Functional characterization of viral auxiliary metabolic genes involved in tetrapyrrole biosynthesis

Bacteriophages are key players in microbial ecosystems, often harboring auxiliary metabolic genes (AMGs) that influence host metabolism during infection. These AMGs are widely distributed in phage genomes and metagenomic sequences originating from phages, but their exact role remains enigmatic. This study explored AMGs from metagenomic datasets of diverse origins related to tetrapyrrole metabolism, a critical pathway for heme and pigment biosynthesis. Within this study, bioinformatic analysis of viral metagenomic data identified a three-gene cassette consisting of ho1, pcyA/X, and alaS genes. While viral ho1 and pcyX have been found before in marine datasets and encode a heme oxygenase and a ferredoxin-dependent bilin reductase for linear tetrapyrrole biosynthesis, the identification of alaS is intriguing. alaS encodes a putative aminolevulinic acid synthase (ALAS), an enzyme that catalyzes the first committed step in tetrapyrrole biosynthesis in alphaproteobacteria to produce aminolevulinic acid (ALA). This so-called C4 pathway is in contrast to the C5 pathway of ALA formation, which is present in all other bacteria and archaea. This is the first report of a phage-encoded alaS in a cassette with linear tetrapyrrole synthesis-associated AMGs (ho1, pcyA/X), suggesting a potential role in increasing the metabolic flux through the tetrapyrrole pathway. The cassette was detected in various metagenomic datasets derived from marine and freshwater habitats, but in freshwater, alaS was not associated directly with this cassette and could be found further up-/downstream on the contig. Using a combination of in silico structure prediction and amino acid sequence alignment, the viral alaS gene seems to be a functional C4 pathway enzyme that uses glycine and succinyl-CoA for ALA synthesis. This was proven by functional complementation using plasmid-encoded viral ALAS (vALAS) that successfully recovered the growth of an ALA-auxotrophic E. coli C5 pathway mutant. Furthermore, the specific enzymatic formation of ALA by vALAS in complemented cell lysate was verified marking the first time a viral ALAS is enzymatically active. Further, the remaining freshwater-derived AMGs were analyzed for their activity. The vHo1 showcases heme conversion to biliverdin IXα, verifying its role as an active heme oxygenase. Surprisingly, vPcyA produces phycocyanobilin (PCB), the chromophore that forms a functional phytochrome, thus verifying it as an active PcyA enzyme. This study elucidates the distribution and roles of these AMGs across various ecological niches, enhancing the understanding of phage-driven metabolic processes.