PhoH2 proteins are highly conserved across bacteria and archaea yet their biological function is poorly characterised. We examined the growth profiles of Mycobacterium smegmatis strains mc 2 155 and mc 2 155 ΔphoH2 and observed the same growth profile and growth rate in a variety of conditions. In light of the comparable growth, we used RNAseq to provide a snapshot of the differences between the transcriptomes of M. smegmatis mc 2 155 and M. smegmatis mc 2 155 ΔphoH2 during normal growth. At 48 hours, elevated expression of the sigF regulon was observed in ΔphoH2 relative to wild type. In biochemical assays, PhoH2 showed activity toward sigF mRNA insinuating a role of PhoH2 in modulating the pool of sigF mRNA in the cell during normal growth, adding further complexity to the repertoire of reported mechanisms of post-translational regulation. Multiple copies of the preferred target site of PhoH2 were identified in loops of the sigF mRNA structure, leading us to propose a mechanism for the activity of PhoH2 that is initiated after assembly on specific singlestranded loops of RNA. We hypothesise that PhoH2 is a toxin-antitoxin that contributes to the regulation of SigF at a post-transcriptional level through targeted activity on sigF mRNA. This work presents the first evidence for post-transcriptional regulation of SigF along with the biological function of PhoH2 from M. smegmatis. This has implications for the highly conserved PhoH2 toxin-antitoxin module across the mycobacteria including the important human pathogen M. tuberculosis.

Microbiota inhabiting the Dry Valleys of Antarctica are subjected to multiple stressors that can damage deoxyribonucleic acid (DNA) such as desiccation, high ultraviolet light (UV) and multiple freeze-thaw cycles. To identify novel or highly-divergent DNA-processing enzymes that may enable effective DNA repair, we have sequenced metagenomes from 30 sample-sites which are part of the most extensive Antarctic biodiversity survey undertaken to date. We then used these to construct wide-ranging sequence similarity networks from protein-coding sequences and identified candidate genes involved in specialized repair processes including unique nucleases as well as a diverse range of adenosine triphosphate (ATP) -dependent DNA ligases implicated in stationary-phase DNA repair processes. In one of the first direct investigations of enzyme function from these unique samples, we have heterologously expressed and assayed a number of these enzymes, providing insight into the mechanisms that may enable resident microbes to survive these threats to their genomic integrity.

DNA ligases, essential enzymes which re-join the backbone of DNA come in two structurallydistinct isoforms, NAD-dependent and ATP-dependent, which differ in cofactor usage. The present view is that all bacteria exclusively use NAD-dependent DNA ligases for DNA replication, while archaea and eukaryotes use ATP-dependent DNA ligases. Some bacteria also possess auxiliary ATP-dependent DNA ligases; however, these are only employed for specialist DNA repair processes. Here we show that in the genomes of high-light strains of the marine cyanobacterium Prochlorococcocus marinus, an ATP-dependent DNA ligase has replaced the NAD-dependent form, overturning the present paradigm of a clear evolutionary split in ligase usage. Genes encoding partial NAD-dependent DNA ligases are found on mobile regions in highlight genomes and lack domains required for catalytic function. This constitutes the first reported example of a bacterium that relies on an ATP-dependent DNA ligase for DNA replication and recommends P. marinus as a model to investigate the evolutionary origins of these essential DNA-processing enzymes.

PhoH2 proteins are highly conserved across bacteria and archaea yet their biological function is poorly characterised. We examined the growth profiles of Mycobacterium smegmatis strains mc 2 155 and mc 2 155 ΔphoH2 and observed the same growth profile and growth rate in a variety of conditions. In light of the comparable growth rates, we used RNAseq to provide a snapshot of the differences between the transcriptomes of M. smegmatis mc 2 155 and M. smegmatis mc 2 155 ΔphoH2 during normal growth. At 48 hours, elevated expression of the sigF regulon and its predicted regulatory cascade was observed in ΔphoH2 relative to wild type. In biochemical assays, PhoH2 showed specific activity toward sigF mRNA insinuating a role of PhoH2 in modulating the pool of sigF mRNA in the cell during normal growth, adding further complexity to the repertoire of reported mechanisms of post-translational regulation. Multiple copies of the preferred target site of PhoH2 were identified in loops of the sigF mRNA structure, leading us to propose a mechanism for the activity of PhoH2 that is initiated after assembly on specific single-stranded loops of RNA. We hypothesise that PhoH2 is a toxin-antitoxin that contributes to the regulation of SigF at a post-transcriptional level through targeted activity on sigF mRNA. This work presents the first evidence for post-transcriptional regulation of SigF along with the biological function of PhoH2 from M. smegmatis. This also has implications for the highly conserved PhoH2 toxin-antitoxin module across the mycobacteria including the important human pathogen M. tuberculosis.