Simultaneous gene inactivation and promoter reporting in cyanobacteria

Abstract

Determining spatiotemporal gene expression and analyzing knockout mutant phenotypes have become powerful tools in elucidating the function of genes; however, genetic approaches for simultaneously inactivating a gene and monitoring its expression have not been reported in the literature. In this study, we designed a dual-functional gene knockout vector pZR606 that contains a multiple cloning site (MCS) for inserting the internal fragment of a target gene, with a gfp gene as its transcriptional marker located immediately downstream of the MCS. By using this gene knockout system, we inactivated ava_2679 from Anabaena variabilis ATCC 29413, as well as all2508, alr2887, alr3608, and all4388 from Anabaena sp. strain PCC 7120. The ava_2679 knockout mutant fails to grow diazotrophically. Morphological analysis of ava_2679 knockout mutant after nitrogen step-down revealed defective junctions between heterocysts and adjacent vegetative cells, and the heterocyst was 1.53-fold longer compared to wild-type heterocysts. The alr2887, all4388, and alr3608 mutant colonies turned yellow and showed lack of protracted growth when deprived of fixed nitrogen, consistent with the previous reports that alr2887, all4388, and alr3608 are Fox genes. The all2508 encodes a GTP-binding elongation factor (EF4/LepA), and its knockout mutant exhibited reduced diazotrophic growth. The heterocyst development of all2508 knockout was significantly delayed, and only about 4.0 % of vegetative cells differentiated to heterocysts after nitrogen deprivation for 72 h, decreased 49.6 % compared to wild-type. Thus, we discovered that All2508 may regulate heterocyst development spatiotemporally. Concurrently, the GFP reporter revealed that all five target gene expressions were up-regulated in response to nitrogen deprivation. We demonstrated that the pZR606-based specific gene knockout approach worked effectively for the five selected genes, including four previously identified Fox genes or Fox gene homolog, and a previously unknown function of gene all2508. Thus, gene expression and phenotypic analysis of mutants can be achieved simultaneously by targeted gene inactivation using the pZR606-based system. This combined approach for targeted gene inactivation and its promoter reporting with GFP may be broadly applicable to the study of gene function in other prokaryotic organisms.

Molecular genetic improvements of cyanobacteria to enhance the industrial potential of the microbe: A review

“…Certain characteristics of cyanobacteria, such as the fact that many strains contain multiple chromosomes per cell (i.e., S. elongatus 7942 63 and Anabaena 7120 64 ), can make gene transfer more difficult in cyanobacteria compared with other bacteria. 63,65,66 Typically, DNA transfer in filamentous cyanobacteria is achieved via conjugation [67][68][69][70][71][72][73][74][75][76] while in unicellular cyanobacteria, natural transformation and electroporation are commonly used techniques. [77][78][79][80][81][82][83] Comprehension of genetic tools available for specific cyanobacterial strains is crucial for any genetic engineering study.…”

Characterization of five putative aspartate aminotransferase genes in the N2-fixing heterocystous cyanobacterium Anabaena sp. strain PCC 7120

“…The resultant expression plasmids bearing the different target genes were transformed into BL21(DE3) for recombinant protein production. For target gene inactivation and promoter reporting in Anabaena, pZR606 (Chen et al, 2015) created by introducing multiple cloning sites into pRL2726 (Zhou & Wolk, 2002) was used. The pZR606-based cargo plasmid bearing the internal fragment of target gene was transferred into NEB 10b carrying the conjugal plasmid pRL443 and helper plasmid pRL623 (Elhai et al, 1997).…”

“…3a). First, the internal fragment of each putative AspATs was PCR amplified and cloned into an integration vector, pZR606 (Chen et al, 2015), to produce cargo plasmids (Table 1). Following conjugal transfer of each cargo plasmid from E. coli into Anabaena and single-crossover recombination, the target gene was interrupted by producing 39 deleted and 59 deleted copies of that target gene (Fig.…”

Bridging Nature and Engineering: Protein-Derived Materials for Bio-Inspired Applications

“…To genetically engineer N 2 -fixing cyanobacteria to produce a high yield of cyanophycin, Zhou’s lab will create a PII mutant of Anabaena sp. PCC 7120 using previously developed molecular genetic approaches [ 239 , 259 , 260 , 261 , 262 , 263 , 264 , 265 ].…”