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• W2135452372 abstract "Quorum-sensing pheromones are signal molecules that are secreted from Gram-positive bacteria and utilized by these bacteria to communicate among individual cells to regulate their activities as a group through a cell density-sensing mechanism. Typically, these pheromones are processed from precursor polypeptides. The mechanisms of trafficking, processing, and modification of the precursor to generate a mature pheromone are unclear. In Staphylococcus aureus, AgrD is the propeptide for an autoinducing peptide (AIP) pheromone that triggers the Agr cell density-sensing system upon reaching a threshold and subsequently regulates expression of virulence factor genes. The transmembrane protein AgrB, encoded in the agr locus, is necessary for the processing of AgrD to produce mature AIP; however, it is not clear how AgrD interacts with AgrB and how this interaction results in the generation of mature AIP. In this study, we found that the AgrD propeptide was integrated into the cytoplasmic membrane by a conserved α-helical amphipathic motif in its N-terminal region. We demonstrated that membrane targeting of AgrD by this motif was required for the stabilization of AgrD and the production of mature AIP, although this region was not specifically involved in the interaction with AgrB. An artificial amphipathic peptide replacing the N-terminal amphipathic motif of AgrD directed the protein to the cytoplasmic membrane and enabled the production of AIP. Analysis of Bacillus ComX precursor protein sequences suggested that the amphipathic membrane-targeting motif might also exist in pheromone precursors of other Gram-positive bacteria. Quorum-sensing pheromones are signal molecules that are secreted from Gram-positive bacteria and utilized by these bacteria to communicate among individual cells to regulate their activities as a group through a cell density-sensing mechanism. Typically, these pheromones are processed from precursor polypeptides. The mechanisms of trafficking, processing, and modification of the precursor to generate a mature pheromone are unclear. In Staphylococcus aureus, AgrD is the propeptide for an autoinducing peptide (AIP) pheromone that triggers the Agr cell density-sensing system upon reaching a threshold and subsequently regulates expression of virulence factor genes. The transmembrane protein AgrB, encoded in the agr locus, is necessary for the processing of AgrD to produce mature AIP; however, it is not clear how AgrD interacts with AgrB and how this interaction results in the generation of mature AIP. In this study, we found that the AgrD propeptide was integrated into the cytoplasmic membrane by a conserved α-helical amphipathic motif in its N-terminal region. We demonstrated that membrane targeting of AgrD by this motif was required for the stabilization of AgrD and the production of mature AIP, although this region was not specifically involved in the interaction with AgrB. An artificial amphipathic peptide replacing the N-terminal amphipathic motif of AgrD directed the protein to the cytoplasmic membrane and enabled the production of AIP. Analysis of Bacillus ComX precursor protein sequences suggested that the amphipathic membrane-targeting motif might also exist in pheromone precursors of other Gram-positive bacteria. Quorum sensing is a way that bacteria communicate with each other (1Bassler B.L. Cell. 2002; 109: 421-424Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). Bacteria produce autoinducing molecules at basal levels. When bacterial population density increases, the autoinducing molecule concentration reaches a threshold that results in the activation of the quorum-sensing system and subsequently alters the cell activity that enables bacteria to change their behavior as a group like multicellular organism. Quorum sensing is involved in the regulation of sporulation, mating, bioluminescence, virulence factor expression, and biofilm formation. Although acylated homoserine lactones with different acyl side chains are common autoinducers for Gram-negative bacteria, Gram-positive bacteria use small peptides as pheromones (2Dunny G.M. Winans S.C. Dunny G.M. Winans S.C. Cell-Cell Signaling in Bacteria. American Society for Microbiology, Washington, D. C.1999: 1-5Google Scholar). Well studied examples of peptide pheromones include the competence-stimulating peptide from Streptococcus pneumoniae (3Havarstein L.S. Coomaraswamy G. Morrison D.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11140-11144Crossref PubMed Scopus (546) Google Scholar, 4Havarstein L.S. Diep D.B. Nes I.F. Mol. Microbiol. 1995; 16: 229-240Crossref PubMed Scopus (451) Google Scholar), lantibiotics and the bacteriocin-inducing peptide of lactic acid bacteria (5Nes I.F. Eijsink V.G. Dunny G.M. Winans S.C. Cell-Cell Signaling in Bacteria. American Society for Microbiology, Washington, D. C.1999: 175-192Google Scholar), the ComX pheromone and competence and sporulation factor of Bacillus subtilis (6Lazazzera B.A. Solomon J.M. Grossman A.D. Cell. 1997; 89: 917-925Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), sex pheromones of Enterococcus faecalis, and the autoinducing peptide (AIP) 1The abbreviations used are: AIP, autoinducing peptide; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine. 1The abbreviations used are: AIP, autoinducing peptide; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine. in staphylococci (7Novick R.P. Dunny G.M. Winans S.C. Cell-Cell Signaling in Bacteria. American Society for Microbiology, Washington, D. C.1999: 129-145Google Scholar). These pheromones are typically processed from precursor polypeptides with modifications in some cases. A leader peptide with a conserved double glycine sequence found in the lactobacterial lantibiotics and bacteriocin-inducing peptides (5Nes I.F. Eijsink V.G. Dunny G.M. Winans S.C. Cell-Cell Signaling in Bacteria. American Society for Microbiology, Washington, D. C.1999: 175-192Google Scholar) is considered important for propeptide trafficking. ATP-binding cassette transporters with proteolytic activity are postulated to be responsible for both processing of the propeptides and secretion of the mature pheromones. It is interesting to note that the precursor of the E. faecalis sex pheromone is first generated from a lipoprotein precursor by a signal peptidase. This precursor molecule, located in the membrane, is then processed through intramembranous proteolysis by a zinc metalloprotease (Eep) (8An F.Y. Sulavik M.C. Clewell D.B. J. Bacteriol. 1999; 181: 5915-5921Crossref PubMed Google Scholar, 9An F.Y. Clewell D.B. J. Bacteriol. 2002; 184: 1880-1887Crossref PubMed Scopus (59) Google Scholar), although the catalytic mechanism has yet to be determined. The leader peptide is also predicted in the competence and sporulation factor precursor, but the trafficking and processing of this precursor are not clearly defined. In Staphylococcus aureus, expression of the virulence factors is coordinately regulated by a quorum-sensing system encoded by the agr (accessory gene regulator) locus (7Novick R.P. Dunny G.M. Winans S.C. Cell-Cell Signaling in Bacteria. American Society for Microbiology, Washington, D. C.1999: 129-145Google Scholar). The agr locus encodes four proteins: AgrA, AgrB, AgrC, and AgrD. AgrC and AgrA compose a two-component signal transduction pathway, in which AgrC is the sensor kinase, and AgrA resembles a response regulator (10Novick R.P. Mol. Microbiol. 2003; 48: 1429-1449Crossref PubMed Scopus (990) Google Scholar, 11Stock A.M. Robinson V.L. Goudreau P.N. Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2398) Google Scholar). AgrD is the precursor of AIP, a peptide pheromone that is secreted from the bacteria into the culture medium (12Ji G. Beavis R.C. Novick R.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12055-12059Crossref PubMed Scopus (501) Google Scholar, 13Ji G. Beavis R. Novick R.P. Science. 1997; 276: 2027-2030Crossref PubMed Scopus (611) Google Scholar) and that is the ligand of AgrC, a membrane protein with its N-terminal half, which has been proposed to contain the AIP-binding site, anchored in the cytoplasmic membrane via five transmembrane helices (14Lina G. Jarraud S. Ji G. Greenland T. Pedraza A. Etienne J. Novick R.P. Vandenesch F. Mol. Microbiol. 1998; 28: 655-662Crossref PubMed Scopus (191) Google Scholar, 15Lyon G.J. Wright J.S. Christopoulos A. Novick R.P. Muir T.W. J. Biol. Chem. 2002; 277: 6247-6253Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) and with its C-terminal half, which composes a histidine kinase domain, located in the cytoplasm (11Stock A.M. Robinson V.L. Goudreau P.N. Annu. Rev. Biochem. 2000; 69: 183-215Crossref PubMed Scopus (2398) Google Scholar). AgrB is required for the production of AIP (13Ji G. Beavis R. Novick R.P. Science. 1997; 276: 2027-2030Crossref PubMed Scopus (611) Google Scholar) and functions as a protease that is involved in the proteolytic cleavage of AgrD (16Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). It has also been proposed that AgrB functions as an oligopeptide transporter that facilitates the secretion of mature AIP (10Novick R.P. Mol. Microbiol. 2003; 48: 1429-1449Crossref PubMed Scopus (990) Google Scholar, 16Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). However, the interaction between AgrD and AgrB and the mechanisms of the processing of AgrD and the secretion of mature AIP by AgrB remain unclear. Based on the DNA sequences of agr loci and the specific interactions between AIP and AgrC and between AgrD and AgrB, four S. aureus groups have been defined (13Ji G. Beavis R. Novick R.P. Science. 1997; 276: 2027-2030Crossref PubMed Scopus (611) Google Scholar, 17Jarraud S. Lyon G.J. Figueiredo A.M. Gerard L. Vandenesch F. Etienne J. Muir T.W. Novick R.P. J. Bacteriol. 2000; 182: 6517-6522Crossref PubMed Scopus (239) Google Scholar). AIP from an S. aureus strain activates the Agr response in itself and in the same group members, but inhibits the Agr response in heterologous group members. The four S. aureus AgrD groups have 46 or 47 residues, and the AIPs have different amino acid sequences and lengths. An alignment of these AgrD groups (group I AgrD, NCBI accession number CAA36782; group II AgrD, accession number AAB63265; group III AgrD, accession number AAB63268; and group IV AgrD, accession number AAG03056) is shown, with the AIP sequences in boldface. The specific interaction between AgrB and AgrD is not so strict. AgrD of S. aureus group II is processed only in the presence of group II AgrB, whereas group I AgrD and group III AgrD are processed by group I AgrB and vice versa (7Novick R.P. Dunny G.M. Winans S.C. Cell-Cell Signaling in Bacteria. American Society for Microbiology, Washington, D. C.1999: 129-145Google Scholar). In this study, we found that the AgrD propeptide was anchored in the cytoplasmic membrane by an N-terminal amphipathic region. We also demonstrate that the membrane targeting of AgrD by the amphipathic region was required for its normal processing to produce mature AIP, but was not involved in the specific interaction with AgrB. This amphipathic region is present in all staphylococcal AgrD sequences available in the GenBank™/EBI Data Bank as revealed by sequence analysis. Furthermore, analysis of B. subtilis ComX sequences revealed the existence of similar amphipathic regions in their N-terminal parts, suggesting that the membrane targeting sequence might play an important role in the processing of the propeptides to generate active mature pheromones in other Gram-positive bacteria. Bacterial Strains and Culture Conditions—The S. aureus plasmids and strains used in this study are listed in Table I. S. aureus cells were grown in CY-GP broth (18Novick R.P. Methods Enzymol. 1991; 204: 587-636Crossref PubMed Scopus (463) Google Scholar), supplemented with antibiotics (5 μg/ml chloramphenicol and 5 μg/ml erythromycin) when necessary. Bacteria grown overnight at 37 °C on GL plates (18Novick R.P. Methods Enzymol. 1991; 204: 587-636Crossref PubMed Scopus (463) Google Scholar) were routinely used to inoculate liquid cultures. Cell growth was monitored with either a Klett-Summerson colorimeter with a green (540 nm) filter (Klett, Long Island City, NY) or a VERSAmax microplate reader (Molecular Devices) at A650 nm. Escherichia coli strain MC1061-5 (19Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000Google Scholar) was grown in LB broth, supplemented with tetracycline (20 μg/ml) when necessary.Table IS. aureus plasmids and strains used in this studyGenotype and descriptionRef.PlasmidpRN5548Vector carrying a staphylococcal inducible bla promoter23Novick R.P. Ross H.F. Projan S.J. Kornblum J. Kreiswirth B. Moghazeh S. EMBO J. 1993; 12: 3967-3975Crossref PubMed Scopus (815) Google ScholarpRN6441Cloning vector23Novick R.P. Ross H.F. Projan S.J. Kornblum J. Kreiswirth B. Moghazeh S. EMBO J. 1993; 12: 3967-3975Crossref PubMed Scopus (815) Google ScholarpRN6683Group I agr P3-blaZ fusion20Novick R.P. Projan S.J. Kornblum J. Ross H.F. Ji G. Kreiswirth B. Vandenesch F. Moghazeh S. Mol. Gen. Genet. 1995; 248: 446-458Crossref PubMed Scopus (319) Google ScholarpLZ2003RN6390B (group I) agrB in pRN644116Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (94) Google ScholarpRN6913RN6390B (group I) agrD in pRN554812Ji G. Beavis R.C. Novick R.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12055-12059Crossref PubMed Scopus (501) Google ScholarpRN6958SA502A (group II) agrD in pRN554813Ji G. Beavis R. Novick R.P. Science. 1997; 276: 2027-2030Crossref PubMed Scopus (611) Google ScholarpLZ4005RN6390B his6-agrD-his6 in pRN554816Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (94) Google ScholarpLZ4009RN6390B agrD-his6 in pRN5548ΔPstIThis studypLZ4010SA502A agrD-his6 in pRN5548ΔPstIThis studypLZ4011RN6390B agrD (txdh) in pRN5548This studypLZ4012RN6390B agrD (tldh) in pRN5548This studypLZ4013RN6390B agrD (amphiNDH) in pRN5548This studypLZ4014Chimeric agrD (dh-IsII) in pRN5548This studypLZ4015Chimeric agrD (dh-IIsI) in pRN5548This studypLZ4017RN6390B agrD (tmdh) in pRN5548This studypLIND5RN6390B agrD (tldh-dn5) in pRN5548This studypLIND10RN6390B agrD (tldh-dn10) in pRN5548This studypLIND12RN6390B agrD (tldh-dn12) in pRN5548This studypLIND14RN6390B agrD (tldh-dn14) in pRN5548This studypLIND18RN6390B agrD (tldh-dn18) in pRN5548This studyStrainGJ2035RN6911 (pI524)16Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (94) Google ScholarLZ0001GJ2035 (pRN5548)16Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (94) Google ScholarLZ4009GJ2035 (pLZ4009)This studyLZ4010GJ2035 (pLZ4010)This studyLZ4011GJ2035 (pLZ4011)This study Open table in a new tab Construction of S. aureus Plasmids—The group I AgrD-His6 expression plasmid pLZ4009 was constructed as follows. A PCR product was generated using primers GJ56 (5′-GCTCTAGAAGCTATTACATTATTACC-3′, before the Shine-Dalgarno sequence of agrD, with the XbaI site underlined) and GJ28 (5′-CTAATGATGATGATGATGATGTTCGTGTAATTGTGTAATTC-3′, with six histidine codons and one stop codon underlined and 3′ of agrD italicized) and pRN6852 (12Ji G. Beavis R.C. Novick R.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12055-12059Crossref PubMed Scopus (501) Google Scholar) as the template. The PCR product was then digested with XbaI and cloned into the pRN5548 XbaI and EcoRI (blunted with Klenow fragment) sites. The group II AgrD-His6 expression plasmid pLZ4010 was made by PCR amplification of the SA502A agrD gene from pRN6958 (13Ji G. Beavis R. Novick R.P. Science. 1997; 276: 2027-2030Crossref PubMed Scopus (611) Google Scholar) with primers GJ44 (5′-CTATTATTCCATGGACTTCATTTAC-3′, sequence around the NcoI site of the pRN5548 plasmid, with the NcoI site underlined) and LZ31 (5′-ATTAATGATGATGATGATGATGTTTGTCGTATAAATTCGTTAATT-3′, with six histidine codons and one stop codon underlined and 3′ of group II agrD italicized). The PCR product was digested with NcoI and ligated to a SmaI- and NcoI-digested 2.2-kb fragment of pRN5548. Plasmids carrying genes encoding various epitope-tagged wild-type or mutant RN6390B (group I) AgrD proteins used in this study were created by PCR-based cloning. Plasmid pLZ4005 contains an NdeI-EcoRI DNA fragment (encoding the His6-T7 tag-Xpress™ epitope and multiple cloning sites sequence) of pRSET-A (Invitrogen) ligated to the 5′-end of agrD (16Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Plasmid pLZ4011 was constructed as follows. A PCR product amplified from pLZ4005 with primer GJ45 (5′-GTAAATGAAGTCCATGGAATAATAG-3′, sequence around the NcoI site of pRN5548, with the NcoI site underlined) and 5′-phosphorylated (T4 polynucleotide kinase, MBI Fermentas) primer LZ8 (5′-ATGGCTAGCATGACT-3′, T7 tag coding sequence) was digested with BspHI and ligated to another BspHI-digested PCR product amplified from pLZ4005 with primer LZ8 and 5′-phosphorylated primer LZ9 (5′-ATGAGAACCCCGCAT-3′, sequence before the His6 coding region in pRSET-A). Plasmid pLZ4012 was made by ligating an NcoI-digested PCR product amplified from pLZ4011 with primers LZ32 (5′-CATGCCATGGTCGTCGTACAGATCCCGAC-3′, sequence in the Xpress™ epitope coding region, with the NcoI site underlined) and GJ44 to the 2.4-kb fragment of NcoI-digested pLZ4011. Plasmids pLIND5, pLIND10, and pLIND18 were constructed as follows. PCR products were amplified from pLZ4012 with primers GJ111 (5′-CATTGAATTCTGAACTTATTTTTTGATTTTATTAC-3′, agrD sequence plus an EcoRI site, with the EcoRI site underlined) and LIN25 (5′-AAAATCATGAAAATTTTAATTTGC-3′, sequence around the BspHI site of pRN5548, with the BspHI site underlined) for pLIND5, with primers GJ112 (5′-CATTGAATTCTGTTTATTACTGGGATTTTAAAAAAC-3′, agrD sequence plus an EcoRI site, with the EcoRI site underlined) and LIN25 for pLIND10, and with primers GJ113 (5′-CATTGAATTCTGATTGGTAACATCGCAGCTTATAGTACTTGTG-3′, agrD sequence plus an EcoRI site, with the EcoRI site underlined) and LIN25 for pLIND18. The PCR products were then digested with EcoRI and BspHI and ligated to an EcoRI- and BspHI-digested 3.1-kb fragment of pLZ4012. Plasmids pLIND12 and pLIND14 were constructed as follows. PCR products were amplified from plasmid pLIND10 using primers GJ56 and GJ120 (5′-AATCCCAGTAATTCCATGGTACCAGCTGC-3′, within agrD, fragment A), primers GJ56 and GJ122 (5′-TTTTAAAATAATTCCATGGTACCAGCTGC-3′, within agrD, fragment B), primers LIN25 and GJ119 (5′-CATGGAATTACTGGGATTTTAAAAAACATTGG-3′, within agrD, fragment C), or primers LIN25 and GJ121 (5′-CATGGAATTATTTTAAAAAACATTGGTAAC-3′, within agrD, fragment D). The PCR products were then prepared using GJ56 and LIN25 as primers and a mixture of fragments A and C or fragments B and D as the template, digested with XbaI and BspHI, and ligated to the XbaI and BspHI sites of pRN5548, generating plasmids pLIND12 and pLIND14, respectively. Plasmid pLZ4013 containing a coding sequence for an artificial amphipathic 11-amino acid peptide followed by the RN6390B AgrD C-terminal region coding sequence (amphiNDH) was constructed by ligating two T4 polynucleotide kinase-phosphorylated and NcoI-digested PCR products amplified from pRN6913: one with primers GJ44 and LZ17 (5′-CATTTTAAGTCCTCCTTA-3′, from the starting codon of agrD) and another with primers GJ45 and LZ49 (5′-ATTACCACTATCATCACTATCATCACTACTATTTTAAAAAACATTGGTAACATC-3′, with the artificial amphipathic 11-amino acid peptide coding sequence underlined and the RN6390B AgrD coding sequence italicized). Plasmid pLZ4014 was made by ligating two HpaII-digested and T4 polynucleotide kinase-phosphorylated PCR products amplified from pLZ4009 using primers LZ42 (5′-ACTGCTGACTTCATAATGGATG-3′, group I agrD sequence) and LZ43 (5′-ACCAATGTTTTTTAAAATCCCAG-3′, group I agrD sequence) and amplified from pLZ4010 using primers LZ44 (5′-ATTGTCGGTGGCGTAAAC-3′, group II agrD sequence) and GJ45. Similarly, plasmid pLZ4015 was constructed by ligating two HpaII-digested and T4 polynucleotide kinase-phosphorylated PCR products amplified from pRN6958 using primers LZ46 (5′-TCCAGCAGTTTATTTGATGAAC-3′, group II agrD sequence) and LZ47 (5′-TCCGATTGCTTTAGCTAAT-3′, group II agrD sequence) and amplified from pLZ4009 using primers LZ48 (5′-AACATCGCAGCTTATAGT-3′, group I agrD sequence) and GJ45. Plasmid pLZ4017 was constructed as follows. A PCR product was amplified from pLZ4011 with primers LZ48 (5′-AACATCGCAGCTTATAGT-3′, group I agrD sequence) and tmR (5′-CAGTGTGGCAATCACCAGAATCAGGGCAAACATATTCGCAATTCCATGGTACCAGC-3′, with the E. coli leader peptidase I gene sequence underlined and the pRSET-A multicloning site sequence italicized), digested with BspHI, and dephosphorylated with shrimp alkaline phosphatase (MBI Fermentas). This fragment was ligated to a BspHI-digested and 5′-phosphorylated PCR fragment amplified from pLZ4011 with primers GJ45 and tmF (5′-GTGACGGGCATTTTATGGTGCGTGCGGCGGATTTTAAAAAACATTGGTAACATCGCAG-3′, with the E. coli leader peptidase I gene and two arginine codons underlined and the agrD sequence italicized). The resulting plasmid contained the coding sequence of the N-terminal transmembrane region of E. coli leader peptidase I (21 amino acids) followed by two arginine codons and the agrD sequence encoded the C-terminal 32 amino acids. This plasmid was used as a template to produce PCR products with primer pairs GJ44/LZ17 and GJ45/lepF (5′-GCGAATATGTTTGCCCTG-3′, the E. coli leader peptidase I coding sequence starting from the second codon). The PCR products were then digested with NcoI, 5′-phosphorylated, and ligated to produce pLZ4017. All PCR products used for plasmid construction were amplified with Pfu Turbo® high fidelity DNA polymerase (Stratagene). The correct sequence of every newly constructed plasmid was confirmed by DNA sequencing. AIP Activity Assays—AIP activities were measured using S. aureus cells containing an agr P3-blaZ fusion on plasmid pRN6683 (20Novick R.P. Projan S.J. Kornblum J. Ross H.F. Ji G. Kreiswirth B. Vandenesch F. Moghazeh S. Mol. Gen. Genet. 1995; 248: 446-458Crossref PubMed Scopus (319) Google Scholar) according to the method described previously (12Ji G. Beavis R.C. Novick R.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12055-12059Crossref PubMed Scopus (501) Google Scholar). PhoA Fusion Plasmid Construction and Expression in E. coli Strains—The AgrD-PhoA fusion protein expression plasmid pLZ5001 was constructed as follows. A PCR product amplified from pGJ4002 (16Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) with primers GJ93 (5′-GAAGATCTTCGTGTAATTGTGTTAATTC-3′, with the BglII site underlined) and GJ94 (5′-CGGGATCCATGAATACATTATTTAACTTATTTTTTG-3′, with the BamHI site underlined) was digested with BglII and BamHI. This product was then inserted into the BglII site of pAWLP-2 (16Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Plasmid pLZ5002, encoding a protein with only the N-terminal region of AgrD fused to PhoA (AgrDN-PhoA), was constructed by deleting the coding sequence for the C-terminal region of AgrD in pLZ5001. A PCR product amplified from pLZ5001 with primers LZ10 (5′-GAAGATCTTTTTTGCAGCTCAG-3′, with the BglII site underlined) and LZ15 (5′-GAAGATCTCTGCGATGTTACCAATGT-3′, with the BglII site underlined) was digested with BglII and DpnI and then self-ligated. E. coli strain MC1061-5 transformed with pLZ5001 or pLZ5002 was cultured overnight for PhoA activity assay or cell fractionation and Western blot hybridization. Alkaline Phosphatase (PhoA) Activity Assays—PhoA activity was measured as described (21Brickman E. Beckwith J. J. Mol. Biol. 1975; 96: 307-316Crossref PubMed Scopus (319) Google Scholar) with modifications. E. coli MC1061-5 cells expressing AgrD-PhoA fusion proteins were grown in LB broth (19Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000Google Scholar) at 37 °C overnight. Cells were collected, washed once with 1 m Tris buffer (pH 8.0), and suspended in the same buffer containing 1% SDS and 10% chloroform, followed by vortexing for 15 min to extract proteins. p-Nitrophenyl phosphate (Sigma 104 solution) was then added to the mixtures. After a 30-min incubation at 37 °C, K2HPO4 solution (13%) was added to stop the reactions. The reaction mixtures were centrifuged, and the clear supernatant was measured at both 420 and 550 nm. Alkaline phosphatase activity units were calculated by a formula described previously (21Brickman E. Beckwith J. J. Mol. Biol. 1975; 96: 307-316Crossref PubMed Scopus (319) Google Scholar). Cell Fractionation—Harvested S. aureus cells were suspended in 1× sucrose/sodium maleate/MgCl2 solution containing 10 μg/ml lysostaphin and incubated for 30 min at 37 °C. The protoplasts were then lysed by addition of phosphate-buffered saline (PBS) or as otherwise indicated and supplemented with 1 mm phenylmethanesulfonyl fluoride and protease inhibitors. The cell lysate was briefly sonicated, and the cell debris was removed by centrifugation at 7000 × g for 10 min at 4 °C. Total protein levels of the lysates were measured with Bio-Rad protein assay dye reagent at A595 nm. Cell membrane fractions were separated by ultracentrifugation at 200,000 × g for 2 h at 4 °C. E. coli MC1061-5 cells containing pLZ5001 or pLZ5002 suspended in PBS with 1 mg/ml lysozyme were incubated at 4 °C for 1 h and then lysed by brief sonication after addition of 1 mm phenylmethanesulfonyl fluoride. Membrane fractions were prepared by ultracentrifugation. The pellets were dissolved in 1× SDS sample buffer (19Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000Google Scholar) or as otherwise indicated. SDS-PAGE and Western Blotting—Protein samples dissolved in SDS sample buffer were incubated at 70 °C for 10 min before being separated by either Tris/glycine/SDS-PAGE (19Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000Google Scholar) or Tris/Tricine/SDS-PAGE (22Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10410) Google Scholar). The separated proteins were then electrophoretically transferred to polyvinylidene difluoride membranes (Millipore). After blocking overnight at 4 °C in Tris-buffered saline (19Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000Google Scholar) containing 0.1% Tween 20 and 5% bovine albumin (Sigma), the polyvinylidene difluoride membranes were incubated in blocking buffer with a primary antibody (1:2000 dilution of mouse anti-tetrahistidine monoclonal antibody (QIAGEN Inc.) or 1:5000 dilution of anti-T7 tag monoclonal antibody (Novagen)) for 1 h at room temperature. The membranes were washed extensively with Tris-buffered saline plus Tween 20 and then probed with horseradish peroxidase-conjugated sheep anti-mouse secondary antibody (Amersham Biosciences). The immunoblots were detected with an ECL Plus Western blot detection system followed by exposure to Hyper-film™ ECL™ (Amersham Biosciences). AgrD Is an Integral Membrane Protein—To determine the subcellular location of AgrD, we made two plasmids in which six histidine codons were added to the 3′-end of either the group I or II agrD gene and inserted downstream of the bla promoter of the pRN5548 expression vector (23Novick R.P. Ross H.F. Projan S.J. Kornblum J. Kreiswirth B. Moghazeh S. EMBO J. 1993; 12: 3967-3975Crossref PubMed Scopus (815) Google Scholar). Plasmids pLZ4009 and pLZ4010 were transformed into agr-null S. aureus strain GJ2035, creating strains LZ4009 and LZ4010, respectively. Western blot hybridization analysis with the anti-tetrahistidine monoclonal antibody used as a probe revealed a band with an estimated size of ∼6 kDa from whole cell lysate of LZ4009 or LZ4010 expressing His6-tagged AgrD (Fig. 1A, lanes 4 and 7). No band was observed from cell lysate of LZ0001 carrying pRN5548 (Fig. 1A, lane 1). The sizes of the proteins detected were consistent with that of the predicted molecular mass of AgrD-His6. These ∼6-kDa bands were detected only in the membrane fractions prepared from LZ4009 and LZ4010 (Fig. 1A, lanes 6 and 9), but not in the cytoplasmic fractions (lanes 5 and 8), indicating that AgrD is a membrane-associated protein. To determine whether AgrD is an integral or peripheral membrane protein, we prepared protoplasts from S. aureus LZ4009 cells and lysed the protoplasts in PBS containing 1 m sodium chloride, in 0.2 m sodium carbonate, or in PBS containing 1% sarcosyl. After incubation at 4 °C for 2 h, the whole cell lysates were fractionated by ultracentrifugation. Fig. 1B showed the results of Western blot hybridization with the anti-tetrahistidine monoclonal antibody as a probe. AgrD-His6 was not extracted from the membrane fraction by 1 m sodium chloride (Fig. 1B, lane 3) and was only partially extracted from the membrane fraction by 0.2 m sodium c" @default.
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• W2135452372 title "Membrane Anchoring of the AgrD N-terminal Amphipathic Region Is Required for Its Processing to Produce a Quorum-sensing Pheromone in Staphylococcus aureus" @default.
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