Identification of Nostoc punctiforme akinete‐expressed genes using differential display

Abstract

SummaryAkinetes are spore-like resting cells formed by certain filamentous cyanobacteria that have increased resistance to environmental stress. They can be found at low frequencies in dense cultures experiencing low light or phosphate limitation, but also form at high frequencies in a zwf mutant strain of Nostoc punctiforme following dark incubation in the presence of fructose. The wild-type strain is capable of facultative heterotrophic growth under these conditions and does not form akinetes. To identify genes associated with akinete development, differential display was used to amplify and compare cDNA from a wild-type and zwf mutant strain of N. punctiforme following a switch to dark heterotrophic conditions. Screening of candidate genes by reverse transcriptase real-time quantitative PCR and subsequent testing for akinetespecific expression using GFP transcriptional reporter plasmids lead to the identification of three novel akinete-expressed genes. The genes identified from the screening encoded for proteins homologous to an aminopeptidase (aapN), a zinc protease (hap) and an ATP-binding cassette (ABC)-type transporter (aet). Expression of hap was also increased in developing hormogonia, a transient type of differentiated filament capable of gliding motility. Transcriptional start sites for akinete-expressed genes were determined using random amplification of cDNA ends (RACE), and promoter regions were compared with orthologues in other filamentous cyanobacteria to identify putative regulatory sequences.

Clear differences in metabolic and morphological adaptations of akinetes of two Nostocales living in different habitats

“…Here, we showed that low light was the best inducer of akinete formation in Anabaena variabilis and confirmed the earlier finding (Argueta & Summers, 2005;Argueta et al, 2006) that phosphate starvation was the best inducer in N. punctiforme. The different inducing conditions also had an effect on the time course of akinete development.…”

“…However, only a few genes involved in akinete formation have been so far identified (e.g. Argueta et al, 2006;Campbell et al, 1996;Leganés et al, 1994Leganés et al, , 1998Lehner et al, 2011;Zhou & Wolk, 2002; see also review by Maldener et al, 2014). Microarray analysis of a zwf mutant of N. punctiforme that is able to differentiate akinetes with similar phenotype characteristics to WT akinetes in a few days (Argueta & Summers, 2005) revealed the upregulation of 255 genes, 41 % of which encode known proteins (Campbell et al, 2007).…”

The Caulobacter crescentus ctrA P1 promoter is essential for the coordination of cell cycle events that prevent the overinitiation of DNA replication

“…RNA was isolated (Argueta et al, 2006) from exponentially growing NA1000 and SM921 in M2G broth. 59 RACE was performed as described by Argueta et al (2006).…”

“…RNA was isolated (Argueta et al, 2006) from exponentially growing NA1000 and SM921 in M2G broth. 59 RACE was performed as described by Argueta et al (2006). The following ctrA-specific primers were used: 59 RACE CtrA Rev, 59-CCGCAGGGTGCGCAGAACATCGAT-39; 59 RACE CtrA Nested Rev, 59-GCAGGATAAGATCGTAGTCGTAGAT-39.…”

(p)ppGpp modulates cell size and the initiation of DNA replication in Caulobacter crescentus in response to a block in lipid biosynthesis

“…The RNA (3 mg) was treated with DNase, and cleaned using RNA Clean & Concentrate (Zymo Research). RNA quality was checked by measuring the A 260 /A 280 ratio and running an RNA sample on a 0.8 % agarose gel (Argueta et al, 2006). Preparation of cDNA.…”