Molecular cloning, nucleotide sequence, and characterization of lppB, encoding an antigenic 40-kilodalton lipoprotein of Haemophilus somnus

Abstract

Haemophilus somnus is a facultative intracellular pathogen which causes a wide range of diseases in cattle. To identify putative virulence determinants, a genomic library of H. somnus in Escherichia coli was screened for Congo red binding, a property associated with virulence in pathogenic bacteria, and subsequently with bovine hyperimmune sera raised against H. somnus HS25. A Congo red-binding clone carrying a 1.8-kb DNA insert was found to encode a strongly seroreactive LppB protein with an apparent molecular weight of 40,000. The nucleotide sequence of the entire DNA insert was determined. Two open reading frames coding for polypeptides with calculated molecular weights of 21,893 and 30,721 were identified. The larger open reading frame encoded LppB, while the smaller reading frame encoded a nonseroreactive protein with a relative molecular mass of approximately 18 kDa. The 16 amino-terminal amino acids of the deduced LppB polypeptide showed strong sequence homology to the signal peptide of secreted bacterial proteins, and the sequence Leu-Ala-Ala-Cys at the putative cleavage site corresponds to the consensus cleavage sequence of bacterial lipoproteins. Synthesis of the mature LppB lipoprotein in H. somnus was inhibited by globomycin, a specific inhibitor of signal peptidase II. LppB was localized to the outer membrane of H. somnus.

Cloning, Sequencing, and Phenotypic Characterization of the rpoS Gene from Pseudomonas putida KT2440

“…The amino acid sequences corresponding to the ORFs were compared with all entries in the nonredundant GenBank CDS translations ϩPDBϩSwissProtϩPIR as described in the BLAST program (1) and with the SwissProt ALL library with the FASTA3 program (29). The data bank sequences that showed the most sequence identity with the partial ORF1 were seven precursor sequences encoding the lipoprotein B in gramnegative bacteria: P. aeruginosa (43), E. coli (42), Haemophilus somnus (45), H. influenzae (9), Salmonella typhimurium (21), Yersinia enterocolitica (17), and S. dublin (SwissProt entry 39700). The PROSITE search for conserved domains in the amino acid sequence derived from ORF2 produced two matches with 70 : sigma70_1 at residue 124 and sigma70_2 at residue 293.…”

Growth-phase-dependent transcriptional regulation of the pcm and surE genes required for stationary-phase survival of Escherichia coli

“…The arrangement was similar to that observed in a closely related species, H . somnus, that has been compared with E. coli (Theisen et al, 1993). In M. jannaschii, a pcm homologue with 45.6% amino acid identity can be identified.…”

Cattle Immunized with a Recombinant Subunit Vaccine Formulation Exhibits a Trend towards Protection against Histophilus somni Bacterial Challenge

“…PCR products of R1, R2, R8, R18, R15, R34, and R35 were double digested with restriction enzyme pairs, (BglII, NcoI), or (XmaI, NcoI) as stated in Table 1 , and inserted into an N-terminal hexa-histidine affinity tag in-house cloning vector pGH433His.2 [ 34 , 40 ] downstream of an Isopropyl-β-d-thiogalactopyranoside (IPTG) inducible tac promoter. As previously stated [ 34 ], pGH433 which is identical to pGH433His.2 except for the exclusion of the histidine tag, was used on double digested PCR products R5 and R13 using restriction enzyme pair (BglII, NcoI) [ 34 , 40 ]. Another in-house cloning vector pAA352 [ 41 ], was used on PCR products R4, R21, R23, R24, R27, R35, R36, and R37.…”