* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Structure-Function Relationship and Regulation of Two Bacillus
Survey
Document related concepts
Protein folding wikipedia , lookup
Homology modeling wikipedia , lookup
Bimolecular fluorescence complementation wikipedia , lookup
Protein purification wikipedia , lookup
Protein mass spectrometry wikipedia , lookup
Protein structure prediction wikipedia , lookup
Western blot wikipedia , lookup
Intrinsically disordered proteins wikipedia , lookup
List of types of proteins wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Transcript
J. Mol. Microbiol. Biotechnol. (2002) 4(3): 323–329. JMMB Symposium Structure-Function Relationship and Regulation of Two Bacillus subtilis DNA-Binding Proteins, HBsu and AbrB Wolfgang Klein1,2, and Mohamed A. Marahiel 1* 1 Philipps Universität Marburg, Biochemie - FB Chemie, Hans-Meerwein-Strasse, D-35032 Marburg, Germany 2 Institut für Pharmazeutische Biologie, Rheinische Friedrich-Wilhelms Universität, Nussallee 6, D-53115 Bonn, Germany Abstract Microorganisms use a number of small basic proteins for organization and compaction of their DNA. By their interaction with the genome, these proteins do have a profound effect on gene expression, growth behavior, and viability. It has to be distinguished between indirect effects as a consequence of the state of chromosome condensation and relaxation that influence the rate of RNA polymerase action as represented by the histone-like proteins, and direct effects by specific binding of proteins to defined DNA segments predominantly located around promoter sequences. This latter class is represented by the transition-state regulators that are involved in integrating various global stimuli and orchestrating expression of the genes under their regulation for a better adaptation to changes in growth rate. In this article we will focus on two different but abundant DNA binding proteins of the gram-positive model organism Bacillus subtilis, the histone-like HBsu as a member of the unspecific and the transition state regulator AbrB as a member of specific classes of DNA binding proteins. The HBsu Protein of Bacillus subtilis is a Member of the Histone-like Protein Superfamily More than 30 members of the family of histone-like proteins, all with a size of about 90 amino acids and an overall basic net charge, have been identified so far in organisms of virtually every branch of the eubacterial kingdom, archaea, cyanobacteria, plant chloroplasts, and bacteriophages (Oberto et al., 1994). The B. subtilis genome encodes for one histone-like protein by the hbs gene (Kunst et al., 1997). This correlates with findings that the hbs gene is essential, and attempts to construct a knock out mutation have failed so far (Micka and Marahiel, 1992). The HBsu protein shows a high level *For correspondence. Email marahiel@ chemie.uni-marburg.de; Tel. +49 +6421 -2825722; Fax. +49 +6421 -2822191. # 2002 Horizon Scientific Press of similarity to the E. coli heterodimeric HU protein, the so far best studied histone-like protein. It shows 57% and 51% identical amino acids with the HU-subunits HU-2 and HU-1, respectively. From the occurrence of a single gene in B. subtilis genome it is concluded that HBsu acts as a homodimer (see Figure 1A), and our data on the DNA-binding activity of HBsu mutant proteins support this idea. When subsaturating protein concentrations of one protein were used, they exhibited full DNA binding activity by the addition of subsaturating amounts of another mutant variant (Köhler and Marahiel, 1998), indicating the formation of a functional complex of both mutant proteins. The construction of a HBsu-GFP fusion protein allowed us to visualize for the first time the cellular localization of this protein as in vitro analysis has shown its ability to bind DNA. Using fluorescence microscopy, HBsu-GFP fluorescence was observed inside the cell at exactly the same location as the DAPI fluorescence that is localizing the nucleoid. Furthermore, additional expression of wildtype HBsu caused the formation of a more condensed nucleoid (Köhler and Marahiel, 1997). These results and investigations using HBsu proteins with reduced DNA binding activity emphasize its principal role in DNA packaging in B. subtilis. The archetype of histone-like proteins is H-NS of Escherichia coli that has been identified several times independently according to the researchers focus on functions like in vitro transcription of phage DNA, phage transposition, and lifestyle (for a review, see Nash, 1996). In vivo localization data of this heterodimeric protein yielded controversial results by showing correlation with metabolically active DNA in immunocytochemical studies and even distribution throughout the entire nucleoid using fluorescein labeled HU, a H-NS homologous protein (Shellman and Pettijohn, 1991). A recent model of nucleoid organization in E. coli suggested that HU and H-NS are located in different domains where an internal domain, thought to contain rarely transcribed DNA, is associated with H-NS while frequently transcribed sequences complexed with HU protein form the coralline appearing outer sphere of the nucleoid (McGovern et al., 1994). Although these two histone-like proteins appear to play special roles in cellular physiology, E. coli strains lacking either HU or H-NS are viable whereas multiple mutations severely impair cellular viability (Yasuzawa et al., 1992). Structural Features of HBsu An understanding of the molecular structure of histonelike proteins is mainly based on X-ray crystallographic Further Reading Caister Academic Press is a leading academic publisher of advanced texts in microbiology, molecular biology and medical research. Full details of all our publications at caister.com • MALDI-TOF Mass Spectrometry in Microbiology Edited by: M Kostrzewa, S Schubert (2016) www.caister.com/malditof • Aspergillus and Penicillium in the Post-genomic Era Edited by: RP Vries, IB Gelber, MR Andersen (2016) www.caister.com/aspergillus2 • The Bacteriocins: Current Knowledge and Future Prospects Edited by: RL Dorit, SM Roy, MA Riley (2016) www.caister.com/bacteriocins • Omics in Plant Disease Resistance Edited by: V Bhadauria (2016) www.caister.com/opdr • Acidophiles: Life in Extremely Acidic Environments Edited by: R Quatrini, DB Johnson (2016) www.caister.com/acidophiles • Climate Change and Microbial Ecology: Current Research and Future Trends Edited by: J Marxsen (2016) www.caister.com/climate • Biofilms in Bioremediation: Current Research and Emerging Technologies Edited by: G Lear (2016) www.caister.com/biorem • Flow Cytometry in Microbiology: Technology and Applications Edited by: MG Wilkinson (2015) www.caister.com/flow • Microalgae: Current Research and Applications • Probiotics and Prebiotics: Current Research and Future Trends Edited by: MN Tsaloglou (2016) www.caister.com/microalgae Edited by: K Venema, AP Carmo (2015) www.caister.com/probiotics • Gas Plasma Sterilization in Microbiology: Theory, Applications, Pitfalls and New Perspectives Edited by: H Shintani, A Sakudo (2016) www.caister.com/gasplasma Edited by: BP Chadwick (2015) www.caister.com/epigenetics2015 • Virus Evolution: Current Research and Future Directions Edited by: SC Weaver, M Denison, M Roossinck, et al. (2016) www.caister.com/virusevol • Arboviruses: Molecular Biology, Evolution and Control Edited by: N Vasilakis, DJ Gubler (2016) www.caister.com/arbo Edited by: WD Picking, WL Picking (2016) www.caister.com/shigella Edited by: S Mahalingam, L Herrero, B Herring (2016) www.caister.com/alpha • Thermophilic Microorganisms Edited by: F Li (2015) www.caister.com/thermophile Biotechnological Applications Edited by: A Burkovski (2015) www.caister.com/cory2 • Advanced Vaccine Research Methods for the Decade of Vaccines • Antifungals: From Genomics to Resistance and the Development of Novel • Aquatic Biofilms: Ecology, Water Quality and Wastewater • Alphaviruses: Current Biology • Corynebacterium glutamicum: From Systems Biology to Edited by: F Bagnoli, R Rappuoli (2015) www.caister.com/vaccines • Shigella: Molecular and Cellular Biology Treatment Edited by: AM Romaní, H Guasch, MD Balaguer (2016) www.caister.com/aquaticbiofilms • Epigenetics: Current Research and Emerging Trends Agents Edited by: AT Coste, P Vandeputte (2015) www.caister.com/antifungals • Bacteria-Plant Interactions: Advanced Research and Future Trends Edited by: J Murillo, BA Vinatzer, RW Jackson, et al. (2015) www.caister.com/bacteria-plant • Aeromonas Edited by: J Graf (2015) www.caister.com/aeromonas • Antibiotics: Current Innovations and Future Trends Edited by: S Sánchez, AL Demain (2015) www.caister.com/antibiotics • Leishmania: Current Biology and Control Edited by: S Adak, R Datta (2015) www.caister.com/leish2 • Acanthamoeba: Biology and Pathogenesis (2nd edition) Author: NA Khan (2015) www.caister.com/acanthamoeba2 • Microarrays: Current Technology, Innovations and Applications Edited by: Z He (2014) www.caister.com/microarrays2 • Metagenomics of the Microbial Nitrogen Cycle: Theory, Methods and Applications Edited by: D Marco (2014) www.caister.com/n2 Order from caister.com/order 324 Klein and Marahiel analysis of the homodimeric HBst protein from B. sterothermophilus (Tanaka et al., 1984); see Figure 1A). As HBsu of the mesophilic B. subtilis and HBst display 87% identity and 94% similarity, a nearly identical 3D-structure for both can be assumed. The amino terminal half of the HBst protein is composed of two a-helices connected by a turn, followed by three b-sheets. Whereas the first sheet is involved in forming the body of the protein structure, the latter two form a b-ribbon extension called the ‘‘arm’’ that was shown to be involved in DNA binding (Köhler and Marahiel, 1998; see Figure 1A). The interface of the dimer is largely dependent upon interactions between several hydrophobic residues scattered along the primary sequence, an attribute that is conserved in all members of this family of proteins. DNA-Binding by HBsu DNA binding by HBsu is independent of cofactors or additional proteins. It has been shown to bind DNA unspecifically but with a preference for curved fragments (Köhler and Marahiel, 1998). Furthermore, HBsu enables b-recombinase-mediated recombination by stabilizing a special DNA structure (Alonso et al., 1995). Even though experimental data are lacking, the preference for curved DNA could be an indication that HBsu might perturb the overall structure of duplex DNA, a fact that has been seen for the E. coli HU protein (Hodges-Garcia et al., 1989). In a model based on the HBst structure, each monomer of the homodimer contributes two DNA binding surfaces – the b-ribbon ‘‘arm’’ and a segment on the flank of the body - both of which contact the minor grove of the DNA. Based on a model that has been deduced from DNA-protein photo-crosslinking experiments with a similar DNA-binding protein (Lee et al., 1992; Yang and Nash, 1994), we have performed the first site-directed mutational analysis of a histone-like protein. Our results strengthened the significance of several basic residues in DNA binding as the mutated proteins showed a dramatically reduced DNA binding activity (see Figure 1C). Furthermore, this in vitro activity is corroborated by in vivo data showing a reduced growth rate, a reduced sporulation efficiency, and a less condensed nucleoid for strains carrying only a mutant hbs allel (Köhler and Marahiel, 1998). Regulation of hbs Expression There are only few data on the regulation of histone-like proteins that may explain the abundance of HU protein in E. coli in response to different growth conditions. Values of 2 to 5 ng of HU per mg total protein might represent an average that varies little, if at all, in the life cycle (Bonnefoy et al., 1994). This might reflect the operation of an autoregulatory circuit, yielding an appropriate amount of HU protein and resulting in a suitably condensed nucleoid (Kohno et al., 1990). Our own data on the regulation of the B. subtilis hbs gene are based on two lines of evidence: two promoter structures in front of the hbs gene were mapped. P1 is 181 nucleotides upstream of the translation start point, shows similarities to sA dependent promoters, and the mRNA derived from this promoter was detected mainly in exponentially growing, non-sporulating cells. P2 localizes only 21 basepairs upstream of the ATG codon, and its sH promoter similarity is corroborated by determining this type of mRNA exclusively in sporulating cells (Micka et al., 1991). Furthermore, different promoter fragments have been cloned in front of the b-galactosidase gene and integrated as single copy into the chromosome for monitoring hbs::lacZ expression (Köhler and Marahiel, 1997). The P1 and P2 comprising fragments triggered a high b-galactosidase activity with a slight maximum around one hour before the transition into stationary phase. Studies on different P1 promoter fragments revealed two facts: a medium level of hbs::lacZ expression during exponential growth but low expression after the transition phase. Deletion of a short DNA segment located between both promoter structures, P1 and P2, selectively reduced expression by promoter1 under all conditions tested. We therefore attributed a P1-specific enhancer function to this DNA segment. In addition, P1 seems to be the target of the HBsu autoregulatory loop as conditional overexpression of wildtype HBsu selectively reduced hbs:lacZ expression directed by P1 but not by P2. Fragments of promoter2 showed only a low activity that is about 12 fold induced around one hour after entry into the sporulation phase. This induction is dependent on sporulation promoting growth conditions and strictly dependent on sH as tested in spo0H mutants (Köhler and Marahiel, 1997). Based on these data, we propose that HBsu is regulated in exponentially growing cells by P1 and an autoregulatory loop. After the transition to stationary phase either expression is promoted by the sH dependent P2 if sporulation is initiated, or expression of hbs is reduced under non-sporulating conditions and HBsu might be in part replaced by proteins like the minor small, acid-soluble spore proteins (Ross and Setlow, 2000). AbrB of Bacillus subtilis is the Major Transition State Regulator At the end of exponential growth in a bacterial culture when conditions for growth become sub-optimal, many genes for alternative catabolic and anabolic pathways to produce substances such as antibiotics, toxins, and polymer degrading enzymes become derepressed. Therefore, the transition state between exponential growth and stationary phase can be looked at as a crossroad where cells still express some growthrelated functions but begin to express new gene products that are necessary for survival in a nutrientdeprived, hostile environment. In Bacillus spp, typical functions of the transition state encompass the development of genetic competence for the uptake of DNA, production of antibiotics and extracellular enzymes, and the synthesis of flagella. Genes encoding such functions are regulated, at least in part, by small, global negative transcriptional factors that have been termed ‘‘transition state regulators’’ as they si- HBsu and AbrB Proteins of Bacillus subtilis 325 Figure 1. Structural and functional features of HBsu as a member of the histone-like protein family. A) The three-dimensional structure of the homodimer HBst protein of B. stearothermophilus was used as a model for site-directed mutagenesis in HBsu of B. subtilis. The basic residues selected for mutagenesis are shown with their side chains and labeled with single-letter amino acid code in both proteins. N- and C-termini are indicated by n and c, respectively, numbers refer to the two subunits. Modelling of the structure was done according to Konradi et al., 1996. B) Comparison of the amino acid sequences of HBsu and HBst which are 86% identical. Mutationally altered amino acid residues are indicated above the sequence, secondary structure elements of HBst are shown schematically below the sequences. C) Gel retardation analysis of HBsu and mutant proteins with curved and non-curved DNA. 326 Klein and Marahiel lence the expression of these functions at inappropriate times (Strauch, 1993). None of the known mutations in these regulators leads to significant sporulation defects, but they are involved in coordinating and transmitting signals received by the sporulation-sensing network and orchestrating the transition state functions. One of these regulators is the product of the abrB gene that controls several transition state genes, some of which are necessary for sporulation (Perego et al., 1988). In this manner, AbrB coordinates the sporulation response with other transition state processes. Furthermore, AbrB is implicated in the regulation of other transition state regulators such as Hpr (originally identified by mutations causing overexpression of extracellular proteases (Perego and Hoch, 1988)) and Sin (inhibitor of sporulation and protease production when overexpressed (Gaur et al., 1988; see Figure 2)). AbrB is looked at as the central transition state regulator in Bacillus subtilis. The abrB gene was isolated as pseudorevertants in a spo0A mutant background (spo0A mutants show impaired antibiotic production, lack of motility, inability to aquire genetic competence and to initiate sporulation) by restoring antibiotic production and motility but not sporulation (Guespin-Michel, 1971). Cloning of this locus revealed a single gene encoding for a 94 amino acid protein (10.5 kDa), and the pseudorevertants isolated resulted in loss of function (Perego et al., 1988) with one class lowering the expression of abrB, while the other class impaired the reading frame (Zuber and Losick, 1987). The abrB gene is transcribed by two promoters, whose physiological significance is not yet fully understood. DNA Binding of AbrB Attempts to define common binding sites for the genes regulated by AbrB have failed so far. The location of AbrB-binding sites relative to the promoters shows some flexibility but suggests that AbrB might interfere or interact with the RNA polymerase (Strauch, 1995a). Footprinting analysis showed variation in size from target to target, with protected regions ranging from 30 to 120 base pairs (Strauch, 1995b). However, from these sequences no common motif can be deduced as an AbrB consensus sequence. Using an in vitro selection of random oligo nucleotides, seemingly optimal AbrB binding sites corresponding to a regularly spaced motif were selected (Xu and Strauch, 1996). This optimally spaced motif, however, is rare within the B. subtilis genome and does not correlate with the footprinting results. Therefore it is not sufficient to explain the selectivity of the AbrB-DNA-interaction. The mapped sequences show a correlation to AT-rich and bent DNA regions, but bending alone is not sufficient for promoting AbrB binding as shown by mobility shift assays using bent DNA (Klein, unpublished data). Therefore, it has been hypothesized that AbrB may actually recognize a type of DNA-three-dimensional structure that can be assumed by a finite subset of differing base sequences (Xu et al., 1996). The 10.5 kDa AbrB protein has been shown to be a hexamer in solution, and that this hexameric state is required for its DNA-binding activity (Strauch et al., 1989). A sequence with slight similarity to the helix-turnhelix motif of DNA-binding proteins that was identified within the AbrB sequence was investigated by mutational analysis and failed to prove this relationship (Fürbaß and Marahiel, 1991). There is no evidence for the presence of any other known DNA-binding motif within the AbrB protein. Therefore at present no explanation can be provided for the specific and co-operative binding behavior of AbrB (Fürbaß et al., 1991; Robertson et al., 1989). To understand the structural basis of AbrB specific DNA-interaction, several attempts to unravel the three dimensional structure of this proteins have been undertaken. These studies were designed to define the residues involved in DNA binding and to shed light on the oligomerization behavior. Full-length recombinant AbrB protein from B. subtilis was purified to homogeneity but failed to crystallize. Recently the structure of the AbrB N-terminal DNA-binding domain was solved by NMR. It was described as a loopedhinged helix fold where two conserved arginine residues required for DNA interaction are located within an a-helix flanked by a double stranded looped-hinge region (Vaughn et al., 2000). This N-terminal domain was shown to retain full sequence specificity and to form dimers that are necessary for DNA interaction, but it failed to show the hexameric state that has been described for the full-length protein. NMR measurements showed that the residues of the hinge-loop region undergo significant concerted motion upon the protein’s interaction with DNA, affecting the disposition of the DNA-binding helix (Zuber, 2000). According to the recent model of gene regulation by AbrB, the ordered structure of AbrB-DNA complexes varies between different target genes, suggesting changes within AbrB’s tertiary and quarternary structure to conform to the site of interaction offered by the DNA target. This might be accomplished by the multimeric state of AbrB as determined by the specific target (Vaughn et al., 2000). The validity of the proposed model can only be tested on the structure of full-length AbrB bound to different DNA-target sites. Such an investigation will also reveal all the amino acid residues mediating direct interaction with the DNA, and those needed for the multimeric organization. As a first step towards structural studies on full length AbrB protein, we isolated the abrB gene from the thermophilic Bacillus stearothermophilus (76% identity to the mesophilic B. subtilis protein). DNA-binding activity and oligomerization behavior of the recombinant protein were studied and displayed a similar behavior as shown for the mesophilic AbrB su. Initial crystallization attempts yielded small crystals that show diffraction at 4 Å (Klein et al., 2000). Regulation of Gene Expression by AbrB AbrB has been characterized to act in three different ways to regulate post-exponential expression of genes HBsu and AbrB Proteins of Bacillus subtilis 327 Figure 2. AbrB regulation of transition state gene expression. +, positive regulation; ! , negative regulation. The formation of the phosphorylated, active form of Spo0A occurs at the end of exponential growth by a phosphorelay which is summarized here. t 0 means time zero according to the sporulation pathway. The components of this regulatory circuit and their cellular functions are summarized. which can be classified according to their cellular function as summarized in Figure 2. It is the sole repressor for genes like spo0E and tycA as abrB mutations cause constitutive expression of these genes (Marahiel et al., 1987; Perego and Hoch, 1991). For spoVG and aprE, AbrB is termed a ‘preventer’: while abrB mutations restore their expression in Spo0 mutants, the absence of AbrB does not cause constitutive expression, indicating additional temporal control acting in concert with AbrB (Ferrari et al., 1988). In a third manner AbrB regulates expression as an activator. Genetic data indicate that AbrB positively affects expression of the transition state regulator Hpr (Strauch and Hoch, 1993), factors of the competence pathway (Dubnau, 1991), and enzymes involved in histidine utilization (Fisher et al., 1994). Whereas in the case of repression the binding of AbrB to DNA segments around the promoter region has been verified, this has not yet been shown clearly for positively regulated genes. By the combination of several levels of macromolecular assembly with the flexibility of the looped-hinge helix DNA recognition fold to alter its orientation, AbrB can serve as specific regulatory protein for diverse promoter elements. This structure-function model sets AbrB as an archetype for a new class of regulatory proteins (Zuber, 2000). To date, abrB expression is known to be controlled by both negative autoregulation and repression by phoshorylated Spo0A protein (Perego et al., 1988). It is believed that the autoregulatory loop ensures an intracellular concentration of AbrB during exponential growth high enough to silence transition state functions and sensitive enough to changes in transcription and/or protein activity. The cooperative DNAbinding activity of AbrB (see above) could magnify the effects of slight decreases in the AbrB levels on its 328 Klein and Marahiel target genes. Phosphorylated Spo0A protein, being the primary sporulation signal as it is phosphorylated by a regulatory phosphorelay cascade in response to nutrient deprivation, seems to be the sole factor responsible for abrB repression (Strauch et al., 1990). This sporulation initiating signal cascade represses transcription of the abrB gene and causes the AbrB level to drop below its threshold, thereby releasing the repression of transition state functions. This genetic model is supported by data showing that the cellular amount of AbrB is indeed growth phase dependent (O’Reilly and Devine, 1997). Both abrB mRNA and AbrB protein accumulate in the early exponential growth phase, but whereas the transcript level sharply decreases in the mid-exponential phase, the AbrB protein is gradually decreasing along the exponential growth with an abrupt reduction at the onset of stationary phase. Furthermore, analysis of abrB (partially deletion) and spo0A mutant strains confirmed the abrB regulation model on the level of mRNA and protein quantification (O’Reilly and Devine, 1997). Even though not related on the amino acid level, the pattern of abrB expression, its size, and overall net charge are similar to that of Fis from E. coli. But both proteins bind to highly degenerate consensus sequences with a bias for A and T residues, both are negatively autoregulated, and both regulate expression of a variety of genes of diverse functions. Interestingly, so far no Fis homolog has been found in gram-positive bacteria, and AbrB seems to be a characteristic of the genus Bacillus. This has been taken as an indication that these two proteins evolved independently but have similar cellular functions (O’Reilly and Devine, 1997). Acknowledgements We would like to thank all the members in our lab that have contributed to the research in these topics as well as all our colleagues that have sheared ideas and results. We apologize to all those whose work was not cited appropriately due to limitations in article size. Our research was supported by grants from the DFG. References Alonso, J.C., Gutierrez, C., and Rojo, F. 1995. The role of chromatinassociated protein Hbsu in beta-mediated DNA recombination is to facilitate the joining of distant recombination sites. Mol. Microbiol. 18: 471–478. Bonnefoy, E., Takahashi, M., and Yaniv, J.R. 1994. DNA-binding parameters of the HU protein of Escherichia coli to cruciform DNA. J. Mol. Biol. 242: 116–129. Dubnau, D. 1991. The regulation of genetic competence in Bacillus subtilis. Mol. Microbiol. 5: 11–18. Ferrari, E., Henner, D.J., Perego, M., and Hoch, J.A. 1988. Transcription of Bacillus subtilis subtilisin and expression of subtilisin in sporulation mutants. J. Bacteriol. 170: 289–295. Fisher, S.H., Strauch, M.A., Atkinson, M.R., and Wray, L.V., Jr. 1994. Modulation of Bacillus subtilis catabolite repression by transition state regulatory protein AbrB. J. Bacteriol. 176: 1903–1912. Fürbaß, R., Gocht, M., Zuber, P., and Marahiel, M.A. 1991. Interaction of AbrB, a transcriptional regulator from Bacillus subtilis with the promoters of the transition state-activated genes tycA and spoVG. Mol. Gen. Genet. 225: 347–354. Fürbaß, R., and Marahiel, M.A. 1991. Mutant analysis of interaction of the Bacillus subtilis transcription regulator AbrB with the antibiotic biosynthesis gene tycA. FEBS 287: 153–156. Gaur, N.K., Cabane, K., and Smith, I. 1988. Structure and expression of the Bacillus subtilis sin operon. J. Bacteriol. 170: 860–869. Guespin-Michel, J.F. 1971. Phenotypic reversion in some early blocked sporulation mutants of Bacillus subtilis. Mol. Gen. Genet. 112: 243–254. Hodges-Garcia, Y., Hagerman, P.J., and Pettijohn, D.E. 1989. DNA ring closure mediated by protein HU. J. Biol. Chem. 264: 14621–14623. Klein, W., Winkelmann, D., Hahn, M., Weber, T., and Marahiel, M.A. 2000. Molecular characterization of the transition state regulator AbrB from Bacillus stearothermophilus. Biochim. Biophys. Acta 1493: 82–90. Köhler, P., and Marahiel, M.A. 1997. Association of the histone-like protein HBsu with the nucleoid of Bacillus subtilis. J. Bacteriol. 179: 2060–2064. Köhler, P., and Marahiel, M.A. 1998. Mutational analysis of the nucleoid-associated protein HBsu of Bacillus subtilis. Mol. Gen. Genet. 260: 487–491. Kohno, K., Wada, M., Kano, Y., and Imamoto, F. 1990. Promoters and autogenous control of the Escherichia coli hupA and hupB genes. J. Mol. Biol. 213: 27–36. Konradi, R., Billeter, M., and Wüthrich, K. 1996. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graphics 14: 51–55. Kunst, F., Ogasawara, N., Moszer, I., Albertini, A.M., Alloni, G., Azevedo, V., Bertero, M.G., Bessieres, P., Bolotin, A., Borchert, S., Borriss, R., Boursier, L., Brans, A., Braun, M., Brignell, S.C., Bron, S., Brouillet, S., Bruschi, C.V., Caldwell, B., Capuano, V., Carter, N.M., Choi, S.K., Codani, J.J., Connerton, I.F., and Danchin, A., et al. 1997. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390: 249–256. Lee, E.C., Hales, L.M., Gumport, R.I., and Gardner, J.F. 1992. The isolation and characterization of mutants of the integration host factor (IHF) of Escherichia coli with altered, expanded DNA-binding specificities. EMBO J. 11: 305–313. Marahiel, M.A., Zuber, P., Czekay, G., and Losick, R. 1987. Identification of the promoter for a peptide antibiotic biosynthesis gene from Bacillus brevis and its regulation in Bacillus subtilis. J. Bacteriol. 169: 2215–2222. McGovern, V., Higgins, N.P., Chiz, R.S., and Jaworski, A. 1994. H-NS over-expression induces an artificial stationary phase by silencing global transcription. Biochimie 76: 1019–1029. Micka, B., Groch, N., Heinemann, U., and Marahiel, M.A. 1991. Molecular cloning, nucleotide sequence, and characterization of the Bacillus subtilis gene encoding the DNA-binding protein HBsu. J. Bacteriol. 173: 3191–3198. Micka, B., and Marahiel, M.A. 1992. The DNA-binding protein HBsu is essential for normal growth and development in Bacillus subtilis. Biochimie 74: 641–650. Nash, H.A. 1996. The Hu and IHF proteins: accessory factors for complex protein-DNA assemblies. In: E.C.C. Lin, and A.S. Lynch (eds.), Regulation of gene expression in Escherichia coli. RG Landes Company, Austin, Texas, pp. 149–179. Oberto, J., Drlica, K., and Rouviere-Yaniv, J. 1994. Histones, HMG, HU, IHF: Meme combat. Biochimie 76: 901–908. O’Reilly, M., and Devine, K.M. 1997. Expression of AbrB, a transition state regulator from Bacillus subtilis, is growth phase dependent in a manner resembling that of Fis, the nucleoid binding protein from Escherichia coli. J. Bacteriol. 179: 522–529. Perego, M., and Hoch, J.A. 1988. Sequence analysis and regulation of the hpr locus, a regulatory gene for protease production and sporulation in Bacillus subtilis. J. Bacteriol. 170: 2560–2567. Perego, M., Spiegelman, G.M., and Hoch, J.A. 1988. Structure of the gene for the transition state regulator, AbrB: regulator synthesis is controlled by the spo0A sporulation gene in Bacillus subtilis. Mol. Microbiol. 2: 689–699. Perego, M., and Hoch, J.A. 1991. Negative regulation of Bacillus subtilis sporulation by the spo0E gene product. J. Bacteriol. 173: 2514–2520. Robertson, J.B., Gocht, M., Marahiel, M.A., and Zuber, P. 1989. AbrB, a regulator of gene expression in Bacillus, interacts with the transcription initiation regions of a sporulation gene and an antibiotic biosynthesis gene. Proc. Natl. Acad. Sci. USA 86: 8457–8461. Ross, M.A., and Setlow, P. 2000. The Bacillus subtilis HBsu protein modifies the effects of alpha/beta-type, small acid-soluble spore proteins on DNA. J. Bacteriol. 182: 1942–1948. Shellman, V.L., and Pettijohn, D.E. 1991. Introduction of proteins into living bacterial cells: distribution of labeled HU protein in Escherichia coli. J. Bacteriol. 173: 3047–3059. Strauch, M.A., Spiegelman, G.B., Perego, M., Johnson, W.C., Burbulys, D., and Hoch, J.A. 1989. The transition state transcription HBsu and AbrB Proteins of Bacillus subtilis 329 regulator abrB of Bacillus subtilis is a DNA binding protein. EMBO J. 8: 1615–1621. Strauch, M., Webb, V., Spiegelman, G., and Hoch, J.A. 1990. The SpoOA protein of Bacillus subtilis is a repressor of the abrB gene. Proc. Natl. Acad. Sci. USA 87: 1801–1805. Strauch, M.A., and Hoch, J.A. 1993. Transition-state regulators: sentinels of Bacillus subtilis post-exponential gene expression. Mol. Microbiol. 7: 337–342. Strauch, M.A. 1993. AbrB, a Transition State Regulator. In: A.L. Sonenshein, J.A. Hoch, and R. Losick (eds.), Bacillus subtilis and other gram-positive bacteria. Am. Soc. Microbiol., Washington DC, pp. 757–763. Strauch, M.A. 1995a. Delineation of AbrB-binding sites on the Bacillus subtilis spo0H, kinB, ftsAZ, and pbpE promoters and use of a derived homology to identify a previously unsuspected binding site in the bsuB1 methylase promoter. J. Bacteriol. 177: 6999–7002. Strauch, M.A. 1995b. In vitro binding affinity of the Bacillus subtilis AbrB protein to six different DNA target regions. J. Bacteriol. 177: 4532–4536. Tanaka, I., Appelt, K., Dijk, J., White, S.W., and Wilson, K.S. 1984. 3-A resolution structure of a protein with histone-like properties in prokaryotes. Nature 310: 376–381. Vaughn, J.L., Feher, V., Naylor, S., Strauch, M.A., and Cavanagh, J. 2000. Novel DNA binding domain and genetic regulation model of Bacillus subtilis transition state regulator AbrB. Nat. Struct. Biol. 7: 1139–1146. Xu, K., Clark, D., and Strauch, M.A. 1996. Analysis of abrB mutations, mutant proteins, and why abrB does not utilize a perfect consensus in the -35 region of its sA promoter. J. Biol. Chem. 271: 2621–2626. Xu, K., and Strauch, M.A. 1996. In vitro selection of optimal AbrBbinding sites: comparison to known in vivo sites indicates flexibility in AbrB binding and recognition of three-dimensional DNA structures. Mol. Microbiol. 19: 145–158. Yang, S.-W., and Nash, H.A. 1994. Specific photocrosslinking of DNA-protein complexes: identification of contacts between integration host factor and its target DNA. Proc. Natl. Acad. Sci. USA 91: 12183–12187. Yasuzawa, K., Hayashi, N., Goshima, N., Kohno, K., Imamoto, F., and Kano, Y. 1992. Histone-like proteins are required for cell growth and constraint of supercoils in DNA. Gene 122: 9–15. Zuber, P., and Losick, R. 1987. Role of AbrB in Spo0A- and Spo0Bdependent utilization of a sporulation promoter in Bacillus subtilis. J. Bacteriol. 169: 2223–2230. Zuber, P. 2000. Specificity through flexibility. Nat. Struct. Biol. 7: 1079–1081.