Extracting high-molecular weight DNA from cyanobacteria using Promega's WizardⓇ HMW DNA extraction kit with a modified protocol, METIS.

Extraction of high molecular weight (HMW) DNA for long read sequencing with little to no fragmentation and high purity is difficult to acquire from cyanobacterial species. Here we describe a modified method of extraction using Promega's WizardⓇ HMW DNA Extraction Kit to acquire high molecular weight DNA from two cyanobacterial species. The protocol used in the kit is the "3.D. Isolating HMW DNA from Gram-Positive and Gram-Negative Bacteria" protocol. During a key step in the protocol, we propose that the lingering remnants of the cellular debris such as the mucilage layer of the cyanobacterial species is removed, preventing it from sticking to the DNA pellet produced. This customized modification is done between steps 11 and 12 and called METIS (maximizing extraction, transfer isopropanol step). This step drastically reduces the remaining mucilage layer, which if kept will stick to the DNA and make the DNA unsuitable for sensitive downstream next generation sequencing, like PacBio Sequencing. This protocol has been used to assemble two genomes from cyanobacteria (Synechococcus sp. and Microcystis aeruginosa) and one from a gram-negative bacterium, Lacibacter. It also allows for HMW DNA to be rapidly extracted without the use of toxic chemicals such as phenol and without extra reagents to be purchased.•Maximizing extraction, transfer isopropanol step (METIS) is the key modification during the step of DNA unraveling•METIS reduces leftover remnants of the mucilage layer in the extraction•High molecular weight DNA is produced with little to no fragmentation, and both a high purity and concentration.

Sound the (Smaller) Alarm: The Triphosphate Magic Spot Nucleotide pGpp.

It has recently become evident that the bacterial stringent response is regulated by a triphosphate alarmone (pGpp) as well as the canonical tetra- and pentaphosphate alarmones ppGpp and pppGpp [together, (p)ppGpp]. Often dismissed in the past as an artifact or degradation product, pGpp has been confirmed as a deliberate endpoint of multiple synthetic pathways utilizing GMP, (p)ppGpp, or GDP/GTP as precursors. Some early studies concluded that pGpp functionally mimics (p)ppGpp and that its biological role is to make alarmone metabolism less dependent on the guanine energy charge of the cell by allowing GMP-dependent synthesis to continue when GDP/GTP has been depleted. However, recent reports that pGpp binds unique potential protein receptors and is the only alarmone synthesized by the intestinal pathogen Clostridioides difficile indicate that pGpp is more than a stand-in for the longer alarmones and plays a distinct biological role beyond its functional overlap (p)ppGpp.