Construction and Characterization of a Lambda Phage Library of Chromosome DNA from Alkaliphilic Bacillus sp. C-125
Hideto TAKAMIa, Kaoru NAKASONEa, Yuka NAKAMURA*a, Chie HIRAMAa, Yoshihiro TAKAKIa, Noriaki MASUIa, Fumie FUJIa, Akira INOUEa, Shinsuke NAKADEb, Naotake OGASAWARAc, and Koki HORIKOSHIa
a The DEEPSTAR Group, Japan Marine Science and Technology Center, 2-15 Natsushima, Yokosuka 237, Japan
b Research and Education Center for the genetic information, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-01, Japan
c Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-01, Japan
Generally, alkaliphilic bacteria can not grow well or only grow slowly at neutral pH values, but grow well at higher pH than pH 9.5. Alkaliphilic Bacillus sp. C-125 is best characterized strain physiologically, biochemically and molecular biologically (1). The size and GC content of the C-125 chromosome are almost identical to those of B. subtilis (4.2 Mb and 43.7 mol%).
Recently, whole genome analysis of Bacillus subtilis which is close to this strain except alkaliphily was completed by the collaboration project between Japan and European Community. All nucleotide sequence data of the B. subtilis chromosome will soon reveal the structure and organization of the genes on the chromosome. Knowledge of organization of the whole genome will provide more information about genes involved in the processes such as transduction, transcriptional regulation and protein secretion. Moreover, these sequence data of B. subtilis will be great helpful to quick genome analysis of similar microbe just like our target, alkaliphilic Bacillus sp. C-125.
From these background, we have initiated the genome analysis of the stain C- 125 based on all information from B. subtilis. As the first step of genome analysis, we attempted to constcut lambda phage library of the C-125 chromosome for large-scale nucleotide sequencing to compare the gene structure and organization with those of B. subtilis.
A recombinant DNA library was constructed from partial SaulIIAI digests of the chromosome of alkaliphilic Bacillus sp. C-125, in the BarnHi site of λ, Dash II. Each DNA fragment of about 20 kb inserted in three lambda clones (lambda 3, 4, and 9) randomly selected was amplified by long accurate PCR. Each amplified DNA fragment was sonicated and the size-fractionated (1-2 kb) fragments were used for construction of M13 phage library. The nucleotide sequence of each lambda clone
