Paired cloning vectors for complementation of mutations in the cyanobacterium Anabaena sp. strain PCC 7120

Abstract

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 (hetF A) of Anabaena sp. are required specifically for fixation of dinitrogen under oxic conditions.

Vibrio coralliilyticus Strain OCN008 Is an Etiological Agent of Acute Montipora White Syndrome

“…Prior to inoculation of corals, the RSF1010-derived vector pRL1383a was introduced into OCN008 through conjugation as previously described (11,33) to genetically tag the bacterium. Fragments infected by tagged OCN008 were crushed in FSW, and serial dilutions from 1:10 to 1:10 6 were plated on solid GASW supplemented with spectinomycin, resistance to which was carried by the plasmid.…”

A Major Facilitator Superfamily Protein, HepP, Is Involved in Formation of the Heterocyst Envelope Polysaccharide in the Cyanobacterium Anabaena sp. Strain PCC 7120

“…Complementation was performed using compatible vectors (63). Plasmid anp00110 is a sequencing clone (chromosomal bp 2047194 to 2056558) that contains all1711 and its neighboring genes cloned into the BamHI site of pUC18 (29).…”

Cyanobacterial defense mechanisms against foreign DNA transfer and their impact on genetic engineering

“…PCC 7524 are also able to replicate in cyanobacteria from subsections II, III and V (Billi et al, 2001, Ravindran et al, 2006, Stucken et al, 2012. Furthermore, broad host range RSF1010 (IncQ) based plasmids replicate in most subsection I species and in fi lamentous strains from subsections III-IV (Elhai and Wolk 1988, Mermet-Bouvier et al, 1993, Haitao and Xudong 2004, Tolonen et al, 2006, Wolk et al, 2007, Min and Sherman 2010, Taton et al, 2012.…”