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

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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.

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Abbreviations

BAC:

Bacterial artificial chromosome

Cm:

Chloramphenicol

Em:

Erythromycin

Gm:

Gentamycin

Km:

Kanamycin

MCS:

Multiple cloning site (polylinker)

Nm:

Neomycin

ORF:

Open reading frame

PCC:

Pasteur Culture Collection

r :

Resistant

s :

Sensitive

Sm:

Streptomycin

Sp:

Spectinomycin

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Acknowledgments

We thank W. J. Buikema (U. Chicago) for Lorist6, W. Reznikoff (U. Wisconsin) for pRZ1107, Haixia He and Yi Li for skilled technical assistance, and S. Tabata and T. Kaneko for sequencing clones that we used for complementation. This work was supported by National Science Foundation grant MCB0090232 and United States Department of Energy grant DOE-FG02-91ER20021. T. Kuritz wishes it to be stated that Oak Ridge National Laboratory is managed for the US Department of Energy by UT-Battelle, LLC under the contract DE-AC05-00OR22725.

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Author notes

  1. Qing Fan

    Present address: Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Chicago, IL, 60611-3008, USA

  2. Ruanbao Zhou

    Present address: College of Life Sciences, Anhui Normal University, Wuhu, Anhui, 241000, People’s Republic of China

  3. Guocun Huang

    Present address: Department of Physiology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX, 75390, USA

  4. Sigal Lechno-Yossef

    Present address: Department of Chemistry, Michigan State University, East Lansing, MI, 48824-1312, USA

  5. Tanya Kuritz

    Present address: Chemical Sciences Division, Oak Ridge National Laboratory, MS-6194, Oak Ridge, TN, 37830, USA

Authors and Affiliations

  1. MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI, 48824-1312, USA

    C. Peter Wolk, Qing Fan, Ruanbao Zhou, Guocun Huang, Sigal Lechno-Yossef, Tanya Kuritz & Elizabeth Wojciuch

  2. Department of Plant Biology, Michigan State University, East Lansing, MI, 48824-1312, USA

    C. Peter Wolk

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  1. C. Peter Wolk
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Correspondence to C. Peter Wolk.

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Communicated by Jack Meeks.

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Wolk, C.P., Fan, Q., Zhou, R. et al. Paired cloning vectors for complementation of mutations in the cyanobacterium Anabaena sp. strain PCC 7120. Arch Microbiol 188, 551–563 (2007). https://doi.org/10.1007/s00203-007-0276-z

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  • DOI: https://doi.org/10.1007/s00203-007-0276-z

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