TY - JOUR
T1 - Paired cloning vectors for complementation of mutations in the cyanobacterium Anabaena sp. strain PCC 7120
AU - Wolk, C. Peter
AU - Fan, Qing
AU - Zhou, Ruanbao
AU - Huang, Guocun
AU - Lechno-Yossef, Sigal
AU - Kuritz, Tanya
AU - Wojciuch, Elizabeth
PY - 2007/12
Y1 - 2007/12
N2 - The clones generated in a sequencing project represent a resource for subsequent analysis of the organism whose genome has been sequenced. We describe an interrelated group of cloning vectors that either integrate into the genome or replicate, and that enhance the utility, for developmental and other studies, of the clones used to determine the genomic sequence of the cyanobacterium, Anabaena sp. strain PCC 7120. One integrating vector is a mobilizable BAC vector that was used both to generate bridging clones and to complement transposon mutations. Upon addition of a cassette that permits mobilization and selection, pUC-based sequencing clones can also integrate into the genome and thereupon complement transposon mutations. The replicating vectors are based on cyanobacterial plasmid pDU1, whose sequence we report, and on broad-host-range plasmid RSF1010. The RSF1010- and pDU1-based vectors provide the opportunity to express different genes from either cell-type-specific or -generalist promoters, simultaneously from different plasmids in the same cyanobacterial cells. We show that pDU1 ORF4 and its upstream region play an essential role in the replication and copy number of pDU1, and that ORFs alr2887 and alr3546 (hetFA ) of Anabaena sp. are required specifically for fixation of dinitrogen under oxic conditions.
AB - The clones generated in a sequencing project represent a resource for subsequent analysis of the organism whose genome has been sequenced. We describe an interrelated group of cloning vectors that either integrate into the genome or replicate, and that enhance the utility, for developmental and other studies, of the clones used to determine the genomic sequence of the cyanobacterium, Anabaena sp. strain PCC 7120. One integrating vector is a mobilizable BAC vector that was used both to generate bridging clones and to complement transposon mutations. Upon addition of a cassette that permits mobilization and selection, pUC-based sequencing clones can also integrate into the genome and thereupon complement transposon mutations. The replicating vectors are based on cyanobacterial plasmid pDU1, whose sequence we report, and on broad-host-range plasmid RSF1010. The RSF1010- and pDU1-based vectors provide the opportunity to express different genes from either cell-type-specific or -generalist promoters, simultaneously from different plasmids in the same cyanobacterial cells. We show that pDU1 ORF4 and its upstream region play an essential role in the replication and copy number of pDU1, and that ORFs alr2887 and alr3546 (hetFA ) of Anabaena sp. are required specifically for fixation of dinitrogen under oxic conditions.
KW - Anabaena sp. strain PCC 7120
KW - Cloning vectors
KW - Complementation of mutations
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U2 - 10.1007/s00203-007-0276-z
DO - 10.1007/s00203-007-0276-z
M3 - Article
C2 - 17639350
AN - SCOPUS:36549045853
SN - 0302-8933
VL - 188
SP - 551
EP - 563
JO - Archives of Microbiology
JF - Archives of Microbiology
IS - 6
ER -