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• W4253388474 abstract "The extracytoplasmic folding of secreted proteins in Gram-positive bacteria is influenced by the microenvironment of the compartment into which they are translocated, namely the negatively charged matrix of the cell wall polymers. In this compartment, the PrsA lipoprotein facilitates correct post-translocational folding or prevents misfolding of secreted proteins. In this study, a secretion mutant of B. subtilis (prsA3) encoding a defective PrsA protein was mutagenized and screened for restored secretion of the AmyQ α-amylase. One mini-Tn10 insertion, which partially suppressed the secretion deficiency, was found to interrupt dlt, the operon involved in thed-alanylation of teichoic acids. The inactivation ofdlt rescued the mutant PrsA3 protein from degradation, and the increased amount of PrsA3 was shown to enhance the secretion of PrsA-dependent proteins. Heterologous or abnormal secreted proteins, which are prone to degradation after translocation, were also stabilized and secreted in increased quantities from a dlt prsA + strain. Furthermore, the dltmutation partially suppressed the lethal effect of PrsA depletion, suggesting that the dlt deficiency also leads to stabilization of an essential cell wall protein(s). Our results suggest that main influence of the increased net negative charge of the wall caused by the absence of d-alanine is to increase the rate of post-translocational folding of exported proteins. The extracytoplasmic folding of secreted proteins in Gram-positive bacteria is influenced by the microenvironment of the compartment into which they are translocated, namely the negatively charged matrix of the cell wall polymers. In this compartment, the PrsA lipoprotein facilitates correct post-translocational folding or prevents misfolding of secreted proteins. In this study, a secretion mutant of B. subtilis (prsA3) encoding a defective PrsA protein was mutagenized and screened for restored secretion of the AmyQ α-amylase. One mini-Tn10 insertion, which partially suppressed the secretion deficiency, was found to interrupt dlt, the operon involved in thed-alanylation of teichoic acids. The inactivation ofdlt rescued the mutant PrsA3 protein from degradation, and the increased amount of PrsA3 was shown to enhance the secretion of PrsA-dependent proteins. Heterologous or abnormal secreted proteins, which are prone to degradation after translocation, were also stabilized and secreted in increased quantities from a dlt prsA + strain. Furthermore, the dltmutation partially suppressed the lethal effect of PrsA depletion, suggesting that the dlt deficiency also leads to stabilization of an essential cell wall protein(s). Our results suggest that main influence of the increased net negative charge of the wall caused by the absence of d-alanine is to increase the rate of post-translocational folding of exported proteins. Spizizen's minimal salts lipoteichoic acid wall teichoic acid isopropyl-β-d-thiogalactopyranoside 4-morpholineethanesulfonic acid Secretory preproteins are transferred across the cytoplasmic membrane of bacteria by a specific translocator or translocase (1Driessen A.J. Fekkes P. van der Wolk J.P. Curr. Opin. Microbiol. 1998; 1: 216-222Crossref PubMed Scopus (148) Google Scholar, 2Meyer T.H. Menetret J.F. Breitling R. Miller K.R. Akey C.W. Rapoport T.A. J. Mol. Biol. 1999; 258: 1789-1800Crossref Scopus (125) Google Scholar). In the major secretion pathway, integral membrane proteins SecY, SecE, SecG, and SecDF form the translocator (3Swaving J. van Wely K.H. Driessen A.J. J. Bacteriol. 1999; 181: 7021-7027Crossref PubMed Google Scholar, 4Bolhuis A. Broekhuizen C.P. Sorokin A. van Roosmalen M.L. Venema G. Bron S. Quax W.J. van Dijl J.M. J. Biol. Chem. 1998; 273: 21217-21224Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), while the energy required for preprotein translocation is provided by a combination of the SecA translocation ATPase and the proton motive force. InBacillus subtilis, two type I signal peptidases (SipS and SipT) with overlapping substrate specificities and one type II signal peptidase (Lsp) are involved in the processing of preproteins (5Tjalsma H. Noback M.A. Bron S. Venema G. Yamane K. van Dijl J.M. J. Biol. Chem. 1997; 272: 25983-25992Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 6Tjalsma H. Zanen G. Venema G. Bron S. van Dijl J.M. J. Biol. Chem. 1999; 274: 28191-28197Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Another signal peptidase, SipW, is required for the processing of a spore-specific TasA protein in the interspore space (7Tjalsma H. Bolhuis A. van Roosmalen M.L. Wiegert T. Schumann W. Broekhuizen C.P. Quax W.J. Venema G. Bron S. van Dijl J.M. Genes Dev. 1998; 12: 2331-2381Crossref Scopus (143) Google Scholar). Two other signal peptidases (SipU and SipV) of unknown specificity have also been identified (5Tjalsma H. Noback M.A. Bron S. Venema G. Yamane K. van Dijl J.M. J. Biol. Chem. 1997; 272: 25983-25992Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). On the trans side of the membrane, translocated secretory proteins fold into their native conformation concomitantly with or following their release from the membrane. In bacteria, several proteins have been identified which facilitate the folding of translocated proteins. In Escherichia coli, these include periplasmic proteins such as PpiD, SurA, and DsbA (8Dartigalongue C. Raina S. EMBO J. 1998; 17: 3968-3980Crossref PubMed Scopus (190) Google Scholar, 9Lazar S.W. Kolter R. J. Bacteriol. 1996; 178: 1770-1773Crossref PubMed Google Scholar, 10Rouviere P.E. Gross C.A. Genes Dev. 1996; 10: 3170-3182Crossref PubMed Scopus (250) Google Scholar, 11Bardwell J.C. Mol. Microbiol. 1994; 14: 199-205Crossref PubMed Scopus (198) Google Scholar), and inB. subtilis they include BdbB, BdbC and PrsA (12Bolhuis A. Venema G. Quax W.J. Bron S. van Dijl J.M. J. Biol. Chem. 1999; 274: 24531-24538Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 13Kontinen V. Sarvas M. Mol. Microbiol. 1993; 8: 727-737Crossref PubMed Scopus (167) Google Scholar, 14Leskelä S. Wahlström E. Kontinen V.P. Sarvas M. Mol. Microbiol. 1999; 31: 1075-1085Crossref PubMed Scopus (74) Google Scholar). The PrsA lipoprotein of B. subtilis appears to be a folding factor that is active during the post-translocational phase of secretion. PrsA is required for many but not all secreted proteins (13Kontinen V. Sarvas M. Mol. Microbiol. 1993; 8: 727-737Crossref PubMed Scopus (167) Google Scholar, 14Leskelä S. Wahlström E. Kontinen V.P. Sarvas M. Mol. Microbiol. 1999; 31: 1075-1085Crossref PubMed Scopus (74) Google Scholar, 15Kontinen V.P. Saris P. Sarvas M. Mol. Microbiol. 1991; 5: 1273-1283Crossref PubMed Scopus (101) Google Scholar, 16Jacobs M. Anderssen J.B. Kontinen V. Sarvas M. Mol. Microbiol. 1993; 8: 957-966Crossref PubMed Scopus (98) Google Scholar) and is essential for viability; depletion of PrsA impairs the synthesis of the cell wall. 1M. Vitikainen, unpublished results.1M. Vitikainen, unpublished results.A search of the TIGR and GenBankTM data bases has revealed that homologues of PrsA occur in other Gram-positive but not in any Gram-negative bacterium. Predictions from protein sequence alignments indicate that PrsA is a putative peptidyl-prolyl cis/trans-isomerase and a membrane-bound lipoprotein, with its protein domain locating at the membrane/wall interface (13Kontinen V. Sarvas M. Mol. Microbiol. 1993; 8: 727-737Crossref PubMed Scopus (167) Google Scholar, 17Rudd K.E. Sofia H.J. Koonin E.V. Plunkett III, G. Lazar S. Rouviere P.E. Trends Biochem. Sci. 1995; 20: 12-14Abstract Full Text PDF PubMed Scopus (79) Google Scholar). YacD is a paralogous protein that is also predicted to be a peptidyl-prolyl cis/trans-isomerase and transmembrane lipoprotein (18Kunst F. Ogasawara N. Moszer I. Albertini A.M. Alloni G. Azevedo V. Bertero M.G. Bessieres P. Bolotin A. Borchert S. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3122) Google Scholar). It remains uncharacterized except for its inability to complement for deficiencies in PrsA and its nonessential nature. Post-translocational folding is rate-limiting for secretion and can lead to misfolded products, which are subsequently removed by proteolytic degradation. This is particularly the case under secretion (e.g. overexpression; Refs. 13Kontinen V. Sarvas M. Mol. Microbiol. 1993; 8: 727-737Crossref PubMed Scopus (167) Google Scholar, 19Leloup L. Haddaoui E.A. Chambert R. Petit-Glatron M.F. Microbiology. 1997; 143: 3295-3303Crossref PubMed Scopus (47) Google Scholar, and 20Danese P.N. Silhavy T.J. Genes Dev. 1997; 11: 1183-1193Crossref PubMed Scopus (211) Google Scholar) or environmental stresses (e.g. heat shock; Refs. 21Missiakas D. Raina S. Trends Biochem. Sci. 1997; 22: 59-63Abstract Full Text PDF PubMed Scopus (56) Google Scholar, 22Raivio T.L. Silhavy T.J. Curr. Opin. Microbiol. 1999; 2: 159-165Crossref PubMed Scopus (155) Google Scholar, 23Pogliano J. Lynch A.S. Belin D. Lin E.C.C. Beckwith J. Genes Dev. 1997; 11: 1169-1182Crossref PubMed Scopus (240) Google Scholar) or when attempting to secrete nonnative proteins with reduced folding rate characteristics (24Stephenson K. Carter N.M. Harwood C.R. Petit-Glatron M.F. Chambert R. FEBS Lett. 1998; 430: 385-389Crossref PubMed Scopus (36) Google Scholar, 25Meens J. Frings E. Klose M. Freudl R. Mol. Microbiol. 1993; 9: 847-855Crossref PubMed Scopus (32) Google Scholar). In addition to PrsA, the folding of certain secretory proteins is dependent on divalent cations such as Ca2+ and Fe3+ (19Leloup L. Haddaoui E.A. Chambert R. Petit-Glatron M.F. Microbiology. 1997; 143: 3295-3303Crossref PubMed Scopus (47) Google Scholar, 24Stephenson K. Carter N.M. Harwood C.R. Petit-Glatron M.F. Chambert R. FEBS Lett. 1998; 430: 385-389Crossref PubMed Scopus (36) Google Scholar, 26Chambert R. Benyahia F. Petit-Glatron M.F. Biochem. J. 1990; 265: 375-382Crossref PubMed Scopus (21) Google Scholar). In Gram-positive bacteria such as B. subtilis, a crucial element determining the properties of the microenvironment immediately outside the membrane is the cell wall matrix, which comprises a complex heteropolymer of peptidoglycan and covalently linked anionic polymers, teichoic acid or teichuronic acid (27Archibald A.R. Hancock I.C. Harwood C.R. Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Other Gram-positive Bacteria. American Society for Microbiology, Washington, D. C.1993: 381-410Google Scholar). These anionic polymers, together with membrane-linked lipoteichoic acids, confer a high density of negative charge to the cell envelope and a high capacity to bind metal ions such as Ca2+ and Fe3+ (28Petit-Glatron M.-F. Grajcar L. Munz A. Chambert R. Mol. Microbiol. 1993; 9: 1097-1106Crossref PubMed Scopus (45) Google Scholar, 29Beveridge T.J. Murray R.G. J. Bacteriol. 1980; 141: 876-887Crossref PubMed Google Scholar) as well as cationic proteins and peptides (30Peschel A. Otto M. Jack R.W. Kalbacher H. Jung G. Götz F. J. Biol. Chem. 1999; 274: 8405-8410Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar). Furthermore, the proton motive force influences this microenvironment by maintaining a low pH at the wall/membrane interface. It is believed that the folding characteristics and kinetics of native secreted proteins have evolved to match the specific conditions found in this environment. In nonsporulating cells, wall and lipoteichoic acids contain ester-linked d-alanine residues, the extent of which modulates the density of negative charge in the wall (31Perego M. Glaser P. Minutello A. Strauch M.A. Leopold K. Fischer W. J. Biol. Chem. 1995; 270: 15598-15606Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar).d-Alanine esterification decreases negative charge, and concomitantly the capacity of teichoic acid for binding cations may decrease (32Lambert P.A. Hancock I.C. Baddiley J. Biochem. J. 1975; 151: 671-676Crossref PubMed Scopus (44) Google Scholar), with consequential effects on the availability of metal cations for post-translocational folding. However, the precise physiological function of the d-alanylation and charge modulation has not been clarified. The dlt operon is responsible for alanylation of teichoic acids. dlt mutants, which are not able to alanylate teichoic acids, have been described for a number of Gram-positive bacteria, and d-alanylation has been shown not to be essential for viability. dlt mutants ofLactobacillus casei exhibit defective cell separation (33Neuhaus F.C. Heaton M.P. Debabov D.V. Zhang Q. Microb. Drug Resist. 1996; 2: 77-84Crossref PubMed Scopus (57) Google Scholar), while those of Staphylococcus aureus show an increased sensitivity to antimicrobial peptides (30Peschel A. Otto M. Jack R.W. Kalbacher H. Jung G. Götz F. J. Biol. Chem. 1999; 274: 8405-8410Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar), the latter suggesting a role in protection against phagosomal defense mechanisms in pathogenic bacteria. In Streptococcus mutants, dlt gene products are associated with the intracellular polysaccharide accumulation (34Spatafora G.A. Sheets M. June R. Luyimbazi D. Howard K. Hulbert R. Barnard D. el Janne M. Hudson M.C. J. Bacteriol. 1999; 181: 2363-2372Crossref PubMed Google Scholar). In contrast, the observed phenotypic effects ofdlt mutations in B. subtilis are relatively minor; the cells exhibit a slightly increased susceptibility to cell lysis, either by endogenous lytic enzymes (autolysins) or β-lactams (35Wecke J. Perego M. Fischer W. Microb. Drug Resist. 1996; 2: 123-129Crossref PubMed Scopus (71) Google Scholar). To help elucidate the role of PrsA in protein secretion and to identify other factors affecting post-translocational folding, we initiated a search for suppressor mutations able to restore protein secretion in a mutant with a defective PrsA protein. As a result, we report a novel phenotype of dlt mutants of B. subtilis and present evidence that the alanylation of teichoic acids, and thus modulation of the negative charge of the wall, protects secretory and cell wall-associated proteins against degradation during post-translocational folding. Modulation of the degree of the alanylation may be an important tool for improving the production of secretory proteins in industrial applications. TableI lists bacterial strains and plasmids. For the determination of the levels of PrsA3 or PrsA and exoproteins in culture medium, bacteria were grown in modified 2xL broth (2% tryptone, 1% yeast extract, 1% NaCl) or in Spizizen's minimal salts (SMS)2 medium (36Anagnostopoulos C. Spizizen J. J. Bacteriol. 1961; 81: 741-746Crossref PubMed Google Scholar) supplemented with 10 mm potassium glutamate, 1 mm CaCl2, 5 mm FeCl3, 1 mm ZnSO4, 0.5% maltose, and 50 μg/ml each of the 20 amino acids except methionine (SMSa medium). L broth also contained 2% starch when the secretion of AmyQ was studied. For the determination of the yields of AmyL and AmyLQS50.5 released into the growth medium, cultures were grown in 2xYT medium (1.6% tryptone, 1% yeast extract, and 0.5% NaCl) buffered to pH 6.5 with 0.2m MES or to pH 8.1 with 0.2 m Tris-HCl. The expression of these α-amylases was induced with 1% (w/v) xylose, using a xylose-inducible promoter (24Stephenson K. Carter N.M. Harwood C.R. Petit-Glatron M.F. Chambert R. FEBS Lett. 1998; 430: 385-389Crossref PubMed Scopus (36) Google Scholar). Pulse-chase labeling experiments were performed either in SMSa or SMS supplemented with 1% (w/v) ribose as a non-catabolite-repressing carbon source.Table IBacterial strains and plasmidsStrain/PlasmidRelevant genotype/CharacteristicsReference/SourceB. subtilis IH6531glyB133 hisA1 trpC2 (pKTH10)Ref. 40Kontinen V.P. Sarvas M. J. Gen. Microbiol. 1988; 134: 2333-2344PubMed Google Scholar IH7052prsA3 (pJBA222)This study IH7053(pJBA222)This study IH7123glyB133 hisA1 trpC2 (pKTH3327)V. P. Kontinen, National Public Health Institute (KTL), Helsinki IH7144glyB133 hisA1 prsA3 (pKTH10)This study IH7163prsA::cat ywlG::P spac -prsA (pKTH10)M. Vitikainen, KTL, Helsinki IH7175glyB133 hisA1 trpC2 (pKTH3327, pKTH3382)V. P. Kontinen, KTL, Helsinki IH7211P spac -prsAU. Airaksinen, KTL, Helsinki IH7231glyB133 hisA1 prsA3 dltD::mini-Tn10 (pKTH10)This study IH7383prsA::cat ywlG::P spac -prsA dltB::pDLT72 (pKTH10)M. Vitikainen, KTL, Helsinki IH7449P spac -prsA dltD::mini-Tn10This study IH7455prsA3 dltD::mini-Tn10(pJBA222)This study IH7458glyB133 hisA1 prsA3 dltD::mini-Tn10 (pKTH3461)This study IH7461glyB133 hisA1 prsA3 (pKTH3461)This study IH7648glyB133 hisA1 prsA3 (pKTH3461, pKTH3382)This study IH7674glyB133 hisA1 prsA3 dltD::mini-Tn10 pKTH3461 (pKTH3382)This study IH7410hisA1 trpC2 prsA3 dltD::pMUTIN4 (pKTH10)This study ECE600(pHV1248)BGSC1-aThe Bacillus Genetic Stock Center, Ohio State University. KS405BamyE xylR::pKS405B, xylose-inducible amyLQS50.5Ref. 24Stephenson K. Carter N.M. Harwood C.R. Petit-Glatron M.F. Chambert R. FEBS Lett. 1998; 430: 385-389Crossref PubMed Scopus (36) Google Scholar KS408amyE xylR::pKS408, xylose-inducibleamyLRef. 24Stephenson K. Carter N.M. Harwood C.R. Petit-Glatron M.F. Chambert R. FEBS Lett. 1998; 430: 385-389Crossref PubMed Scopus (36) Google Scholar KS405BdltB::pDLT72amyE dltB::pDLT72 xylR::pKS405B, xylose-inducible amyLQS50.5This study KS408dltB::pDLT72amyE dltB::pDLT72 xylR::pKS408, xylose-inducible amyLThis studyPlasmid pDG148AprKmr, P spac promoter,B. subtilis-E. coli shuttle vectorRef. 37Stragier P. Bonamy C. Karmazyn-Campelli C. Cell. 1988; 52: 697-704Abstract Full Text PDF PubMed Scopus (306) Google Scholar pHV1248AprEmrCmr mini-Tn10ori-pE194tsRef. 39Petit M.-A. Bruand L. Janniere L. Ehrlich D. J. Bacteriol. 1990; 172: 6736-6740Crossref PubMed Google Scholar pJBA222Emrφ(P xyn -subC(prepro)-phoA)Ref.16Jacobs M. Anderssen J.B. Kontinen V. Sarvas M. Mol. Microbiol. 1993; 8: 957-966Crossref PubMed Scopus (98) Google Scholar pKTH10Kmr amyQRef. 47Palva I. Gene (Amst.). 1982; 19: 81-87Crossref PubMed Scopus (178) Google Scholar pKTH3327Kmr P spac -prsAT. Pummi, KTL, Helsinki pKTH3382Emr amyQV. P. Kontinen, KTL, Helsinki pKTH3461KmrP spac -prsA3-HIS6This study pMUTIN4AprEmr, integrable vectorRef.38Vagner V. Dervyn E. Ehrlich S.D. Microbiology. 1998; 144: 3097-3104Crossref PubMed Scopus (572) Google Scholar1-a The Bacillus Genetic Stock Center, Ohio State University. Open table in a new tab Plasmid pKTH3461 was constructed by polymerase chain reaction-amplifying theprsA3 gene, containing the ribosome binding site, the protein-encoding region, and the transcription terminator, with 5′-GCCGAAGCTTTGGAATGATTAGGAGTGTTT as a forward primer and 5′-GCACGTCGACTTAATGGTGATGGTGATGGTGTTTAGAATTGCTTG as a reverse primer. The amplified fragment was inserted between the SalI andHindIII sites on the pDG148 shuttle vector (37Stragier P. Bonamy C. Karmazyn-Campelli C. Cell. 1988; 52: 697-704Abstract Full Text PDF PubMed Scopus (306) Google Scholar). pKTH3461 expresses PrsA3 protein with a C-terminal extension of six histidine residues (His tag) from the inducible P spac promoter. ThedltD gene was interrupted with the integrative plasmid pMUTIN4 (38Vagner V. Dervyn E. Ehrlich S.D. Microbiology. 1998; 144: 3097-3104Crossref PubMed Scopus (572) Google Scholar). An internal fragment of dltD (0.5 kilobases) was polymerase chain reaction-amplified with the primers 5′-CGGTAAGCTTAGCAGCAGATCAACCTTCAC and 5′-TCAAGGATCCTACCTGCAAGGCAAGAATGG, and inserted between theBamHI and HindIII sites in pMUTIN4. Transformation of a B. subtilis strain with the constructed plasmid and subsequent integration into the dlt locus by a Campbell-type recombination event resulted in the interruption of thedltD gene. The knockout mutation was verified by Southern hybridization. The dltB gene of strains DN1885xylR::pKS408 and DN1885xylR::pKS405B (24Stephenson K. Carter N.M. Harwood C.R. Petit-Glatron M.F. Chambert R. FEBS Lett. 1998; 430: 385-389Crossref PubMed Scopus (36) Google Scholar) was insertionally inactivated with an erythromycin resistance cassette by transforming naturally competent cells with chromosomal DNA isolated from JH642::pDLT72 (31Perego M. Glaser P. Minutello A. Strauch M.A. Leopold K. Fischer W. J. Biol. Chem. 1995; 270: 15598-15606Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), and the insertion was confirmed by polymerase chain reaction. A prsA3 mutant overexpressing α-amylase from pKTH10 and harboring plasmid pHV1248 with the temperature-sensitive replication origin (ori-pE194ts) and the mini-Tn10 transposon (39Petit M.-A. Bruand L. Janniere L. Ehrlich D. J. Bacteriol. 1990; 172: 6736-6740Crossref PubMed Google Scholar) was grown in modified 2xL-broth at 30 °C up to the cell density of Klett 50, after which the temperature was shifted to 50 °C. Incubation at the nonpermissive temperature was continued for 3 h, and then appropriately diluted cell samples were plated on agar plates containing chloramphenicol (5 μg/ml) for the selection of insertion mutants and 5% starch (40Kontinen V.P. Sarvas M. J. Gen. Microbiol. 1988; 134: 2333-2344PubMed Google Scholar) for screening for mutants affected in α-amylase production by halo formation. The plates were incubated at 51 °C for 16 h. Increased halo size around a colony was indicative of a suppressor mutant. A library of chromosomal DNA of the suppressor mutant was constructed in bacteriophage λ as described previously (15Kontinen V.P. Saris P. Sarvas M. Mol. Microbiol. 1991; 5: 1273-1283Crossref PubMed Scopus (101) Google Scholar). Clones carrying a DNA fragment with the mini-Tn10 were screened by hybridization with plasmid pHV1248, labeled with digoxigenin-dUTP. Purification of λ particles and isolation of their DNA was carried out with the methods described previously (41Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar, 42Thomas M. Davis R.W. J. Mol. Biol. 1975; 91: 315-328Crossref PubMed Scopus (550) Google Scholar). DNA sequencing was performed with the dideoxy sequencing method of Sanger et al.(43Sanger F. Nicklen S. Caulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52610) Google Scholar). The pellet from 1 ml of culture was washed in 500 ml of 0.1m MES, pH 6.0, at room temperature, resuspended in 250 ml of 0.4% SDS and 250 ml of 0.1 m MES, pH 6.0, and then boiled for 15 min. The insoluble cell wall material was washed four times with 0.1 m MES (pH 6.0) dried, resuspended in 0.5 ml of sodium pyrophosphate (pH 8.3), and then incubated at 60 °C for 3 h to release ester-linked alanine residues. The alanine content of the supernatant was determined by mixing 100 ml of supernatant with 250 ml of fresh assay reagent (4 volumes of 0.1 m sodium pyrophosphate (pH 8.3), 2 volumes of 0.2 mg/ml FAD in 0.1 msodium pyrophosphate (pH 8.3), 1 volume of 50 mg/ml horseradish peroxidase (200 units/mg), 1 volume of 5 mg/ml dianisidine sulfate, and 0.1 volume of 5 mg/ml d-amino acid oxidase (15 units/mg). The assay mix was incubated at 37 °C for 15 min, the reaction was stopped by the addition of 1 ml of 0.1% SDS, and the absorbance was determined at 460 nm. Cell samples for immunoblotting were prepared as follows. Cells were separated from culture medium by centrifugation, resuspended in 50 μl of protoplast buffer (20 mm potassium phosphate, pH 7.5, 15 mm MgCl2 20% sucrose, and 1 mg/ml lysozyme), and incubated at 37 °C for 20 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nylon membranes by Mini Trans-Blot system (Bio-Rad), and treated with anti-PrsA antibodies, and cross-reacting proteins were visualized by ECL (Amersham Pharmacia Biotech). Pulse-chase labeling was performed as described previously (24Stephenson K. Carter N.M. Harwood C.R. Petit-Glatron M.F. Chambert R. FEBS Lett. 1998; 430: 385-389Crossref PubMed Scopus (36) Google Scholar, 44Leskelä S. Wahlström E. Hyyryläinen H.-L. Jacobs M. Palva A. Sarvas M. Kontinen V.P. Mol. Microbiol. 1999; 31: 533-543Crossref PubMed Scopus (29) Google Scholar). α-Amylase released into the culture medium was determined using the Phadebas kit (Amersham Pharmacia Biotech) as described previously (24Stephenson K. Carter N.M. Harwood C.R. Petit-Glatron M.F. Chambert R. FEBS Lett. 1998; 430: 385-389Crossref PubMed Scopus (36) Google Scholar, 44Leskelä S. Wahlström E. Hyyryläinen H.-L. Jacobs M. Palva A. Sarvas M. Kontinen V.P. Mol. Microbiol. 1999; 31: 533-543Crossref PubMed Scopus (29) Google Scholar). Alkaline phosphatase was assayed using 4-nitrophenyl phosphate (Merck) as a substrate; 50 μl of culture supernatant was mixed with 410 μl of 50 mmTris-HCl, pH 8.0, and 300 μl of 4-nitrophenyl phosphate (1 mg/ml), followed by incubation at 30 °C for 8 min. The reaction was stopped by adding 300 μl of 2 m NaOH. Absorbance was determined at 410 nm and converted to enzyme concentrations (mg/liter) by usingE. coli alkaline phosphatase (Fluka) of known specific activity as a standard. Mutants such asprsA3, encoding a defective PrsA protein, exhibit secretion defects for several exoproteins due to their post-translocational misfolding and subsequent proteolytic degradation. In order to identify putative cell components that interact with PrsA, we screened for suppressors in which the secretion deficiency of a prsA3mutant was fully or partially restored. Transposon mutagenesis was used to interrupt genes on the chromosome of a prsA3 mutant. Transposon-induced mutants were screened for increased secretion of AmyQ, a PrsA-dependent α-amylase from B. amyloliquefaciens, encoded by multicopy plasmid pKTH10. The mini-Tn10 transposon, encoded by the temperature-sensitive plasmid pHV1248 (ori-pE194ts; Ref. 39Petit M.-A. Bruand L. Janniere L. Ehrlich D. J. Bacteriol. 1990; 172: 6736-6740Crossref PubMed Google Scholar), was transformed into a prsA3 mutant (IH7144), and transformants were selected by virtue of their resistance to erythromycin. Upshift to the nonpermissive temperature (51 °C) under selection for chloramphenicol resistance (39Petit M.-A. Bruand L. Janniere L. Ehrlich D. J. Bacteriol. 1990; 172: 6736-6740Crossref PubMed Google Scholar) allowed selection for derivatives in which the mini-Tn10 had integrated into the chromosome. Screening for suppressor mutants was carried out on starch plates using an α-amylase halo assay (40Kontinen V.P. Sarvas M. J. Gen. Microbiol. 1988; 134: 2333-2344PubMed Google Scholar). After screening about only 100 transposon-induced mutants, a single mutant was identified in which increased halo size was 100% co-transformable with the integrated transposon. The properties of this mutant suggested that interruption of a chromosomal gene(s) was capable of suppressing, at least in part, the secretion defect of the prsA3. The suppression was confirmed in liquid culture, the suppressor mutant (IH7231) secreting about 10-fold more α-amylase than its prsA3 parent (Fig.1 A). However, this level is only about 30% of that of the wild type, indicating that the suppression, although significant, did not allow full restoration of α-amylase production. The transposon mutation had no effect on the growth of IH7231 as compared with IH7144 (Fig. 1 A). In order to determine the identity of the gene(s) interrupted by mini-Tn10, the integrated transposon with flanking chromosomal regions was cloned into LambdaGEM-11 (Promega) and sequenced over the ends of the transposon. Comparison of the obtained sequence with the genome sequence of B. subtilis (18Kunst F. Ogasawara N. Moszer I. Albertini A.M. Alloni G. Azevedo V. Bertero M.G. Bessieres P. Bolotin A. Borchert S. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3122) Google Scholar) revealed the location of the mini-Tn10 transposon indltD, the fourth of five genes in the dlt operon (dltA-dltE), responsible ford-alanine esterification of both lipoteichoic acid (LTA) and wall teichoic acid (WTA). Previously, it has been shown that insertional inactivation of any of the first four genes in the operon (i.e. dltA–dltD) resulted in the absence of alanylation of both LTA and WTA, whereas inactivation ofdltE has no effect on the alanylation of these polymers (31Perego M. Glaser P. Minutello A. Strauch M.A. Leopold K. Fischer W. J. Biol. Chem. 1995; 270: 15598-15606Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Targeted interruption of dltD with pMUTIN4 (38Vagner V. Dervyn E. Ehrlich S.D. Microbiology. 1998; 144: 3097-3104Crossref PubMed Scopus (572) Google Scholar), an integrative plasmid in which the polar effect of the integration on downstream genes can be suppressed by an IPTG-induced promoter, and interruption of dltB with pDLT72 (31Perego M. Glaser P. Minutello A. Strauch M.A. Leopold K. Fischer W. J. Biol. Chem. 1995; 270: 15598-15606Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) similarly suppressed the secretion defect of AmyQ in the prsA3 mutant (data not shown). These results suggest that the absence ofd-alanyl residues on teichoic acids is responsible for the observed suppression of the PrsA3 phenotype. dltD::mini-Tn10 was introduced into aprsA3 mutant, producing another PrsA-dependent exoprotein, namely the SubC(prepro)-PhoA fusion protein (16Jacobs M. Anderssen J.B. Kontinen V. Sarvas M. Mol. Microbiol. 1993; 8: 957-966Crossref PubMed Scopus (98) Google Scholar). This protein is secreted from the prsA3 mutant at a level that is less than 5% that of the wild-type strain (Fig. 1 B). Like that of AmyQ, the accumulation of SubC(prepro)-PhoA in the medium was enhanced ∼10-fold by the dltD mutation (Fig.1 B), indicating that the effect of the dltmutation was pleiotropic with respect to PrsA-dependent proteins. Thed-alanine ester content of WTA was analyzed to determine the activity o" @default.
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• W4253388474 title "d-Alanine Substitution of Teichoic Acids as a Modulator of Protein Folding and Stability at the Cytoplasmic Membrane/Cell Wall Interface of Bacillus subtilis" @default.
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