Crystallization of the IIA domain of the glucose permease of Bacillus subtilis

The IIA domain of the glucose permease of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) from Bacillus subtilis has been crystallized. Crystals are obtained from ammonium sulfate solution. They diffract to at least 2.2 A resolution, and belong to space group C222(1), with unit cell dimensions: a = 74.2 A; b = 54.9 A; c = 67.0 A.     

Structure of the IIA domain of the glucose permease of Bacillus subtilis at 2.2-A resolution

The crystal structure of the IIA domain of the glucose permease of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) from Bacillus subtilis has been determined at 2.2-A resolution. Refinement of the structure is in progress, and the current R-factor is 0.201 (R = sigma h parallel Fo magnitude of - Fc parallel/sigma h magnitude of Fo, where magnitude of Fo and magnitude of Fc are the observed and calculated structure factor amplitudes, respectively) for data between 6.0- and 2.2-A resolution for which F greater than or equal to 2 sigma (F). This is an antiparallel beta-barrel structure that incorporates "Greek key" and "jellyroll" topological motifs. A shallow depression is formed at the active site by part of the beta-sheet and an omega-loop flanking one side of the sheet. His83, the histidyl residue which is the phosphorylation target of HPr and which transfers the phosphoryl group to the IIB domain of the permease, is located at the C-terminus of a beta-strand. The N epsilon atom is partially solvated and also interacts with the N epsilon atom of a second histidyl residue, His68, located at the N-terminus of an adjacent beta-strand, suggesting they share a proton. The geometry of the hydrogen bond is imperfect, though. Electrostatic interactions with other polar groups and van der Waals contacts with the side chains of two flanking phenylalanine residues assure the precise orientation of the imidazole rings. The hydrophobic nature of the surface around the His83-His68 pair may be required for protein-protein recognition by HPr or/and by the IIB domain of the permease. The side chains of two aspartyl residues, Asp31 and Asp87, are oriented toward each other across a narrow groove, about 7 A from the active-site His83, suggesting they may play a role in protein-protein interaction. A model of the phosphorylated form of the molecule is proposed, in which oxygen atoms of the phosphoryl group interact with the side chain of His68 and with the main-chain nitrogen atom of a neighboring residue, Val89. The model, in conjunction with previously reported site-directed mutagenesis experiments, suggests that the phosphorylation of His83 may be accompanied by the protonation of His68. This may be important for the interaction with the IIB domain of the permease and/or play a catalytic role in the phosphoryl transfer from IIA to IIB.     

The immobilization antigen of Paramecium aurelia is a single polypeptide chain.

The immobilization antigen (i-antigen) fraction of Paramecium aurelia syngen 4 is shown to contain a protease that is activated by mercaptoethaneol. After the protease has been heat-inactivated, the molecular weight of the i-antigen (similar to 250,000 daltons) cannot be decreased by mercaptoethanol treatment. It is demonstrated that the i-antigen is a single polypeptide chain. Reasons are also given why low molecular weight subunits were previously reported by other authors.     

Reconstitution studies show that rifampicin resistance is determined by the largest polypeptide of Bacillus subtilis RNA polymerase.

A procedure has been developed to separate the subunits of Bacillus subtilis RNA polymerase rapidly and in good yield. The method involved the use of a blue dextran-Sepharose column which bound the beta' subunit. A phosphocellulose column was used to separate the alpha and beta subunits. During purification, the enzyme eluted from the DNA-cellulose column in three separate forms in the order alpha2betabeta'deltaomega1,alpha2betabeta'omega1, and alpha2betabeta'omega1sigma. Subunit reconstitution studies with RNA polymerase subunits from wild type and a rifampicin-resistant mutant indicated that the largest polypeptide was responsible for rifampicin resistance. Thus, this subunit is referred to as beta. The mobility of the subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis cannot be used as the sole criterion for designating the functions of the subunits of RNA polymerase.     

The Bacillus subtilis division protein DivIC is a highly abundant membrane-bound protein that localizes to the division site

The Bacillus subtilis divIC gene is involved in the initiation of cell division. It encodes a 14.7 kDa protein, with a potential transmembrane region near the N-terminus. In this paper, we show that DivIC is associated with the cell membrane and, in conjunction with previously published sequence data, conclude that it is oriented such that its small N-terminus is within the cytoplasm and its larger C-terminus is external to the cytoplasm. DivIC is shown to be a highly abundant division protein, present at approximately 50 000 molecules per cell. Using immunofluorescence microscopy, DivIC was seen to localize at the division site of rapidly dividing cells between well-segregated nucleoids. Various DivIC immunostaining patterns were observed, and these correlated with different cell lengths, suggesting that the DivIC localization takes on various forms during the cell cycle. The DivIC immunolocalization patterns are very similar to those of another membrane-bound B . subtilis division protein, DivIB.     

Backbone dynamics of the Bacillus subtilis glucose permease IIA domain determined from 15N NMR relaxation measurements.

The backbone dynamics of the uniformly 15N-labeled IIA domain of the glucose permease of Bacillus subtilis have been characterized using inverse-detected two-dimensional 1H-15N NMR spectroscopy. Longitudinal (T1) and transverse (T2) 15N relaxation time constants and steady-state (1H)-15N NOEs were measured, at a spectrometer proton frequency of 500 MHz, for 137 (91%) of the 151 protonated backbone nitrogens. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau e), and 15N exchange broadening contributions (Rex) for each residue, as well as the overall molecular rotational correlation time (tau m). The T1 and T2 values for most residues were in the ranges 0.45-0.55 and 0.11-0.15 s, respectively; however, a small number of residues exhibited significantly slower relaxation. Similarly, (1H)-15N NOE values for most residues were in the range 0.72-0.80, but a few residues had much smaller positive NOEs and some exhibited negative NOEs. The molecular rotational correlation time was 6.24 +/- 0.01 ns; most residues had order parameters in the range 0.75-0.90 and tau e values of less than ca. 25 ps. Residues found to be more mobile than the average were concentrated in three areas: the N-terminal residues (1-13), which were observed to be highly disordered; the loop from P25 to D41, the apex of which is situated adjacent to the active site and may have a role in binding to other proteins; and the region from A146 to S149. All mobile residues occurred in regions close to termini, in loops, or in irregular secondary structure.     

The flaA locus of Bacillus subtilis is part of a large operon coding for flagellar structures, motility functions, and an ATPase-like polypeptide.

We cloned and sequenced 8.3 kb of Bacillus subtilis DNA corresponding to the flaA locus involved in flagellar biosynthesis, motility, and chemotaxis. The DNA sequence revealed the presence of 10 complete and 2 incomplete open reading frames. Comparison of the deduced amino acid sequences to data banks showed similarities of nine of the deduced products to a number of proteins of Escherichia coli and Salmonella typhimurium for which a role in flagellar functioning has been directly demonstrated. In particular, the sequence data suggest that the flaA operon codes for the M-ring protein, components of the motor switch, and the distal part of the basal-body rod. The gene order is remarkably similar to that described for region III of the enterobacterial flagellar regulon. One of the open reading frames was translated into a protein with 48% amino acid identity to S. typhimurium FliI and 29% identity to the beta subunit of E. coli ATP synthase.     

The PatB protein of Bacillus subtilis is a C-S-lyase

The PatB protein of Bacillus subtilis had both cystathionine beta-lyase and cysteine desulfhydrase activities in vitro. The apparent K(m) value of the PatB protein for cystathionine was threefold higher than that of the MetC protein, the previously characterized cystathionine beta-lyase of B. subtilis. In the presence of cystathionine as sole sulfur source, the patB gene present on a multicopy plasmid restored the growth of a metC mutant. In addition, the patB metC double mutant was unable to grow in the presence of sulfate or cystine while the patB or metC single mutants grew similarly to the wild-type strains in the presence of the same sulfur sources. In a metC mutant, the PatB protein can replace the MetC enzyme in the methionine biosynthetic pathway.     

The composition of the Bacillus subtilis aerobic respiratory chain supercomplexes

Bacillus subtilis has a bifurcated respiratory chain composed of a cytochrome branch and a quinol oxidase branch. The respiratory complexes of this bacterium have been elucidated mostly by the analysis of the genome and by the isolation of individual complexes. The supramolecular organization of this respiratory chain is not known. In this work, we have analyzed the organization of the supercomplex in membranes isolated from B. subtilis grown in aerobic conditions in a medium with 3?% succinate. We used two different native electrophoretic techniques, clear native electrophoresis (CNE) and blue native electrophoresis (BNE). Using a heme-specific stain and Coomassie blue stain with in-gel activity assays followed by mass spectrometry, we identified the proteins resolved in both the first and second dimensions of the electrophoreses to detect the supercomplexes. We found that complexes b ( 6 ) c and caa ( 3 ) form a very high molecular mass supercomplex with the membrane-bound cytochrome c ( 550 ) and with ATP synthase. Most of the ATP synthase was found as a monomer. Succinate dehydrogenase was identified within a high molecular band between F(0)F(1) and F(1) and together with nitrate reductase. The type-2 NADH dehydrogenase was detected within a low molecular mass band. Finally, the quinol oxidase aa ( 3 ) seems to migrate as an oligomer of high molecular mass.     

The putative Bacillus subtilis l,d-transpeptidase YciB is a lipoprotein that localizes to the cell poles in a divisome-dependent manner

Cell wall synthesis in bacteria is spatially organized by cytoskeletal structures. Common to all cell wall-bearing bacteria, the cytokinetic machinery localizes the cell wall synthesis to the site of septation. Recently, MinJ, a new component of the cytokinetic machinery, or divisome, of Bacillus subtilis has been described. MinJ is part of the division site selection system but also essential for correct assembly of the divisome. Here, I used the isolated PDZ domain of MinJ for co-elution experiments. One of the proteins that co-eluted was the so far uncharacterized, putative l,d-transpeptidase protein YciB. Evidence is shown that YciB localizes to the cell poles. YciB localization depends on the existence of a mature divisome, suggesting that l,d-transpeptidases are, like penicillin-binding proteins, part of the divisome.     

Colicin B consists of a single polypeptide chain

Abstract Colicin B was isolated in pure form from Escherichia coli Cl139 and was shown to consist of a single polypeptide chain with an apparent M _r of 70000. Therefore, it does not differ from other colicins where the toxic activity resides in one polypeptide.     

Evidence that the Bacillus subtilis SpoIIGA protein is a novel type of signal-transducing aspartic protease

The bacterium Bacillus subtilis undergoes endospore formation in response to starvation. sigma factors play a key role in spatiotemporal regulation of gene expression during development. Activation of sigma factors is coordinated by signal transduction between the forespore and the mother cell. sigma(E) is produced as pro-sigma(E), which is activated in the mother cell by cleavage in response to a signal from the forespore. We report that expression of SpoIIR, a putative signaling protein normally made in the forespore, and SpoIIGA, a putative protease, is necessary and sufficient for accurate, rapid, and abundant processing of pro-sigma(E) to sigma(E) in Escherichia coli. Modeling and mutational analyses provide evidence that SpoIIGA is a novel type of aspartic protease whose C-terminal half forms a dimer similar to the human immunodeficiency virus type 1 protease. Previous studies suggest that the N-terminal half of SpoIIGA is membrane-embedded. We found that SpoIIGA expressed in E. coli is membrane-associated and that after detergent treatment SpoIIGA was self-associated. Also, SpoIIGA interacts with SpoIIR. The results support a model in which SpoIIGA forms inactive dimers or oligomers, and interaction of SpoIIR with the N-terminal domain of SpoIIGA on one side of a membrane causes a conformational change that allows formation of active aspartic protease dimer in the C-terminal domain on the other side of the membrane, where it cleaves pro-sigma(E).     

The Bacillus subtilis menCD promoter is responsive to extracellular pH

Activation kinetics of a Bacillus subtilis menaquinone biosynthetic gene promoter (the menCD promoter) were measured during growth and sporulation, with the aid of a menCD-lacZ translational gene fusion. Transient maximal activation was seen shortly after the end of exponential growth in unbuffered complex medium containing a low glucose concentration. These activation kinetics were correlated with transient acidification of the medium under conditions permitting TCA cycle function during the post-exponential period, while mutations that blocked TCA cycle function (cit mutants) were associated with sustained acidification and promoter activation during this period. In cit ⁺ strains, buffering of the medium to pH 5.7 caused sustained maximal activation, while buffering to pH 7.2 prevented enhancement of activation. The menCD promoter appears to be responsive to extracellular acidic pH.     

A gene required for nutritional repression of the Bacillus subtilis dipeptide permease operon

An insertion mutation was isolated that resulted in derepressed expression of the Bacillus subtilis dipeptide transport operon (dpp) during the exponential growth phase in rich medium. DNA flanking the site of insertion was found to encode an operon (codVWXY) of four potential open reading frames (ORFs). The deduced product of the codV ORF is similar to members of the λ Int family; CodW and CodX are homologous to HsIV and HsIU, two putative heat-shock proteins from Escherichia coli, and to LapC and LapA, two gene products of unknown function from Pasteurella haemolytica. CodX also shares homology with a family of ATPases, including CIpX, a regulatory subunit of the E. coli ClpP protease. CodY does not have any homologues in the databases. The insertion mutation and all previously isolated spontaneous cod mutations were found to map In codY. In-frame deletion mutations in each of the other cod genes revealed that only codY is required for repression of dpp in nutrient-rich medium. The cody mutations partially relieved amino acid repression of the histidine utilization (hut) operon but had no effect on regulation of certain other early stationary phase-induced genes, such as spoVG and gsiA.     

The N-terminal region of Escherichia coli lactose permease mediates membrane contact of the nascent polypeptide chain

Plasmids encoding N-terminal segments of the Escherichia coli lactose permease (also referred to as lactose carrier) have been used to analyze the biosynthesis and membrane insertion of this complex integral protein of the cytoplasmic membrane. Such truncated polypeptides were found to be stably associated with the membrane and to resemble the full-length protein with respect to their solubilization characteristics. Membrane-bound and free cytoplasmic polysomes were prepared from plasmid-bearing cells and incubated in the presence of [35S]methionine to permit completion of polypeptides initiated in vivo. Under these conditions, lactose permease was found to be radiolabeled in the fraction of membrane-bound polysomes; beta-galactosidase, used as a control, was translated almost exclusively by free polysomes. From similar experiments with N-terminal segments of lactose permease, we estimate that at most a polypeptide of 120 amino acid residues emerging from the ribosome is needed to target the nascent chain to the lipid bilayer and to mediate attachment of the ribosome to the membrane during elongation. Additional data support the idea that even shorter N-terminal sequences of 50 and 71 amino acid residues contain sufficient 'information' to provide contact with the membrane.