Browsing by Subject "Bacillus subtilis"
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Item Open Access A DNA mimic: the structure and mechanism of action for the anti-repressor protein AbbA.(Journal of molecular biology, 2014-05) Tucker, Ashley T; Bobay, Benjamin G; Banse, Allison V; Olson, Andrew L; Soderblom, Erik J; Moseley, M Arthur; Thompson, Richele J; Varney, Kristen M; Losick, Richard; Cavanagh, JohnBacteria respond to adverse environmental conditions by switching on the expression of large numbers of genes that enable them to adapt to unfavorable circumstances. In Bacillus subtilis, many adaptive genes are under the negative control of the global transition state regulator, the repressor protein AbrB. Stressful conditions lead to the de-repression of genes under AbrB control. Contributing to this de-repression is AbbA, an anti-repressor that binds to and blocks AbrB from binding to DNA. Here, we have determined the NMR structure of the functional AbbA dimer, confirmed that it binds to the N-terminal DNA-binding domain of AbrB, and have provided an initial description for the interaction using computational docking procedures. Interestingly, we show that AbbA has structural and surface characteristics that closely mimic the DNA phosphate backbone, enabling it to readily carry out its physiological function.Item Open Access Classification and genetic characterization of pattern-forming Bacilli.(Mol Microbiol, 1998-02) Rudner, R; Martsinkevich, O; Leung, W; Jarvis, EDOne of the more natural but less commonly studied forms of colonial bacterial growth is pattern formation. This type of growth is characterized by bacterial populations behaving in an organized manner to generate readily identifiable geometric and predictable morphologies on solid and semi-solid surfaces. In our first attempt to study the molecular basis of pattern formation in Bacillus subtilis, we stumbled upon an enigma: some strains used to describe pattern formation in B. subtilis did not have the phenotypic or genotypic characteristics of B. subtilis. In this report, we show that these strains are actually not B. subtilis, but belong to a different class of Bacilli, group I. We show further that commonly used laboratory strains of B. subtilis can co-exist as mixed cultures with group I Bacilli, and that the latter go unnoticed when grown on frequently used laboratory substrates. However, when B. subtilis is grown under more stringent semiarid conditions, members of group I emerge in the form of complex patterns. When B. subtilis is grown under less stringent and more motile conditions, B. subtilis forms its own pattern, and members of group I remain unnoticed. These findings have led us to revise some of the mechanistic and evolutionary hypotheses that have been proposed to explain pattern growth in Bacilli.Item Open Access Co-orientation of replication and transcription preserves genome integrity.(PLoS Genet, 2010-01-15) Srivatsan, Anjana; Tehranchi, Ashley; MacAlpine, David M; Wang, Jue DIn many bacteria, there is a genome-wide bias towards co-orientation of replication and transcription, with essential and/or highly-expressed genes further enriched co-directionally. We previously found that reversing this bias in the bacterium Bacillus subtilis slows replication elongation, and we proposed that this effect contributes to the evolutionary pressure selecting the transcription-replication co-orientation bias. This selection might have been based purely on selection for speedy replication; alternatively, the slowed replication might actually represent an average of individual replication-disruption events, each of which is counter-selected independently because genome integrity is selected. To differentiate these possibilities and define the precise forces driving this aspect of genome organization, we generated new strains with inversions either over approximately 1/4 of the chromosome or at ribosomal RNA (rRNA) operons. Applying mathematical analysis to genomic microarray snapshots, we found that replication rates vary dramatically within the inverted genome. Replication is moderately impeded throughout the inverted region, which results in a small but significant competitive disadvantage in minimal medium. Importantly, replication is strongly obstructed at inverted rRNA loci in rich medium. This obstruction results in disruption of DNA replication, activation of DNA damage responses, loss of genome integrity, and cell death. Our results strongly suggest that preservation of genome integrity drives the evolution of co-orientation of replication and transcription, a conserved feature of genome organization.Item Open Access Electrostatic Energetics of Bacillus subtilis Ribonuclease P Protein Determined by Nuclear Magnetic Resonance-Based Histidine pKa Measurements.(Biochemistry, 2015-09-08) Mosley, Pamela L; Daniels, Kyle G; Oas, Terrence GThe pKa values of ionizable groups in proteins report the free energy of site-specific proton binding and provide a direct means of studying pH-dependent stability. We measured histidine pKa values (H3, H22, and H105) in the unfolded (U), intermediate (I), and sulfate-bound folded (F) states of RNase P protein, using an efficient and accurate nuclear magnetic resonance-monitored titration approach that utilizes internal reference compounds and a parametric fitting method. The three histidines in the sulfate-bound folded protein have pKa values depressed by 0.21 ± 0.01, 0.49 ± 0.01, and 1.00 ± 0.01 units, respectively, relative to that of the model compound N-acetyl-l-histidine methylamide. In the unliganded and unfolded protein, the pKa values are depressed relative to that of the model compound by 0.73 ± 0.02, 0.45 ± 0.02, and 0.68 ± 0.02 units, respectively. Above pH 5.5, H22 displays a separate resonance, which we have assigned to I, whose apparent pKa value is depressed by 1.03 ± 0.25 units, which is ∼0.5 units more than in either U or F. The depressed pKa values we observe are consistent with repulsive interactions between protonated histidine side chains and the net positive charge of the protein. However, the pKa differences between F and U are small for all three histidines, and they have little ionic strength dependence in F. Taken together, these observations suggest that unfavorable electrostatics alone do not account for the fact that RNase P protein is intrinsically unfolded in the absence of ligand. Multiple factors encoded in the P protein sequence account for its IUP property, which may play an important role in its function.Item Open Access FtsZ filament capping by MciZ, a developmental regulator of bacterial division.(Proceedings of the National Academy of Sciences of the United States of America, 2015-04-06) Bisson-Filho, Alexandre W; Discola, Karen F; Castellen, Patrícia; Blasios, Valdir; Martins, Alexandre; Sforça, Maurício L; Garcia, Wanius; Zeri, Ana Carolina M; Erickson, Harold P; Dessen, Andréa; Gueiros-Filho, Frederico JCytoskeletal structures are dynamically remodeled with the aid of regulatory proteins. FtsZ (filamentation temperature-sensitive Z) is the bacterial homolog of tubulin that polymerizes into rings localized to cell-division sites, and the constriction of these rings drives cytokinesis. Here we investigate the mechanism by which the Bacillus subtilis cell-division inhibitor, MciZ (mother cell inhibitor of FtsZ), blocks assembly of FtsZ. The X-ray crystal structure reveals that MciZ binds to the C-terminal polymerization interface of FtsZ, the equivalent of the minus end of tubulin. Using in vivo and in vitro assays and microscopy, we show that MciZ, at substoichiometric levels to FtsZ, causes shortening of protofilaments and blocks the assembly of higher-order FtsZ structures. The findings demonstrate an unanticipated capping-based regulatory mechanism for FtsZ.Item Open Access L form bacteria growth in low-osmolality medium.(Microbiology (Reading, England), 2019-08) Osawa, Masaki; Erickson, Harold PL form bacteria do not have a cell wall and are thought to require medium of high osmolality for survival and growth. In this study we tested whether L forms can adapt to growth in lower osmolality medium. We first tested the Escherichia coli L form NC-7, generated in 1987 by Onoda following heavy mutagenesis. We started with growth in osmoprotective medium (~ 764 mOsm kg-1) and diluted it stepwise into medium of lower osmolality. At each step the cells were given up to 10 days to adapt and begin growing, during which they apparently acquired multiple new mutations. We eventually obtained a strain that could grow in LB containing only 34 mM NaCl, 137 mOsm kg-1 total. NC-7 showed a variety of morphologies including spherical, angular and cylindrical cells. Some cells extruded a bud that appeared to be the outer membrane enclosing an enlarged periplasm. Additional evidence for an outer membrane was sensitivity of the cells to the compound CHIR-090, which blocks the LPS pathway, and to EDTA which chelates Mg that may stabilize and rigidify the LPS in the outer membrane. We suggest that the mechanical rigidity of the outer membrane enables the angular shapes and provides some resistance to turgor in the low-osmolality media. Interestingly, cells that had an elongated shape underwent division shortly after addition of EDTA, suggesting that reducing the rigidity of the outer membrane under some turgor pressure induces division before lysis occurs. We then tested a well-characterized L form from Bacillus subtilis. L form strain LR-2L grew well with sucrose at 1246 and 791 mOsm kg-1. It survived when diluted directly into 440 mOsm kg-1 but grew poorly, achieving only 1/10 to 1/5 the density. The B. subtilis L form apparently adapted to this direct dilution by rapidly reducing cytoplasmic osmolality.Item Open Access Long range dynamic effects of point-mutations trap a response regulator in an active conformation.(FEBS letters, 2010-10) Bobay, Benjamin G; Thompson, Richele J; Hoch, James A; Cavanagh, JohnWhen a point-mutation in a protein elicits a functional change, it is most common to assign this change to local structural perturbations. Here we show that point-mutations, distant from an essential highly dynamic kinase recognition loop in the response regulator Spo0F, lock this loop in an active conformation. This 'conformational trapping' results in functionally hyperactive Spo0F. Consequently, point-mutations are seen to affect functionally critical motions both close to and far from the mutational site.Item Open Access Osmolyte-induced folding of an intrinsically disordered protein: folding mechanism in the absence of ligand.(Biochemistry, 2010-06-29) Chang, Yu-Chu; Oas, Terrence GUnderstanding the interconversion between thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of the bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was previously shown to be unfolded in the absence of its cognate RNA or other anionic ligands. P protein was used in this study as a model system to explore general features of intrinsically disordered protein (IDP) folding mechanisms. The use of trimethylamine N-oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic traces at various final TMAO concentrations exhibited multiphasic kinetics. Equilibrium "cotitration" experiments were performed using both TMAO and urea during the titration to produce a urea-TMAO titration surface of P protein. Both kinetic and equilibrium studies show evidence of a previously undetected intermediate state in the P protein folding process. The intermediate state is significantly populated, and the folding rate constants are relatively slow compared to those of intrinsically folded proteins similar in size and topology. The experiments and analysis described serve as a useful example for mechanistic folding studies of other IDPs.Item Open Access Structure and DNA-binding traits of the transition state regulator AbrB.(Structure (London, England : 1993), 2014-11) Olson, Andrew L; Tucker, Ashley T; Bobay, Benjamin G; Soderblom, Erik J; Moseley, M Arthur; Thompson, Richele J; Cavanagh, JohnThe AbrB protein from Bacillus subtilis is a DNA-binding global regulator controlling the onset of a vast array of protective functions under stressful conditions. Such functions include biofilm formation, antibiotic production, competence development, extracellular enzyme production, motility, and sporulation. AbrB orthologs are known in a variety of prokaryotic organisms, most notably in all infectious strains of Clostridia, Listeria, and Bacilli. Despite its central role in bacterial response and defense, its structure has been elusive because of its highly dynamic character. Orienting its N- and C-terminal domains with respect to one another has been especially problematic. Here, we have generated a structure of full-length, tetrameric AbrB using nuclear magnetic resonance, chemical crosslinking, and mass spectrometry. We note that AbrB possesses a strip of positive electrostatic potential encompassing its DNA-binding region and that its C-terminal domain aids in DNA binding.Item Open Access The Solution Structures and Interaction of SinR and SinI: Elucidating the Mechanism of Action of the Master Regulator Switch for Biofilm Formation in Bacillus subtilis.(Journal of molecular biology, 2020-01) Milton, Morgan E; Draughn, G Logan; Bobay, Benjamin G; Stowe, Sean D; Olson, Andrew L; Feldmann, Erik A; Thompson, Richele J; Myers, Katherine H; Santoro, Michael T; Kearns, Daniel B; Cavanagh, JohnBacteria have developed numerous protection strategies to ensure survival in harsh environments, with perhaps the most robust method being the formation of a protective biofilm. In biofilms, bacterial cells are embedded within a matrix that is composed of a complex mixture of polysaccharides, proteins, and DNA. The gram-positive bacterium Bacillus subtilis has become a model organism for studying regulatory networks directing biofilm formation. The phenotypic transition from a planktonic to biofilm state is regulated by the activity of the transcriptional repressor, SinR, and its inactivation by its primary antagonist, SinI. In this work, we present the first full-length structural model of tetrameric SinR using a hybrid approach combining high-resolution solution nuclear magnetic resonance (NMR), chemical cross-linking, mass spectrometry, and molecular docking. We also present the solution NMR structure of the antagonist SinI dimer and probe the mechanism behind the SinR-SinI interaction using a combination of biochemical and biophysical techniques. As a result of these findings, we propose that SinI utilizes a residue replacement mechanism to block SinR multimerization, resulting in diminished DNA binding and concomitant decreased repressor activity. Finally, we provide an evidence-based mechanism that confirms how disruption of the SinR tetramer by SinI regulates gene expression.