High-throughput sequencing of sorted expression libraries reveals inhibitors of bacterial cell division

Abstract

BackgroundBacterial filamentation occurs when rod-shaped bacteria grow without dividing. To identify genetically encoded inhibitors of division that promote filamentation, we used cell sorting flow cytometry to enrich filamentous clones from an inducible expression library, and then identified the cloned DNA with high-throughput DNA sequencing. We applied the method to an expression library made from fragmented genomic DNA of uropathogenic E. coli UTI89, which undergoes extensive reversible filamentation in urinary tract infections and might encode additional regulators of division.ResultsWe identified 55 genomic regions that reproducibly caused filamentation when expressed from the plasmid vector, and then further localized the cause of filamentation in several of these to specific genes or sub-fragments. Many of the identified genomic fragments encode genes that are known to participate in cell division or its regulation, and others may play previously-unknown roles. Some of the prophage genes identified were previously implicated in cell division arrest. A number of the other fragments encoded potential short transcripts or peptides.ConclusionsThe results provided evidence of potential new links between cell division and distinct cellular processes including central carbon metabolism and gene regulation. Candidate regulators of the UTI-associated filamentation response or others were identified amongst the results. In addition, some genomic fragments that caused filamentation may not have evolved to control cell division, but may have applications as artificial inhibitors. Our approach offers the opportunity to carry out in depth surveys of diverse DNA libraries to identify new genes or sequences encoding the capacity to inhibit division and cause filamentation.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5187-7) contains supplementary material, which is available to authorized users.

Tunable transcription factor library for robust quantification of regulatory properties in Escherichia coli

“…For instance, some TFs such as Crp, ArgR, CysB, and MetJ are critical for essential metabolic pathways, and under‐producing or deleting these TFs may seriously affect the fitness of the corresponding strains under certain induction conditions. On the other hand, some TFs such as Nac and PdhR may be toxic when expressed at high concentrations (Mediati et al , 2018 ). The physiological effects of different TFs might be influenced by the growth media (for instance, in Appendix Fig S7 shows the impact of different carbon sources on the growth of the library strain expressing H‐NS).…”

The role of bacterial size, shape and surface in macrophage engulfment of uropathogenicE. colicells

“…FtsZ was produced (as a YFP fusion) from pLau80 [38] under P BAD control, in addition to endogenous FtsZ in UTI89. This induced strong filamentation [39] on addition of arabinose (herein referred to as FtsZ filaments) [40]. FtsZ filaments were 5.5-fold longer than their rod counterpart (11.4 μm ± 0.4 SEM vs. FtsZ rods; 2.1 μm ± 0.04 SEM; Fig 1A; Fig S1E-F) and were as viable as FtsZ rods (Fig S2C) but showed a moderate decrease (29%, p = 0.002) in ATP production compared to FtsZ rods (Fig S3C).…”

“…FtsZ was produced (as a YFP fusion) from pLau80 [38] under P BAD control, in addition to endogenous FtsZ in UTI89. This induced strong filamentation [39] on addition of arabinose (herein referred to as FtsZ filaments) [40]. FtsZ filaments were 5.5-fold longer than their rod counterpart (11.4 The combined results above demonstrate that the actual length of the UTI89 filament is at least one key factor in providing this protection.…”

Tunable Transcription Factor Library for Robust Quantification of Gene Expression Dynamics in E. coli

“…For instance, some TFs such as Crp, ArgR, CysB and MetJ are critical for essential metabolic pathways and under-producing or deleting these TFs may seriously affect the fitness of the corresponding strains under certain induction conditions. On the other hand, some TFs such as Nac and PdhR may be toxic when expressed at high concentrations 33 . Here, we will use growth rate in glucose minimal media as a proxy to evaluate the fitness or physiological effects due to the titration of each TF.…”