Identification of Protein-Protein Contacts between α/β-Type Small, Acid-soluble Spore Proteins of Bacillus Species Bound to DNA (1998)

Small, acid-soluble spore proteins (SASP) of the α/β-type from several Bacillus species were cross-linked into homodimers, heterodimers and homooligomers with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of linear plasmid DNA. Significant protein cross-linking was not detected in the absence of DNA. In all four α/β-type SASP examined, the amino donor in the EDC induced amide cross-links was the α-amino group of the protein. However, the carboxylate containing amino acid residues involved in cross-linking varied. In SASP-A and SASP-C ofBacillus megaterium two conserved glutamate residues, which form part of the germination protease recognition sequence, were involved in cross-link formation. In SspC from Bacillus subtilis and Bce1 from Bacillus cereus the acidic residues involved in cross-link formation were not in the protease recognition sequence, but at a site closer to the N terminus of the proteins. These data indicate that, although there are likely to be subtle structural differences between different α/β-type SASP, the N-terminal regions of these proteins are involved in protein-protein interactions while in the DNA bound state. Small, acid-soluble spore proteins (SASP) of the α/β-type from several Bacillus species were cross-linked into homodimers, heterodimers and homooligomers with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of linear plasmid DNA. Significant protein cross-linking was not detected in the absence of DNA. In all four α/β-type SASP examined, the amino donor in the EDC induced amide cross-links was the α-amino group of the protein. However, the carboxylate containing amino acid residues involved in cross-linking varied. In SASP-A and SASP-C ofBacillus megaterium two conserved glutamate residues, which form part of the germination protease recognition sequence, were involved in cross-link formation. In SspC from Bacillus subtilis and Bce1 from Bacillus cereus the acidic residues involved in cross-link formation were not in the protease recognition sequence, but at a site closer to the N terminus of the proteins. These data indicate that, although there are likely to be subtle structural differences between different α/β-type SASP, the N-terminal regions of these proteins are involved in protein-protein interactions while in the DNA bound state. Between 5 and 10% of the total protein in spores of theBacillus and Clostridium species of bacteria is α/β-type small, acid-soluble spore protein (α/β-type SASP) 1The abbreviations used are: SASP, small, acid-soluble spore proteins; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis; GPR, germination protease. (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar, 2Setlow P. Annu. Rev. Microbiol. 1995; 49: 29-54Crossref PubMed Scopus (331) Google Scholar). These proteins are encoded by four to seven monocistronic genes in each species, and their amino acid sequences are highly conserved both within and betweenBacillus species (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar, 2Setlow P. Annu. Rev. Microbiol. 1995; 49: 29-54Crossref PubMed Scopus (331) Google Scholar). The α/β-type SASP are nonspecific DNA-binding proteins which are synthesized only within the forespore compartment during sporulation (3Mason J.M. Hackett R.H. Setlow P. J. Bacteriol. 1988; 170: 239-244Crossref PubMed Scopus (88) Google Scholar, 4Sun D. Stragier P. Setlow P. Genes Dev. 1989; 3: 141-149Crossref PubMed Scopus (107) Google Scholar). Typically, two major α/β-type SASP accumulate to high levels within the spore, while the minor α/β-type SASP are found at much lower levels. The level of total α/β-type SASP in spores is sufficient to saturate the spore chromosome, and the binding of these proteins to spore DNA is the major determinant of spore resistance to UV radiation and a significant determinant of spore heat resistance (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar, 2Setlow P. Annu. Rev. Microbiol. 1995; 49: 29-54Crossref PubMed Scopus (331) Google Scholar). Bacillus subtilis spores which lack the two major α/β-type SASP (α and β) are much more sensitive to UV radiation and heat than are wild type spores (5Mason J.M. Setlow P. J. Bacteriol. 1986; 167: 174-178Crossref PubMed Google Scholar). During the first few minutes of spore germination, α/β-type SASP are quickly degraded by a sequence-specific protease termed germination protease (GPR) (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar, 2Setlow P. Annu. Rev. Microbiol. 1995; 49: 29-54Crossref PubMed Scopus (331) Google Scholar). Structural studies of purified α/β-type SASP and α/β-type SASP·DNA complexes have shown that significant changes in these proteins' structure occur upon binding to DNA, as α/β-type SASP are predominantly unfolded in solution but acquire significant α-helical content upon binding to DNA 2S. C. Mohr and P. Setlow, unpublished results. (6Rao H. Mohr S.C. Fairhead H. Setlow P. FEBS Lett. 1992; 305: 115-120Crossref PubMed Scopus (9) Google Scholar). The α/β-type SASP cover 4–6 base pairs of DNA, and binding of these proteins to DNA is highly cooperative, particularly to DNAs bound with low affinity. (7Nicholson W.L. Setlow B. Setlow P. J. Bacteriol. 1990; 172: 6900-6906Crossref PubMed Google Scholar). Electron micrographs of α/β-type SASP·DNA complexes indicate that the protein forms a helical coat along the DNA (8Griffith J. Makhov A. Santiago-Lara L. Setlow P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8224-8228Crossref PubMed Scopus (36) Google Scholar), suggesting that there are extensive interactions between α/β-type SASP when bound to DNA, although these proteins are monomers in solution 3B. Setlow and P. Setlow, unpublished results. (9Setlow P. J. Biol. Chem. 1975; 250: 8168-8173Abstract Full Text PDF PubMed Google Scholar). Consequently, it is possible that interactions between adjacent α/β-type SASP along the DNA backbone may be important for the α/β-type SASP/DNA binding interaction. To determine which regions of the proteins are involved in interactions between α/β-type SASP bound to DNA, we have performed protein cross-linking studies with the zero-length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). We have identified EDC-catalyzed protein cross-links in four different α/β-type SASP from Bacillus species, and the identification of these cross-links has yielded new insights into the interaction of α/β-type SASP on DNA. TheEscherichia coli strains used include: JM107 (F′traD36 proA+ proB+ lacIq lacZ ΔM15/endA1 gyrA96(Nalr) thi hsdR17 supE44 relA1Δ(lac-proAB) mcrA) (Life Technologies, Inc.), JM83 (ara Δ(lac-proAB) rpsL φ80lacZΔM15) (10Yanisch-Perron C. Vieira J. Messing J. Gene ( Amst. ). 1985; 33: 103-119Crossref PubMed Scopus (11462) Google Scholar), BL21(DE3) (T7 RNA polymerase under control of the lac promoter) (11Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4831) Google Scholar), and BMH 71-18 (F′ proAB lacIq lacZΔM15 thi supEΔ(lac-proAB) mutS::Tn10) (CLONTECH Laboratories, Inc.). TheBacillus strains used were Bacillus cereus T (originally obtained from H. O. Halvorson) and Bacillus megaterium QMB1551, ATCC no. 12872 (originally obtained from H. Levinson). E. coli strains were routinely grown in 2× YT medium (16 g of tryptone, 10 g of yeast extract, 5 g of NaCl per liter) at 37 °C with shaking. For the overexpression of cloned genes encoding α/β-type SASP, the medium was supplemented with 100 μg/ml ampicillin (JM107) or 200 μg/ml ampicillin and 0.5% glucose (BL21).B. megaterium was sporulated at 30 °C in supplemented nutrient broth, and spores were harvested and cleaned as described previously (12Setlow P. Kornberg A. J. Bacteriol. 1969; 100: 1155-1160Crossref PubMed Google Scholar). Oligonucleotides were designed to polymerase chain reaction amplify a 512-base pair fragment containing the gene encoding Bce1 (13Loshon C.A. Fliss E.R. Setlow B. Foerster H.F. Setlow P. J. Bacteriol. 1986; 167: 168-173Crossref PubMed Google Scholar) from B. cereus genomic DNA; the amplified fragment contained the gene's ribosome binding site and transcription terminator (13Loshon C.A. Fliss E.R. Setlow B. Foerster H.F. Setlow P. J. Bacteriol. 1986; 167: 168-173Crossref PubMed Google Scholar). The upstream primer, BCE1–1 (5′-AAAGGATCCTTATTATTTCATAATTTGTAGC; complementary to nucleotides 119–140) (13Loshon C.A. Fliss E.R. Setlow B. Foerster H.F. Setlow P. J. Bacteriol. 1986; 167: 168-173Crossref PubMed Google Scholar) and downstream primer, BCE1–2 complementary to nucleotides (13Loshon C.A. Fliss E.R. Setlow B. Foerster H.F. Setlow P. J. Bacteriol. 1986; 167: 168-173Crossref PubMed Google Scholar), each contained and sequences for The polymerase chain reaction was and plasmid (11Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4831) Google Scholar), plasmid in which that the gene encoding Bce1 was under the control of the was used to E. coli to ampicillin The form of Bce1 was with the to for designed to the site of and the of the gene encoding Bce1 (13Loshon C.A. Fliss E.R. Setlow B. Foerster H.F. Setlow P. J. Bacteriol. 1986; 167: 168-173Crossref PubMed Google Scholar) were used with plasmid as a to a plasmid the and with a the for glutamate 10 of The plasmid was with to into the E. coli with the plasmid was by plasmid with and coli was and the of the termed was by DNA SspC and SASP-C were in E. coli JM107 from containing the α/β-type SASP genes under control of as described previously B. Setlow P. J. Bacteriol. PubMed Google Scholar, P. C. C. 1988; Full Text PDF PubMed Scopus Google Scholar). Bce1 and were in E. coli from and and from E. coli with as described previously Setlow P. J. Bacteriol. Google Scholar). SASP-C and SASP-A were with acid (9Setlow P. J. Biol. Chem. 1975; 250: 8168-8173Abstract Full Text PDF PubMed Google Scholar) from coli and spores of B. megaterium QMB1551, α/β-type SASP were purified as described previously (9Setlow P. J. Biol. Chem. 1975; 250: 8168-8173Abstract Full Text PDF PubMed Google Scholar). SASP with or plasmid DNA were in of 5 at °C for to of EDC to The of protein to DNA is sufficient to saturate the DNA with α/β-type SASP, although SASP-A more than the α/β-type SASP B. D. Setlow P. J. Bacteriol. 1992; PubMed Google Scholar). these of the α/β-type SASP are bound to the DNA. The cross-linking were for 30 at by in of 10 at °C for cross-linking were in and on H. PubMed Scopus Google Scholar). were with and and cross-linked proteins were with a were from polyacrylamide gel into purified proteins were in 100 of and with of proteins were with of and in to proteins were with 5 in 100 of 10 at 37 °C for were on high performance liquid a two a and a protein were the in by 5 of with A. were at a of with a linear as acid in B. were detected by their UV at with a and containing were with and of were by matrix-assisted laser desorption/ionization-time of a of two and was used for all in from were and on the with of acid in acid was as described previously (9Setlow P. J. Biol. Chem. 1975; 250: 8168-8173Abstract Full Text PDF PubMed Google Scholar). sequences were with protein In to the regions of α/β-type SASP bound to DNA, we to protein cross-linking to amino acid residues for α/β-type SASP not residues (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar) and several protein cross-linking which upon not be used for The α/β-type SASP are proteins which are in (9Setlow P. J. Biol. Chem. 1975; 250: 8168-8173Abstract Full Text PDF PubMed Google Scholar) and to with only when bound to DNA. We were in regions of between α/β-type SASP and to cross-linking with or zero-length EDC, a cross-linking of α/β-type SASP only in the presence of DNA Consequently, we reagent for cross-linking with EDC the formation of amide between N-terminal α-amino or group and the chain of in to that several in EDC induced cross-links occur only between residues that are in to The proteins for were SASP-A and SASP-C from B. SspC from B. and Bce1 cereus of the between these proteins the N which both in and amino acid (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar). α/β-type SASP lack the N-terminal which is the only that is (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar). SASP-A and SASP-C are major α/β-type SASP in SspC and Bce1 are minor proteins. For all four proteins or protein-protein cross-linking was detected in the absence of DNA, while significant protein-protein cross-linking was detected in containing α/β-type SASP and DNA and data not The of protein-protein cross-link formation and the of for each protein polyacrylamide be detected in cross-linking with Bce1 and DNA, only of were detected in with SASP-A and DNA and data not The of protein cross-linking to the of each protein for linear plasmid DNA SspC SASP-C as by B. D. Setlow P. J. Bacteriol. 1992; PubMed Google Scholar) not although the cross-linking of SASP-A was lower than cross-linking of α/β-type SASP is with or linear plasmid DNA was with EDC for 30 at by 10 at °C as described under were in and on and with on the the of in The DNA of cross-link formation between α/β-type SASP that the protein-protein cross-links are only between α/β-type SASP that are adjacent to on the DNA These in used only a α/β-type However, there are α/β-type SASP in with two proteins at high levels. Consequently, is the different α/β-type SASP when bound to DNA. To data to we protein-protein cross-link formation in with two different α/β-type SASP bound to DNA. SASP-A and SASP-C megaterium were for the are the two major α/β-type SASP found in spores of B. megaterium (9Setlow P. J. Biol. Chem. 1975; 250: 8168-8173Abstract Full Text PDF PubMed Google Scholar). These proteins in and SASP-C to of the possible forms by of cross-linking containing and DNA the presence of a new that at the for a and is a that the of SASP-A to SASP-C that the is not the levels of these two proteins megaterium spores (9Setlow P. J. Biol. Chem. 1975; 250: 8168-8173Abstract Full Text PDF PubMed Google Scholar). were between SASP-A and SspC from B. subtilis not However, in to the cross-linking reaction in which the was the cross-linked formation was much than formation of the SspC in these not SspC and SASP-C have for linear plasmid DNA in the in their formation of heterodimers with SASP-A is to differences in the amino acid sequences of SspC and is structural on α/β-type SASP or the form with DNA. identification of the amino acid residues involved in cross-link formation was to determine which regions of α/β-type SASP are involved in protein-protein interactions that occur in the DNA bound state. and α/β-type SASP from EDC cross-linking were with and the by of differences be detected between the of and EDC α/β-type the of α/β-type SASP in the of as with the amino acid residues within be in a cross-linked in the there be a new in of α/β-type SASP which be the containing the amino acid and amino acid of the both the identification of the in the as as the amino acid residues cross-links be by EDC, as α/β-type SASP from unfolded to a more structure on binding to DNA. cross-links be found within both and and be detected by of EDC monomers and protein. However, we for cross-link formation in these not identified two in the of SASP-A and which were not in the of the SASP-A in the of was in the of SASP-A when with that of SASP-A significant differences were between of the and the of all to be the between of and these data that the cross-link between amino acid in and a within a which has UV The of the SASP-C and differences that were to with SASP-A not of the of the identified two which were not in of SASP-A or SASP-C monomers not of these from the that from of the cross-linked identified from the SASP-A and SASP-C not two significant were detected in the Bce1 that were not in the Bce1 and of these 5 with was form of Bce1 which contained a not The the cross-linked in the 5 in the level of major was when the of the of Bce1 was with that of suggesting that the cross-link between amino acid residues from two In to and of the of the SspC identified only in to the of the SspC not However, as was found with SASP-A and the of major was in the of the SspC as with the of the not is involved in cross-link formation with a The high of SspC and Bce1 cross-linking the and of cross-linked and species of these proteins. The of the of the and species of both SspC and Bce1 were to the of the with the of in the in the cross-link in the of SspC not These data that EDC cross-links occur between each protein in of cross-linked α/β-type of were used to determine the amino acid residues involved in cross-link formation in the different α/β-type For and and amino acid identified the level was in of the species as and identified in the major cross-link in these proteins. of the of each identified as a cross-linked species from of both and heterodimers as as amino acid not of the two in the In all the site of cross-linking was identified as between the α-amino group of the protein and acidic group on a The in the two new from of cross-linked of SASP-A or SASP-C suggesting that each contained the two but cross-linked at a different of the two from the of the that the cross-links were between the α-amino group of protein and a in the protein of cross-linked by were on the are and to the of their during and 5 and data not are and to the of their during and 5 and data not are and to the of their during and 5 and data not are and to the of their during and 5 and data not are and to the of their during and 5 and data not are and to the of their during and 5 and data not was performed as described under were on the are (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar) and P. Annu. Rev. Microbiol. 1995; 49: 29-54Crossref PubMed Scopus (331) Google Scholar) to the of their during and 5 and data not in a new was performed as described under acid identified the cross-linked from the four α/β-type SASP as as the amino acid was obtained from the cross-linked with the of the α-amino group of each protein in these of the two cross-linked from SASP-A that of the involved in the cross-link was The differences in and amino acid between the cross-linked and were with the cross-linking of to of SASP-A and data not from the SASP-A a of glutamate in while a of glutamate in both of these glutamate residues are involved in cross-links to the amino terminus of the and of these glutamate residues were of the two cross-linked were not from by was obtained in from the SASP-C as a of glutamate was found in We that from SASP-C which the and amino acid as a cross-link to the glutamate at in of the two from the cross-links at the residues as in the SASP-A and SASP-C However, the in glutamate were not as as with the cross-linked from the SASP-A and SASP-C homodimers, that each of the from the heterodimers were of two different cross-linked acid of cross-linked and amino acid the amino acid involved in cross-link amino acid was obtained in amino acid was obtained in are as described in The the amino acid involved in cross-link amino acid was obtained in in a new are as described in was found with the cross-linked from SASP-A and only a amino acid was obtained with the cross-linked from SspC and Bce1 is with the of the α-amino group of the two proteins in cross-link formation. However, the that the glutamate residues involved in cross-link formation in SASP-A and SASP-C are not involved in formation of the major cross-link found in SspC and were obtained at to of SspC and glutamate 10 of Bce1 and These residues are at in α/β-type SASP amino acid However, the residues at are not as highly conserved as are residues in α/β-type SASP (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar). The acidic residues identified as of cross-link formation in Bce1 and SspC are not highly conserved α/β-type SASP (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar), and their in EDC cross-linking was Consequently, we to the these acidic residues in DNA binding and to determine in Bce1 the cross-link between the α-amino group and glutamate 10 was the only by form of Bce1 was in which glutamate 10 was to a not α/β-type SASP a SASP-A and or a at (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar). type of was not with SASP-A or has shown that changes in the glutamate residues involved in cross-link formation in these proteins DNA binding Setlow P. J. Bacteriol. 1994; PubMed Google Scholar). bound to DNA and the to linear plasmid as of Bce1 not EDC but with much lower on than wild type Bce1 The in cross-linking between Bce1 and that glutamate 10 is the major site of EDC cross-link formation in but that there is at cross-linking cross-linked was purified and to the residues involved in of a of the two when with the of not of these contained but not a while the was identified as a cross-linked by and amino acid The cross-link to be between the α-amino group of the protein and of the two conserved glutamate residues which in the cross-links in SASP-A and SASP-C not studies have that α/β-type SASP to DNA in a and that protein-protein interactions occur during binding (7Nicholson W.L. Setlow B. Setlow P. J. Bacteriol. 1990; 172: 6900-6906Crossref PubMed Google Scholar). The data in that protein-protein interactions occur between α/β-type SASP bound to DNA. The protein-protein interaction detected by EDC cross-linking is not in the absence of DNA, which is with data that that α/β-type SASP as monomers in (9Setlow P. J. Biol. Chem. 1975; 250: 8168-8173Abstract Full Text PDF PubMed Google Scholar). that α/β-type SASP/DNA binding the of the proteins along the backbone of the DNA is both by of α/β-type SASP·DNA complexes by (8Griffith J. Makhov A. Santiago-Lara L. Setlow P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8224-8228Crossref PubMed Scopus (36) Google Scholar) and by the of α/β-type SASP to the DNA backbone from by and B. D. Setlow P. J. Bacteriol. 1992; PubMed Google Scholar). that α/β-type SASP in a along the DNA EDC cross-linked and of SspC and Bce1 to only type of which is with a However, it is not DNA has in α/β-type SASP The DNA binding of α/β-type SASP has to be within the of these proteins. of α/β-type SASP is more highly conserved than the N-terminal and a acid to residues of SspC has shown to to DNA and the UV (6Rao H. Mohr S.C. Fairhead H. Setlow P. FEBS Lett. 1992; 305: 115-120Crossref PubMed Scopus (9) Google Scholar). However, the binding of to DNA was much than that of SspC (6Rao H. Mohr S.C. Fairhead H. Setlow P. FEBS Lett. 1992; 305: 115-120Crossref PubMed Scopus (9) Google Scholar). The EDC cross-link found in α/β-type SASP that the N-terminal of these proteins is involved in protein-protein These protein-protein interactions the binding of α/β-type SASP for DNA, and at in for the in DNA binding between the and The N-terminal of α/β-type SASP is the of these with in both and (1Setlow P. Annu. Rev. Microbiol. 1988; 42: 319-338Crossref PubMed Scopus (176) Google Scholar). In α/β-type SASP are proteins with the of their N-terminal is by SASP-A and SASP-C of B. which are with the of the N terminus of SASP-C However, their in sequence, SASP-C has a for DNA in solution (9Setlow P. J. Biol. Chem. 1975; 250: 8168-8173Abstract Full Text PDF PubMed Google Scholar). In of the it that the N-terminal regions of α/β-type SASP are involved in protein-protein and be a major in the binding of these proteins for DNA in is important to that regions of α/β-type SASP may be involved in protein-protein which may not be detected the reagent used in cross-links only amino and The identification of cross-linked acidic residues within the recognition sequences of SASP-A and SASP-C is particularly in studies have that α/β-type SASP are to when bound to DNA B. Setlow P. J. Bacteriol. 1995; PubMed Google Scholar). The data in that the N of SASP-A and SASP-C are to the while these proteins are in the DNA bound state. the site may be to the protease to by the N terminus of adjacent protein. However, structural changes are important purified cross-linked and SspC both are to not EDC cross-linking may a protein of α/β-type SASP in which is The identification of two different of DNA cross-link formation in the α/β-type SASP was upon the of between of protein the to and in SspC and is from the recognition site in sequence, it is possible that these two regions are in the structure of α/β-type SASP bound to DNA. SASP-A and SASP-C a at the to these proteins are to form cross-links with the α-amino group at and form cross-links with the glutamate residues of the recognition The cross-links between the α-amino group and the two glutamate residues of the recognition in SASP-A and indicate that the N terminus of each protein is and may with acidic that is by the glutamate residues of the recognition acidic may and in SspC and and the in the cross-link formation site from in Bce1 to the recognition site in we that we have identified the major of EDC cross-linking in each α/β-type SASP we of the that minor cross-linking which were not detected by of α/β-type SASP that has in is that different α/β-type SASP protein-protein with in the DNA bound state. proteins from different species were found to as by cross-linking of SspC from B. subtilis to SASP-A from B. However, it that the interaction between SspC and SASP-A is not as as the interaction between SASP-C and In heterodimers of SASP-A and SASP-C were the cross-linking when the of the two proteins the in formation may be to of the two proteins while bound to DNA, or to a of cross-linking between these two proteins. SASP-A and SASP-C EDC cross-linking which were different from of SspC and it is to that the of the protein-protein interactions in these two of α/β-type SASP is from subtle in protein structure may α/β-type SASP to with more than These minor differences in between α/β-type SASP may be important for binding to different regions of the spore chromosome, and for the to α/β-type SASP in each We are cross-linking to amino acid residues in α/β-type SASP which with DNA. However, the structural obtained during these of studies is and to a high structure of α/β-type SASP·DNA are high structure of α/β-type SASP·DNA the obtained in cross-linking and the of the in DNA which the in UV of spore DNA and spore UV We Setlow and for of SASP-A and and amino acid were performed by at the for