Microcin B17 (MccB17) is a ribosomally synthesized peptide antibiotic of 43 amino acids that induces double-strand breaking of DNA in a DNA gyrase-dependent reaction. As a consequence, the SOS regulon is induced and massive DNA degradation occurs. In this work we have characterized an Escherichia coli gene, sbmC, that in high copy number determines high cell resistance to MccB17. sbmC encodes a cytoplasmic polypeptide of 157 amino acids (M(r), 18,095) that has been visualized in SDS-polyacrylamide gels. The gene is located at min 44 of the E. coli genetic map, close to the sbcB gene. sbmC expression is induced by DNA-damaging agents and, also, by the entry of cells into the stationary growth phase. A G-->T transversion at the fifth nucleotide of the quasicanonical LexA-box preceding the gene makes recA cells 16-fold more resistant to exogenous MccB17. The gene product, SbmC, also blocks MccB17 export from producing cells. Altogether, our results suggest that SbmC recognizes and sequesters MccB17 in a reversible way.

Pal amidase, encoded by pneumococcal bacteriophage Dp-1, represents one step beyond in the modular evolution of pneumococcal murein hydrolases. It exhibits the choline-binding module attaching pneumococcal lysins to the cell wall, but the catalytic module is different from those present in the amidases coded by the host or other pneumococcal phages. Pal is also an effective antimicrobial agent against Streptococcus pneumoniae that may constitute an alternative to antibiotic prophylaxis. The structural implications of Pal singular structure and their effect on the choline-amidase interactions have been examined by means of several techniques. Pal stability is maximum around pH 8.0 (T m Х 50.2°C; ⌬H t ‫؍‬ 183 ؎ 4 kcal mol ؊1 ), and its constituting modules fold as two tight interacting cooperative units whose denaturation merges into a single process in the free amidase but may proceed as two well resolved events in the choline-bound state. Choline titration curves reflect low energy ligand-protein interactions and are compatible with two sets of sites. Choline binding strongly stabilizes the cell wall binding module, and the conformational stabilization is transmitted to the catalytic region. Moreover, the high proportion of aggregates formed by the unbound amidase together with choline preferential interaction with Pal dimers suggest the existence of marginally stable regions that would become stabilized through choline-protein interactions without significantly modifying Pal secondary structure. This structural rearrangement may underlie in vitro "conversion" of Pal from the low to the full activity form triggered by choline. The Pal catalytic module secondary structure could denote folding conservation within pneumococcal lytic amidases, but the number of functional choline binding sites is reduced (2-3 sites per monomer) when compared with pneumococcal LytA amidase (4 -5 sites per monomer) and displays different intermodular interactions.Dp-1, the first described pneumococcal bacteriophage, belongs to the Siphoviridae family (1). Its peptidoglycan-degrading enzyme, Pal, 1 was biochemically characterized as a cholinedependent amidase (2) synthesized as a low activity form that requires in vitro incubation with choline or choline-containing cell walls to achieve full activity (3), a process designated as "conversion." Pal shows the modular organization characteristic of pneumococcal cell wall lysins (3), but represents one step beyond in the modular evolution of pneumococcal murein hydrolases. Its N-terminal region has no similarity with the amidases coded by the host or other pneumococcal phages (4, 5), 2 but is highly similar to the murein hydrolase coded by Lactococcus phage BK5-T. 3 The C-terminal region comprises a choline binding module (ChBM) homologous to that found in the pneumococcal lytic system 4 (6) that attaches the enzyme to choline residues present in pneumococcal envelope (7). As in most pneumococcal lysins so far known, six repeats of about 20 amino acids (p1-p6) and a short C-terminal tail fo...

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HSP-3 is a member of the cysteine-rich secretory protein (CRISP) family from stallion seminal plasma. We report a large-scale purification protocol for native HSP-3. This protein is a non-glycosylated polypeptide chain with a pI of 8^9 and an isotope-averaged molecular mass of 24 987 þ 3 Da. The molecular mass of HSP-3, determined by equilibrium sedimentation, is 26 kDa, showing that the protein exists in solution as a monomer. The concentration of HSP-3 in the seminal plasma of different stallions ranged from 0.3 to 1.3 mg/ml. On average, 0.9^9 million HSP-3 molecules/cell coat the postacrosomal and mid-piece regions of an ejaculated, washed stallion spermatozoon, suggesting a role in sperm physiology. Conformational characterisation of purified HSP-3 was assessed by combination of circular dichroism and Fourier-transform infrared spectroscopies and differential scanning microcalorimetry. Based on secondary structure assignment, HSP-3 may belong to the K K+L L class of proteins. Thermal denaturation of HSP-3 is irreversible and follows a non-two state transition characterised by a T m of 64³C, an enthalpy change of 75 kcal/mol, and a van 't Hoff enthalpy of 184 kcal/mol. Analysis of the spectroscopic and calorimetric data indicates the occurrence of aggregation of denatured HSP-3 molecules and suggests the monomer as the cooperative unfolding unit.z 1997 Federation of European Biochemical Societies.

The Ejl amidase is coded by Ej-1, a temperate phage isolated from the atypical pneumococcus strain 101/87. Like all the pneumococcal cell-wall lysins, Ejl has a bimodular organization; the catalytic region is located in the N-terminal module, and the C-terminal module attaches the enzyme to the choline residues of the pneumococcal cell wall. The structural features of the Ejl amidase, its interaction with choline, and the structural changes accompanying the ligand binding have been characterized by CD and IR spectroscopies, differential scanning calorimetry, analytical ultracentrifugation, and FPLC. According to prediction and spectroscopic (CD and IR) results, Ejl would be composed of short ␤-strands (ca. 36%) connected by long loops (ca. 17%), presenting only two well-predicted ␣-helices (ca. 12%) in the catalytic module. Its polypeptide chain folds into two cooperative domains, corresponding to the N-and C-terminal modules, and exhibits a monomer ↔ dimer self-association equilibrium. Choline binding induces small rearrangements in Ejl secondary structure but enhances the amidase self-association by preferential binding to Ejl dimers and tetramers. Comparison of LytA, the major pneumococcal amidase, with Ejl shows that the sequence differences (15% divergence) strongly influence the amidase stability, the organization of the catalytic module in cooperative domains, and the self-association state induced by choline. Moreover, the ligand affinity for the choline-binding locus involved in regulation of the amidase dimerization is reduced by a factor of 10 in Ejl. Present results evidence that sequence differences resulting from the natural variability found in the cell wall amidases coded by pneumococcus and its bacteriophages may significantly alter the protein structure and its attachment to the cell wall.Keywords: Pneumococcal cell-wall amidases; pneumococcal bacteriophages; Ejl; Ej-1; choline binding proteins; DSC; CD; IR The cell-wall lytic enzymes encoded by pneumococcus are involved in a variety of physiological functions associated with cell-wall growth, wall turnover, and cell separation (Tomasz 1984). Their polypeptide chains have evolved from the fusion of two independent modules, which are responsible for either the catalytic activity or the attachment to the choline residues of pneumococcal cell wall, which act as an antenna where lysins dock by the so-called cholinebinding module, ChBM (García et al. 1990;López et al. 1995López et al. , 2000. The cell-wall lytic enzymes coded by many Reprint requests to: M. Menéndez, Instituto de Química-Física "Rocasolano," CSIC, Serrano 119, 28006 Madrid, Spain; e-mail: mmenendez@ iqfr.csic.es; fax: 34-91-5642431.Abbreviations: ChBM, choline-binding module; Ejl, amidase encoded by the Ejl gene from Ej-1 phage; LytA, major autolysin from S. pneumoniae; IR, Fourier transform infrared spectroscopy; X, aromatic residue; DSC, differential scanning calorimetry; ⌬H, calorimetric enthalpy change; ⌬H vH , van't Hoff enthalpy change; 〈⌬H i (T)〉, excess enthalpy chan...