TY - JOUR
T1 - Cloning and characterization of the Neisseria meningitidis asd gene
AU - Hatten, Lee Ann
AU - Schweizer, Herbert P.
AU - Averill, Nuzhat
AU - Wang, Ling
AU - Schryvers, Anthony B.
N1 - Funding Information:
Financial supportt o H.P.S. is provided by a Medical Scholarshipo f the Medical ResearchC ouncil (MRC) of Canadaa nd by MRC operatingg rant MA-l 1245.
PY - 1993/7/15
Y1 - 1993/7/15
N2 - The asd mutants of Gram- and some Gram+ bacteria have an obligate requirement for diaminopimelic acid (DAP), an essential constituent of the cell wall of these organisms. In environments deprived of DAP, i.e., mammalian tissues, they will undergo lysis. This has previously been exploited to develop vaccine strains of Salmonella typhimurium and Streptococcus mutaons. As a first step for the development of a biosafe Neisseria meningitidis laboratory strain, we have cloned the asd from wild-type strain B16B6 by complementation of an Eschirichia coli asd mutant. By subcloning and insertion mutagenesis, the N. meningitidis asd was localized to a 1.5-kb DNA fragment. In a T7 RNA polymerase-T7 promoter expression system, a 38-kDa protein was strongly expressed from this DNA fragment. The N-terminal amino acid (aa) sequence was deduced from the nucleotide sequence, which was determined with the help of an in-frame Asd'::'LacZ protein fusion. A comparison of the N-terminal aa of the Asd proteins from N. meningitidis and E. coli revealed 70% identity, suggesting that the Asd protein may be highly conserved among Gram- bacteria.
AB - The asd mutants of Gram- and some Gram+ bacteria have an obligate requirement for diaminopimelic acid (DAP), an essential constituent of the cell wall of these organisms. In environments deprived of DAP, i.e., mammalian tissues, they will undergo lysis. This has previously been exploited to develop vaccine strains of Salmonella typhimurium and Streptococcus mutaons. As a first step for the development of a biosafe Neisseria meningitidis laboratory strain, we have cloned the asd from wild-type strain B16B6 by complementation of an Eschirichia coli asd mutant. By subcloning and insertion mutagenesis, the N. meningitidis asd was localized to a 1.5-kb DNA fragment. In a T7 RNA polymerase-T7 promoter expression system, a 38-kDa protein was strongly expressed from this DNA fragment. The N-terminal amino acid (aa) sequence was deduced from the nucleotide sequence, which was determined with the help of an in-frame Asd'::'LacZ protein fusion. A comparison of the N-terminal aa of the Asd proteins from N. meningitidis and E. coli revealed 70% identity, suggesting that the Asd protein may be highly conserved among Gram- bacteria.
KW - biosafety
KW - Cell wall
KW - gene fusion
KW - reading frame
KW - recombinant DNA
KW - β-galactosides
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U2 - 10.1016/0378-1119(93)90707-A
DO - 10.1016/0378-1119(93)90707-A
M3 - Article
C2 - 8101504
AN - SCOPUS:0027248704
SN - 0378-1119
VL - 129
SP - 123
EP - 128
JO - Gene
JF - Gene
IS - 1
ER -