Arthur J. G. Moir

How do spores germinate

Spore germination, as defined as those events that result in the loss of the spore‐specific properties, is an essentially biophysical process. It occurs without any need for new macromolecular synthesis, so the apparatus required is already present in the mature dormant spore. Germination in response to specific chemical nutrients requires specific receptor proteins, located at the inner membrane of the spore. After penetrating the outer layers of spore coat and cortex, germinant interacts with its receptor: one early consequence of this binding is the movement of monovalent cations from the spore core, followed by Ca2+ and dipicolinic acid (DPA). In some species, an ion transport protein is also required for these early stages. Early events – including loss of heat resistance, ion movements and partial rehydration of the spore core – can occur without cortex hydrolysis, although the latter is required for complete core rehydration and colony formation from a spore. In Bacillus subtilis two crucial cortex lytic enzymes have been identified: one is CwlJ, which is DPA‐responsive and is located at the cortex‐coat junction. The second, SleB, is present both in outer layers and at the inner spore membrane, and is more resistant to wet heat than is CwlJ. Cortex hydrolysis leads to the complete rehydration of the spore core, and then enzyme activity within the spore protoplast resumes. We do not yet know what activates SleB activity in the spore, and neither do we have any information at all on how the spore coat is degraded.

Genes of Bacillus cereus and Bacillus anthracis Encoding Proteins of the Exosporium

The exosporium is the outermost layer of spores of Bacillus cereus and its close relatives Bacillus anthracis and Bacillus thuringiensis. For these pathogens, it represents the surface layer that makes initial contact with the host. To date, only the BclA glycoprotein has been described as a component of the exosporium; this paper defines 10 more tightly associated proteins from the exosporium of B. cereus ATCC 10876, identified by N-terminal sequencing of proteins from purified, washed exosporium. Likely coding sequences were identified from the incomplete genome sequence of B. anthracis or B. cereus ATCC 14579, and the precise corresponding sequence from B. cereus ATCC 10876 was defined by PCR and sequencing. Eight genes encode likely structural components (exsB, exsC, exsD, exsE, exsF, exsG, exsJ, and cotE). Several proteins of the exosporium are related to morphogenetic and outer spore coat proteins of B. subtilis, but most do not have homologues in B. subtilis. ExsE is processed from a larger precursor, and the CotE homologue appears to have been C-terminally truncated. ExsJ contains a domain of GXX collagen-like repeats, like the BclA exosporium protein of B. anthracis. Although most of the exosporium genes are scattered on the genome, bclA and exsF are clustered in a region flanking the rhamnose biosynthesis operon; rhamnose is part of the sugar moiety of spore glycoproteins. Two enzymes, alanine racemase and nucleoside hydrolase, are tightly adsorbed to the exosporium layer; they could metabolize small molecule germinants and may reduce the sensitivity of spores to these, limiting premature germination.

Binding sites involved in the interaction of actin with the N-terminal region of dystrophin

Two actin‐binding sites have been identified on human dystrophin by proton NMR spectroscopy of synthetic peptides corresponding to defined regions of the polypeptide sequence. These are Actin‐Binding Site 1 (ABS1) located at residues 17–26 and Actin‐Binding Site 2 (ABS2) in the region of residues 128–156. Using defined fragments of the actin amino acid sequence, ABS1 has been shown to bind to actin in the region represented by residues 83–117 and ABS2 to the C‐terminal region represented by residues 350–375. These dystrophin‐binding sites lie on the exposed domain in the actin filament.

Characterization of the exosporium of Bacillus cereus

Exosporium components from endospores of Bacillus cereus ATCC 10876 were purified and separated by gel electrophoresis. Several of the proteins for which N‐terminal sequences were recovered were found to have homologues in protein databases which have been demonstrated to have enzymic activity in other organisms. Amongst these is a zinc metalloprotease, immune inhibitor A, already described in B. thuringiensis. This has been shown to be present in an active 73 kDa form on the exosporium of B. cereus. Other proteins associated with the exosporium include the molecular chaperone GroEL and a homologue of RocA (1‐pyrroline‐5‐carboxylate dehydrogenase (EC 1.5.1.12)) of B. subtilis. Although these are unlikely to represent integral structural proteins of the exosporium, the observation that they are selectively present in the spore surface layer suggests that this layer may have functional significance.

The interaction of actin with dystrophin.

Proton NMR spectroscopy of synthetic peptides corresponding to defined regions of human dystrophin has been employed to study the interaction with F‐actin. No evidence of interaction with a C‐terminal region corresponding to amino acid residues 3429–3440 was obtained. F‐actin restricted the mobility of residues 19–27 in a synthetic peptide corresponding to residues 10–32. This suggests that this is a site of F‐actin interaction in the intact dystrophin molecule. Identical sequences to that of residues 19—22 in dystrophin, namely Lys‐Thr‐Phe‐Thr are also present in the N‐terminal regions of the α‐actinins implying this is also a site of F‐actin interaction with α‐actinin.

Cysteine is exported from the Escherichia coli cytoplasm by CydDC, an ATP-binding cassette-type transporter required for cytochrome assembly.

Assembly of Escherichia colicytochrome bd and periplasmic cytochromes requires the ATP-binding cassette transporter CydDC, whose substrate is unknown. Two-dimensional SDS-PAGE comparison of periplasm from wild-type andcydD mutant strains revealed that the latter was deficient in several periplasmic transport binding proteins, but no single major protein was missing in the cydD periplasm. Instead, CydDC exports from cytoplasm to periplasm the amino acid cysteine, demonstrated using everted membrane vesicles that transported radiolabeled cysteine inward in an ATP-dependent, uncoupler-independent manner. New pleiotropic cydDphenotypes are reported, including sensitivity to benzylpenicillin and dithiothreitol, and loss of motility, consistent with periplasmic defects in disulfide bond formation. Exogenous cysteine reversed these phenotypes and affected levels of periplasmic c-type cytochromes in cydD and wild-type strains but did not restore cytochrome d. Consistent with CydDC being a cysteine exporter, cydD mutant growth was hypersensitive to high cysteine concentrations and accumulated higher cytoplasmic cysteine levels, as did a mutant defective inorf299, encoding a transporter of the major facilitator superfamily. A cydD orf299 double mutant was extremely cysteine-sensitive and had higher cytoplasmic cysteine levels, whereas CydDC overexpression conferred resistance to high extracellular cysteine concentrations. We propose that CydDC exports cysteine, crucial for redox homeostasis in the periplasm.

SALMONELLA TYPHIMURIUM RESPONSES TO A BACTERICIDAL PROTEIN FROM HUMAN NEUTROPHILS

Bactericidal/permeability‐increasing protein [BPI] is a cationic antimicrobial protein from neutrophils that specifically binds to the surfaces of Gram‐negative bacteria via the lipid A component of lipopolysaccharide. To obtain information about the responses of Salmonella typhimurium to cell‐surface damage by BPI, two‐dimensional gel electrophoresis and N‐terminal microsequencing were used to identify proteins that were induced or repressed following BPI treatment. The majority of the affected proteins are involved in central metabolic processes. Upon addition of BPI, the β‐subunit of the F1 portion of Escherichia coli ATP synthase was repressed threefold whereas six proteins were induced up to 11‐fold. Three of the latter were identified as lipoamide dehydrogenase, enoyl‐acyl carrier protein reductase, and the heat‐shock protein HtpG. Additionally, a novel protein, BipA, was identified that is induced over sevenfold by BPI; sequence analysis suggests that it belongs to the GTPase superfamily and interacts with ribosomes. A conserved direct‐repeat motif is present in the regulatory regions of several BPI‐inducible genes, including the bipA gene. Only one of the BPI‐responsive proteins was induced when cells were treated with polymyxin B, which also binds to lipid A. We therefore conclude that BPI and polymyxin B affect different global regulatory networks in S. typhimurium even though they bind with high affinity to the same cell‐surface component.

Specificity of a milk clotting enzyme extracted from the thistleCynara cardunculus L.: Action on oxidised insulin and k-casein

SummaryK-casein and oxidised insulin were digested with an acid protease extracted fromCynara cardunculus L. The fragments produced were isolated and characterised. In k-casein cleavage occured specifically at Phe 105-Met 106 bond. In oxidised insulin seven fragments were obtained and cleavage was found to occur at the carboxylic side of (Phe, Leu, He)-X, where X was preferentially Val or Tyr. The results obtained with insulin B chain suggest thatCynara cardunculus L. protease possesses a greater specificity than other acid proteases reported.

Purification and characterization of a new peptide antibiotic produced by a thermotolerant Bacillus licheniformis strain

A Bacillus licheniformis strain, I89, isolated from a hot spring environment in the Azores, Portugal, strongly inhibited growth of Gram-positive bacteria. It produced a peptide antibiotic at 50 °C. The antibiotic was purified and biochemically characterized. It was highly resistant to several proteolytic enzymes. Additionally, it retained its antimicrobial activity after incubation at pH values between 3.5 and 8; it was thermostable, retaining about 85% and 20% of its activity after 6 h at 50 °C and 100 °C, respectively. Its molecular mass determined by mass spectrometry was 3249.7 Da.

Indicain, a dimeric serine protease from Morus indica cv. K2

A high molecular mass serine protease has been purified to homogeneity from the latex of Morus indica cv. K2 by the combination of techniques of ammonium sulfate precipitation, hydrophobic interaction chromatography, and size-exclusion chromatography. The protein is a dimer with a molecular mass of 134.5 kDa and with two monomeric subunits of 67.2 kDa and 67.3 (MALDI-TOF), held by weak bonds susceptible to disruption on exposure to heat and very low pH. Isoelectric point of the enzyme is pH 4.8. The pH and temperature optima for caseinolytic activity were 8.5 and 80 degrees C, respectively. The extinction coefficient (epsilon280(1%)) of the enzyme was estimated as 41.24 and the molecular structure consists of 52 tryptophan, 198 tyrosine and 42 cysteine residues. The enzyme activity was inhibited by phenylmethylsulfonylflouride, chymostatin and mercuric chloride indicating the enzyme to be a serine protease. The enzyme is fairly stable and similar to subtilases in its stability toward pH, strong denaturants, temperature, and organic solvents. Polyclonal antibodies specific to enzyme and immunodiffusion studies reveal that the enzyme has unique antigenic determinants. The enzyme has activity towards broad range of substrates comparable to those of subtilisin like proteases. The N-terminal residues of indicain (T-T-N-S-W-D-F-I-G-F-P) exhibited considerable similarity to those of other known plant subtilases, especially with cucumisin, a well-characterized plant subtilase. This is the first report of purification and characterization of a subtilisin like dimeric serine protease from the latex of M. indica cv. K2. Owing to these unique properties the reported enzyme would find applications in food and pharma industry.