueno, E., Maitournam, A., Vicente, J.M., Ng, E., Nedjari, H., Nordsiek, G., Novella, S., de Pablos, B., Perez-Diaz, J.C., Purcell, R., Remmel, B., Rose, M., Schlueter, T., Simoes, N., Tierrez, A., Vazquez-Boland, J.A., Voss, H., Wehland, J., and Cossart, P. 2001. Comparative genomics of Listeria species. Science 294: 849-852.

Gründling, A., Burrack, L.S., Bouwer, H.G., and Higgins, D.E. 2004. Listeria monocytogenes regulates flagellar motility gene expression through MogR, a transcriptional repressor required for virulence. Proc. Natl. Acad. Sci. USA 101: 12318-12323.

Higgins, D.E., Buchrieser,C., Freitag,N.E. 2006. Genetic tools for use with Listeria monocytogenes. In Gram-positive pathogens (eds. V.A. Fischetti, R.P. Novick, J.J. Ferretti, D.A. Portnoy, and J.I. Rood), pp. 620-633. ASM Press, Washington, D.C.

Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K., and Pease, L.R. 1989. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77: 61-68.

Hughes, T.R., Marton, M.J., Jones, A.R., Roberts, C.J., Stoughton, R., Armour, C.D., Bennett, H.A., Coffey, E., Dai, H., He, Y.D., Kidd, M.J., King, A.M., Meyer, M.R., Slade, D., Lum, P.Y., Stepaniants, S.B., Shoemaker, D.D., Gachotte, D., Chakraburtty, K., Simon, J., Bard, M., and Friend, S.H. 2000. Functional discovery via a compendium of expression profiles. Cell 102: 109-126.

Kuhn, M. and Goebel, W. 1989. Identification of an extracellular protein of Listeria monocytogenes possibly involved in intracellular uptake by mammalian cells. Infect. Immun. 57: 55-61.

Lauer, P., Chow, M.Y.N., Loessner, M.J., Portnoy, D.A., and Calendar, R. 2002. Construction, characterization, and use of two Listeria monocytogenes site-specific phage integration vectors. J. Bacteriol. 184: 4177-4186.

Shen, A. and Higgins, D.E. 2005. The 5' untranslated region-mediated enhancement of intracellular listeriolysin O production is required for Listeria monocytogenes pathogenicity. Mol. Microbiol. 57: 1460-1473.

Shen, A. and Higgins, D.E. 2006. The MogR transcriptional repressor regulates non-hierarchal expression of flagellar motility genes and virulence in Listeria monocytogenes. PLoS Pathog. 2: e30.

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