The Genome Copy Number of the Thermophilic Cyanobacterium Thermosynechococcus elongatus E542 Is Controlled by Growth Phase and Nutrient Availability

Abstract

Recently isolated thermophilic cyanobacterium Thermosynechococcus E542 is a promising strain for the fundamental and applied research. Here, we used several improved ploidy estimation approaches, which include qPCR, spectrofluorometry and flow cytometry to precisely determine the ploidy level in Thermosynechococcus E542 across different growth stages, nutritional, and stress conditions. The distribution of genome copies per cell among the populations of Thermosynechococcus E542 was also analysed. The strain tends to maintain 3 to 4 genome copies per cell in lag phase, early growth phase or stationary phase under standard conditions. Increased ploidy (5.5 ± 0.3) was observed in exponential phase; hence ploidy level is growth phase regulated. Nearly no monoploid cells were detected in all growth phases, and prolonged stationary phase could not yield ploidy level lower than 3 under standard conditions. During the late growth phase, significantly higher ploidy level was observed in the presence of bicarbonate (7.6 ±0.7) and high phosphate (6.9 ±0.2) at the expense of the reduced percentage of di- and triploid cells. Meanwhile, the reduction in phosphates decreased the average ploidy level by increasing the percentage of mono- and diploid cells. In contrast, temperature and antibiotic stress reduced the percentage of mono-, di and triploid cells, yet maintaining average ploidy. The results indicate a possible causality between growth rate, stress and genome copy number across the conditions tested, but the exact mechanism is yet to be elucidated. Furthermore, the spectrofluorometric approach presented here is a quick and straightforward ploidy estimation method with reasonable accuracy. Importance Present study revealed the genome copy number (ploidy) status in thermophilic cyanobacterium Thermosynechococcus E542 is regulated by growth phase and various environmental parameters to give us a window into understanding the role of polyploidy. Increased ploidy level is found associated with the higher metabolic activity and increased vigor by acting as backup genetic information to compensate for damage to the other chromosomal copies. Several improved ploidy estimation approaches are presented in this work that may upgrade the ploidy estimation procedure for cyanobacteria in the future. Furthermore, a new spectrofluorometric method presented here is a rapid and straightforward method of ploidy estimation with reasonable accuracy compared to other laborious methods.

Debottlenecking Thermophilic Cyanobacteria Cultivation and Harvesting through the Application of Inner-Light Photobioreactor and Chitosan

“…Thermosynechococcaceae , including the E542 strain [ 29 ], are typically less than 1 μm in diameter and 2–3 μm in length, sizes typical for picocyanobacteria. This is the main reason why their harvesting is particularly challenging.…”

Generation of miniploid cells and improved natural transformation procedure for a model cyanobacterium Synechococcus elongatus PCC 7942

“…The distribution of genome copy numbers in populations of Synechococcus 7942 was determined using flow cytometry as described previously ( Pope et al, 2020 ; Riaz et al, 2021 ). Briefly, around 5 × 10 7 cells were collected from each test condition during the mid to late growth phase (OD 730 : 0.4–0.5) by centrifugation and washed two times with PBS.…”

“…Analysis of the data was performed on FlowJo v.10. To analyse the ploidy level by spectrofluorometer, the DNA amount was quantified using the Quant-IT dsDNA HS Assay kit (Invitrogen, Carlsbad, CA, United States) as described before ( Riaz et al, 2021 ). Briefly, 5 × 10 7 cells were collected by centrifugation and resuspended in distilled water to obtain a final OD 730 of 0.2.…”

“…The ploidy level of cyanobacteria is highly variable and influenced by the growth phase, growth rate, nutritional conditions, and stress (Zerulla et al, 2016;Ohbayashi et al, 2019;Pope et al, 2020;Riaz et al, 2021). Most cyanobacteria, including Synechococcus 7942, possess multiple genome copies irrespective of the growth rate or condition (Griese et al, 2011;Ohbayashi et al, 2019).…”

Molecular Components of Nitrogen Fixation Gene Cluster and Associated Enzymatic Activities of Non-Heterocystous Thermophilic Cyanobacterium Thermoleptolyngbya sp.

“…The increased sequence pool allows for a better understanding of nitrogen fixation in the thermal niches [ 16 ]. Simultaneously, as new cyanobacterial strains are isolated from thermal sites worldwide [ 17 ], and their genetic modification systems are being developed [ 18 , 19 ], experimental validation of sequence data is also increasingly possible. Interestingly, complete nitrogen fixation gene clusters have been identified in the genomes of Thermoleptolyngbya , which could be particularly interesting considering their ability to thrive in thermal environments.…”