Next-generation microbiology : from comparative genomics to gene function

Microbiology is at a turning point in its 120-year history. Widespread next-generation sequencing has revealed genetic complexity among bacteria that could hardly have been imagined by pioneers such as Pasteur, Escherich and Koch. This data cascade brings enormous potential to improve our understanding of individual bacterial cells and the genetic basis of phenotype variation. However, this revolution in data science cannot replace established microbiology practices, presenting the challenge of how to integrate these new techniques. Contrasting comparative and functional genomic approaches, we evoke molecular microbiology theory and established practice to present a conceptual framework and practical roadmap for next-generation microbiology

Regulation of heterologous subtilin production in Bacillus subtilis W168

Background Subtilin is a peptide antibiotic (lantibiotic) natively produced by Bacillus subtilis ATCC6633. It is encoded in a gene cluster spaBTCSIFEGRK (spa-locus) consisting of four transcriptional units: spaS (subtilin pre-peptide), spaBTC (modification and export), spaIFEG (immunity) and spaRK (regulation). Despite the pioneer understanding on subtilin biosynthesis, a robust platform to facilitate subtilin research and improve subtilin production is still a poorly explored spot. Results In this work, the intact spa-locus was successfully integrated into the chromosome of Bacillus subtilis W168, which is the by far best-characterized Gram-positive model organism with powerful genetics and many advantages in industrial use. Through systematic analysis of spa-promoter activities in B. subtilis W168 wild type and mutant strains, our work demonstrates that subtilin is basally expressed in B. subtilis W168, and the transition state regulator AbrB strongly represses subtilin biosynthesis in a growth phase-dependent manner. The deletion of AbrB remarkably enhanced subtilin gene expression, resulting in comparable yield of bioactive subtilin production as for B. subtilis ATCC6633. However, while in B. subtilis ATCC6633 AbrB regulates subtilin gene expression via SigH, which in turn activates spaRK, AbrB of B. subtilis W168 controls subtilin gene expression in SigH-independent manner, except for the regulation of spaBTC. Furthermore, the work shows that subtilin biosynthesis in B. subtilis W168 is regulated by the two-component regulatory system SpaRK and strictly relies on subtilin itself as inducer to fulfill the autoregulatory circuit. In addition, by incorporating the subtilin-producing system (spa-locus) and subtilin-reporting system (PpsdA-lux) together, we developed “online” reporter strains to efficiently monitor the dynamics of subtilin biosynthesis. Conclusions Within this study, the model organism B. subtilis W168 was successfully established as a novel platform for subtilin biosynthesis and the underlying regulatory mechanism was comprehensively characterized. This work will not only facilitate genetic (engineering) studies on subtilin, but also pave the way for its industrial production. More broadly, this work will shed new light on the heterologous production of other lantibiotics

Loss of Pde1 function acts as an evolutionary gateway to penicillin resistance in Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen and rising resistance to β-lactam antibiotics, such as penicillin, is a significant threat to global public health. Mutations occurring in the penicillin-binding proteins (PBPs) can confer high-level penicillin resistance but other poorly understood genetic factors are also important. Here, we combined strictly controlled laboratory experiments and population analyses to identify a new penicillin resistance pathway that is independent of PBP modification. Initial laboratory selection experiments identified high-frequency pde1 mutations conferring S. pneumoniae penicillin resistance. The importance of variation at the pde1 locus was confirmed in natural and clinical populations in an analysis of >7,200 S. pneumoniae genomes. The pde1 mutations identified by these approaches reduce the hydrolytic activity of the Pde1 enzyme in bacterial cells and thereby elevate levels of cyclic-di-adenosine monophosphate and penicillin resistance. Our results reveal rapid de novo loss of function mutations in pde1 as an evolutionary gateway conferring low-level penicillin resistance. This relatively simple genomic change allows cells to persist in populations on an adaptive evolutionary pathway to acquire further genetic changes and high-level penicillin resistance