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Cloning and characterization of the Neisseria meningitidis asd gene
L A Hatten1, H P Schweizer, N Averill
1Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, Alberta, Canada.
Abstract:
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 mutans. 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 Escherichia 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.
Insights
This study cloned the asd gene from Neisseria meningitidis to create a safer laboratory strain. The cloned gene
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Genetics
Background:
- Certain bacterial mutants lacking the asd gene require diaminopimelic acid (DAP) for survival.
- DAP is crucial for bacterial cell wall integrity.
- The DAP auxotrophy of asd mutants has been used to create attenuated vaccine strains.
Purpose of the Study:
- To clone the asd gene from Neisseria meningitidis.
- To develop a genetically modified, biosafe laboratory strain of N. meningitidis.
- To investigate the potential for using asd mutants in bacterial research.
Main Methods:
- Cloning of the N. meningitidis asd gene by complementation of an Escherichia coli asd mutant.
- Subcloning and insertion mutagenesis to localize the asd gene.
- Expression of the asd gene in a T7 RNA polymerase-T7 promoter system.
- Nucleotide sequencing and N-terminal amino acid sequence determination using an Asd'::'LacZ fusion.
Main Results:
- The N. meningitidis asd gene was successfully cloned and localized to a 1.5-kb DNA fragment.
- A 38-kDa protein was expressed from this fragment.
- The N-terminal amino acid sequence showed 70% identity with E. coli Asd protein.
- This suggests high conservation of the Asd protein in Gram-negative bacteria.
Conclusions:
- The asd gene from N. meningitidis has been cloned and characterized.
- The high conservation of the Asd protein supports its potential as a target for genetic manipulation.
- This work is a foundational step towards developing a biosafe N. meningitidis laboratory strain.