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Levoglucosan is produced in the pyrolysis of cellulose and starch, including from bushfires or the burning of biofuels, and is deposited from the atmosphere across the surface of the earth. We describe two levoglucosan degrading Paenarthrobacter spp. (Paenarthrobacter nitrojuajacolis LG01 and Paenarthrobacter histidinolovorans LG02) that were isolated from soil by metabolic enrichment using levoglucosan as the sole carbon source. Genome sequencing and proteomics analysis revealed the expression of a series of genes encoding known levoglucosan degrading enzymes, levoglucosan dehydrogenase (LGDH, LgdA), 3-keto-levoglucosan β -eliminase (LgdB1) and glucose 3-dehydrogenase (LgdC), along with an ABC transporter cassette and an associated solute binding protein. However, no homologues of 3-ketoglucose dehydratase (LgdB2) were evident, while the expressed genes contained a range of putative sugar phosphate isomerases/xylose isomerases with weak similarity to LgdB2. Sequence similarity network analysis of genome neighbours of LgdA revealed that homologues of LgdB1 and LgdC are generally conserved in a range of bacteria in the phyla Firmicutes, Actinobacteria and Proteobacteria. One group of sugar phosphate isomerase/xylose isomerase homologues (named LgdB3) was identified with limited distribution that is mutually exclusive with LgdB2, and we propose that they may fulfil a similar function. LgdB1, LgdB2 and LgdB3 adopt similar predicted 3D folds, suggesting overlapping function in processing intermediates in LG metabolism. Our findings highlight diversity within the LGDH pathway, through which bacteria utilize levoglucosan as a nutrient source.

Staphylococcus aureusinfections are associated with high mortality rates. Often considered an extracellular pathogen,S. aureuscan persist and replicate within host cells, evading immune responses and causing host cell death. Classical methods for assessingS. aureuscytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic diversity of intracellular bacteria. Using a well-established epithelial cell line model, we have developed a platform calledInToxSa(IntracellularToxicity ofS. aureus) to quantify intracellula cytotoxicS. aureusphenotypes. Studying a panel of 387S. aureusbacteraemia isolates, and combined with comparative, statistical and functional genomics, our platform identified mutations inS. aureusclinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations inausA,encoding the aureusimine non-ribosomal peptide synthetase, reducedS. aureuscytotoxicity and increased intracellular persistence.InToxSais a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevantS. aureuspathoadaptive mutations that promote intracellular residency.