Brunson, D. N. | Search bioRxiv

bioRxiv2023

Optimized replication of arrayed bacterial mutant libraries increase access to biological resources

Biological collections, including arrayed libraries of single transposon or deletion mutants, greatly accelerate the pace of bacterial genetics research. Despite the importance of these resources, few protocols exist for the replication and distribution of these materials. Here, we describe a protocol for creating multiple replicates of an arrayed bacterial Tn library consisting of approximately 6,800 mutants in 73 x 96-well plates. Our protocol provides multiple checkpoints to guard against contamination and minimize genetic drift caused by freeze/thaw cycles. This approach can also be scaled for arrayed culture collections of various sizes. Overall, this protocol is a valuable resource for other researchers considering the construction and distribution of arrayed culture collection resources for the benefit of the greater scientific community. ImportanceArrayed mutant collections drive robust genetic screens, yet few protocols exist for replication of these resources and subsequent quality control. Increasing distribution of arrayed biological collections will increase accessibility to and use of these resources. Developing standardized techniques for replication of these resources is essential for ensuring their quality and usefulness to the scientific community.

microbiology↗

bioRxiv2022

Identification of Multiple Iron Uptake Mechanisms in Enterococcus faecalis and Their Relationship to Virulence

Among the unfavorable conditions bacteria encounter within the host is restricted access to essential trace metals such as iron. To overcome iron deficiency, bacteria deploy multiple strategies to scavenge iron from host tissues with abundant examples of iron acquisition systems being implicated in bacterial pathogenesis. Yet, the mechanisms utilized by the major nosocomial pathogen Enterococcus faecalis to maintain intracellular iron balance are poorly understood. In this report, we conducted a systematic investigation to identify and characterize the iron acquisition mechanisms of E. faecalis and to determine their contribution to virulence. Bioinformatic analysis and literature surveys revealed that E. faecalis possesses three conserved iron uptake systems. Through transcriptomics, we discovered two novel ABC-type transporters that mediate iron uptake. While inactivation of a single transporter had minimal impact on the ability of E. faecalis to maintain iron homeostasis, inactivation of all five systems ({Delta}5Fe strain) disrupted intracellular iron homeostasis and considerably impaired cell growth under iron-deficiency. Virulence of the {Delta}5Fe strain was generally impaired in different animal models but showed niche-specific variations in mouse models, leading us to suspect that heme can serve as an iron source to E. faecalis during mammalian infections. Indeed, heme supplementation restored growth of {Delta}5Fe under iron-depletion and virulence in an invertebrate infection model. Collectively, this study reveals that the collective contribution of five iron transporters promotes E. faecalis virulence and that the ability to acquire and utilize heme as an iron source is critical to the systemic dissemination of E. faecalis.

microbiology↗