Nucleotide sequence of the pepB region and predicted amino acid sequence of PepB. The sequence for a potential ς⁷⁰ promoter is indicated by a double underline, and the putative ribosome binding site is in boldface type. The N-terminal sequence of the purified PepB protein and the 22-kDa peptide (the N-terminal fragment of cleaved PepB) is underlined. The N-terminal sequence of the 24-kDa peptide (the C-terminal fragment of cleaved PepB) is indicated by a dashed underline.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of PepB purification. Four micrograms from each step of the purification of serovar Typhimurium PepB was loaded on a 10% Tris-tricine gel. Lanes: 1, molecular mass standards; 2, crude extract of TN5179; 3, Q Sepharose; 4, 70°C heat step; 5, Superdex. The arrows indicate purified PepB and the N-terminal and C-terminal cleavage products.

Heat stability of PepB. Crude extracts of strain TN5179 (●) and partially purified PepB (after the Q Sepharose column purification step) (○) were heated at 70°C. Samples were withdrawn at the indicated times and assayed for peptidase activity.

Effect of pH on PepB activity. The activity of PepB was determined in 50 mM bis-Tris at pH 6.0 and 6.5, in 50 mM Tris at pH 7.0 to 9.5, and in 50 mM 2-(cyclohexylamine) ethanesulfonic acid at pH 8.5 to 10.0 in the standard assay using 1 mM Leu-Leu as a substrate.

Effect of salt on PepB activity. The activity of PepB was determined by using the standard assay in 0.05 M Tris-HCl (pH 8.5) and 1 mM MgCl₂ with 1 mM Leu-Leu as a substrate.

Sequence alignment of the C-terminal domains of a subset of the LAP family. All members of the proposed PepB subfamily and its close relatives are presented, and a diverse group of other LAPs chosen to represent some of the major subgroupings predicted by the phylogenetic tree (Fig. 7) is included for comparison. Positions that are identical for all seven PepBs are indicated by black shading. The conserved residues in the PepB subfamily are indicated in the consensus sequence above the alignment, and the residues that are identical in all members of the LAP family are underlined. The asterisks indicate the positions of K310 and the CSATYRK motif. The arrows indicate the metal binding sites. The reported sequence for A. actinomycetemcomitans PepB shows a G rather than an E in the universally conserved NTDAEGR metal binding site (line 3 of the alignment). We believe that this is likely to be a sequencing error in this incomplete genome sequence and have replaced the G with an E. A.aPepB, A. actinomycetemcomitans PepB (NUMG); P.mPepB, P. multocida PepB (NUMG); H.iPepB, H. influenzae PepB (P45334); S.tiPepB, serovar Typhi PepB (SSC); S.tmPepB, serovar Typhimurium PepB (AF201078 [this work]); E.cPepB, E. coli PepB (P37095); Y.pPepB, Y. pestis PepB (SSC); S.pLAP3, S. putrefaciens LAP3 (NUMG); C.cLAP3, C. crescentus LAP3 (NUMG); L.pLAP, L. pneumophila LAP (NUMG); R.cLAP3, R. capsulatus LAP3 (blast search of WIT at http://www.wit.mcs.anl.gov); E.cPepA, E. coli PepA (P11648); P.aPepA, Pseudomonas aeruginosa PepA (AAD04821); B.tLAP, Bos taurus kidney (P00727); A.pLAP, Aeropyrum pernix LAP (NUMG); SynecLAP, Synechocystis sp. LAP (P73971); B.sLAP, Bacillus subtilis LAP (Z99120); S.aLAP, Staphylococcus aureus LAP (NUMG); R.pPepA, Rickettsia prowazekii PepA (P27888); R.cLAP2, R. capsulatus LAP2 (WIT).

Phylogenetic tree of the C-terminal domain of the LAP family. The tree is rooted with the Aeropyrum pernix sequence as an outgroup. The scale bar represents 10 amino acid substitutions per 100 positions. The numbers near each node are the bootstrap proportions estimated by the RELL method from candidate trees. The nodes without bootstrap values cannot be assigned with confidence. A.actn PepA, A. actinomycetemcomitans PepA; A.aPepB, A. actinomycetemcomitans PepB; A.aeol, Aquifex aeolicus (AAC07829); A.pLAP, A. pernix LAP (NUMG); A.thal, Arabidopsis thaliana (P30184); B.sLAP, Bacillus subtilis LAP; B.pert, Bordetella pertussis (NUMG); B.tLAP, Bos taurus kidney (P00727); C.cLAP2, C. crescentus LAP2; C.jejun, Campylobacter jejuni (NUMG); C.pneum, Chlamydia pneumoniae (AE001623); C.trach, Chlamydia trachomatis (AE001279); C.tepid, Chlorobium tepidum (NUMG); C.diff, Clostridium difficile (NUMG); C.dipth, Corynebacterium diptheriae (NUMG); D.radio, Deinococcus radiodurans (AE001928); E.cPepA, E. coli; PepA; H.inflPepA, Haemophilus influenzae PepA (AAC23347); H.iPepB, H. influenzae PepB; H.pylor, Helicobacter pylori (AE001485); H.sapi, Homo sapiens (AAD17527); L.escu, Lycopersicon esculentum (Q42876); L.escuChl, L. esculentum Chloroplast (Q10712); L.pLAP, L. pneumophila LAP; M.lepr, Mycobacterium leprae (CAB11379); M.tube, Mycobacterium tuberculosis (Q10401); M.gent, Mycoplasma genitalium (P47631); M.pneu, Mycoplasma pneumoniae (P75206); N.gonor, Neisseria gonorrhoeae (NUMG); P.aPepA, Pseudomonas aeruginosa PepA; P.mPepB, P. multocida PepB; P.multPepA, P. multocida PepA (NUMG); P.falc, Plasmodium falciparum (NUMG); P.putiPepA, Pseudomonas putida PepA (CAA09054); R.cLAP1, R. capsulatus LAP1; R.pPepA, Rickettsia prowazekii PepA (P2788); S.typiPepA, serovar Typhi PepA (SSC); S.pomb, Schizosaccharomyces pombe (Q09735); S.melil, Sinorhizobium meliloti (NUMG); S.tiPepB, serovar Typhi PepB; S.tmPepB, serovar Typhimurium PepB; S.tube, Solanum tuberosum (P31427); S.aLAP, Staphylococcus aureus LAP (NUMG); S.coel, Streptomyces coelicolor (CAB51263); S.pLAP2, S. putrefaciens LAP2; SynecLAP, Synechocystis sp. LAP; T.ferr, Thiobacillus ferrooxidans (NUMG); V.chol, Vibrio cholerae (NUMG); Y.pestPepA, Y. pestis PepA (SSC); Y.PepB, Y. pestis PepB. In organisms where there is more than one LAP, a numerical designation has been arbitrarily assigned. In several cases, we have assigned names that have not been previously assigned in the literature based on location in the tree: A.actnPepA and -PepB and P.multPepA and -PepB.