Gene transfer to the desiccation-tolerant cyanobacterium Chroococcidiopsis

Abstract

The coccoid cyanobacterium Chroococcidiopsis dominates microbial communities in the most extreme arid hot and cold deserts. These communities withstand constraints that result from multiple cycles of drying and wetting and/or prolonged desiccation, through mechanisms which remain poorly understood. Here we describe the first system for genetic manipulation of Chroococcidiopsis. Plasmids pDUCA7 and pRL489, based on the pDU1 replicon of Nostoc sp. strain PCC 7524, were transferred to different isolates of Chroococcidiopsis via conjugation and electroporation. This report provides the first evidence that pDU1 replicons can be maintained in cyanobacteria other than Nostoc and Anabaena. Following conjugation, both plasmids replicated in Chroococcidiopsis sp. strains 029, 057, and 123 but not in strains 171 and 584. Both plasmids were electroporated into strains 029 and 123 but not into strains 057, 171, and 584. Expression of P(psbA)-luxAB on pRL489 was visualized through in vivo luminescence. Efficiencies of conjugative transfer for pDUCA7 and pRL489 into Chroococcidiopsis sp. strain 029 were approximately 10(-2) and 10(-4) transconjugants per recipient cell, respectively. Conjugative transfer occurred with a lower efficiency into strains 057 and 123. Electrotransformation efficiencies of about 10(-4) electrotransformants per recipient cell were achieved with strains 029 and 123, using either pDUCA7 or pRL489. Extracellular deoxyribonucleases were associated with each of the five strains. Phylogenetic analysis, based upon the V6 to V8 variable regions of 16S rRNA, suggests that desert strains 057, 123, 171, and 029 are distinct from the type species strain Chroococcidiopsis thermalis PCC 7203. The high efficiency of conjugative transfer of Chroococcidiopsis sp. strain 029, from the Negev Desert, Israel, makes this a suitable experimental strain for genetic studies on desiccation tolerance.

FIG. 1

Typical result of spot matings using Chroococcidiopsis sp. strains 029, 057, 123, 171, and 584. Rows: A, mobilization of plasmid pDUCA7 using conjugative plasmid pK2013 in the presence of helper plasmid pRL528; B, mobilization of plasmid pDUCA7 using conjugative plasmid pK2013 in the absence of helper plasmid pRL528; C, presumptive transconjugants of Chroococcidiopsis cells bearing plasmid pRL489 obtained using conjugative plasmid pRL443 and helper plasmid pRL528; D, luminescence was observed in colonies (see Fig. 1C) obtained from strains 029, 057, and 123 and not in the areas where strains 171 and 584 were used as the recipients. Filters shown were collected after 30 days of incubation.

FIG. 2

Southern analysis of Chroococcidiopsis sp. strain 029 transconjugants (isolate CH91B1) obtained after mobilization of pDUCA7. The probe was labelled pDUCA7. Lanes: 1, 1-kb DNA ladder (LTI); 2, total DNA from Chroococcidiopsis sp. strain 029 (wild type); 3, total DNA from Chroococcidiopsis strain 029 isolate CH91B1; 4, total DNA from Chroococcidiopsis strain 029 isolate CH91B1 digested with PstI; 5, authentic pDUCA7 digested with PstI; 6, undigested pDUCA7 from E. coli DH10B transformed with plasmid DNA extracted from Chroococcidiopsis sp. strain 029 isolate CH91B1; 7, authentic pDUCA7.

FIG. 3

Electron micrograph of ultrathin section of 2-month-old Chroococcidiopsis strain 029 showing multicellular aggregate containing cells of different sizes. An electron-translucent layer (white) and a fibrillar electron-dense envelope of capsular polysaccharide (grey) surround the aggregate. Bar = 0.5 μm.

FIG. 4

Phylogenetic analysis of partial 16S rDNA. An unrooted consensus tree is shown. The numbers at the tree forks indicate the number of times out of 100 data sets that the strains to the right of the fork clustered (only numbers greater than 50 are shown).