Developmentally Regulated Genome Editing in Terminally Differentiated N₂-Fixing Heterocysts ofAnabaena cylindricaATCC 29414

Abstract

1Some vegetative cells of Anabaena cylindrica are programed to differentiate semi-regularly spaced, 2 single heterocysts along filaments. Since heterocysts are terminally differentiated non-dividing 3 cells, with the sole known function for solar-powered N2-fixation, is it necessary for a heterocyst 4 to retain the entire genome (7.1 Mbp) from its progenitor vegetative cell? By sequencing the 5 heterocyst genome, we discovered and confirmed that at least six DNA elements (0.12 Mbp) are 6 deleted during heterocyst development. The six-element deletions led to the restoration of five 7 genes (nifH1, nifD, hupL, primase P4 and a hypothetical protein gene) that were interrupted in 8 vegetative cells. The deleted elements contained 172 genes present in the genome of vegetative 9 cells. By sequence alignments of intact nif genes (nifH, nifD and hupL) from N2-fixing 10 cyanobacteria (multicellular and unicellular) as well as other N2-fixing bacteria (non-11 cyanobacteria), we found that interrupted nif genes all contain the conserved core sequences that 12 may be required for phage DNA insertion. Here, we discuss the nif genes interruption which 13 uniquely occurs in heterocyst-forming cyanobacteria. To our best knowledge, this is first time to 14 sequence the genome of heterocyst, a specially differentiated oxic N2-fixing cell. This research 15 demonstrated that (1) different genomes may occur in distinct cell types in a multicellular 16 bacterium; and (2) genome editing is coupled to cellular differentiation and/or cellular function in 17 a heterocyst-forming cyanobacterium. 18 19 Keywords 20 cyanobacteria, heterocysts, oxic nitrogen fixation, genome editing, phage DNA insertion, 21 multicellular bacterium 22 Bioinformatics analysis. Reads trimming, assembly and mapping were carried out using CLC 132 Genomics Workbench 10.1.1 (Qiagen). Trimming was carried out using a Q of 20 as the cutoff, 133 eliminating any read with any ambiguous nucleotide and removing the 5 and 15 terminal 134 nucleotides in the 3' and 5' ends respectively. Assembly of the trimmed reads was accomplished 135 by setting an arbitrary minimum contig length of 3,000 bp and using the automated function to 136 select a word size of 23 and a bubble size of 50; finally, reads were mapped (mismatch cost: 2, 137 insertion cost: 3, deletion cost: 3, minimum length fraction: 0.5 and minimum similarity fraction:

Molecular circuit of heterocyst differentiation in cyanobacteria

“…It is clear that genome editing is coupled to cellular differentiation in heterocyst-forming cyanobacteria. It is reported that at least six DNA elements (about 0.12 Mbp) are deleted, leading to the restoration of five genes (nifH1, nifD, hupL, primase P4, and a hypothetical protein gene) in a heterocyst along with loss of function of 172 genes in Anabaena cylindrica [38].…”