The Capsule Regulatory Network of Klebsiella pneumoniae Defined by density-TraDISort

Abstract

Capsule production is essential for K. pneumoniae to cause infections, but its regulation and mechanism of synthesis are not fully understood in this organism. We have developed and applied a new method for genome-wide identification of capsule regulators. Using this method, many genes that positively or negatively affect capsule production in K. pneumoniae were identified, and we use these data to propose an integrated model for capsule regulation in this species. Several of the genes and biological processes identified have not previously been linked to capsule synthesis. We also show that the methods presented here can be applied to other species of capsulated bacteria, providing the opportunity to explore and compare capsule regulatory networks in other bacterial strains and species.

A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impactKlebsiella pneumoniaefitness

“…The forward transposon screen identified six genes previously identified to support CPS production, including five hmv 0 hits ( wzi, wza, rmpA, kvrB , and pgi ) and one hmv low hit ( ompR ) ( Fig 2C ) [23, 24, 30]. These findings serve as an internal experimental validation, which provide confidence in our results obtained from screening the transposon library.…”

“…A recent study used density-TraDISort to identify K. pneumoniae transposon mutants with altered buoyancy as a surrogate for CPS production and hmv [23]. This work identified transposon mutants that increased NTUH-K2044 buoyancy, and mutants that decrease NTUH-K2044 and/or ATCC 43816 buoyancy [23]. We sought to integrate the results of the forward screen ( Fig 1–2 and Table 2 ) with these results by systematically exploring the hmv and CPS production of KPPR1 transposon mutants in genes identified to have altered buoyancy.…”

“…Despite CPS being a key fitness factor for K. pneumoniae , the regulatory network that directly controls CPS biosynthesis is not fully understood. A recent study reported the K. pneumoniae CPS biosynthesis regulatory network using density-TraDISort [23]. Transposon mutant pools were separated over a discontinuous Percoll gradient to separate populations with altered buoyancy as a surrogate measure of CPS production in two hvKp strains, NTUH-K2044 and ATCC 43816 [23].…”

“…A recent study reported the K. pneumoniae CPS biosynthesis regulatory network using density-TraDISort [23]. Transposon mutant pools were separated over a discontinuous Percoll gradient to separate populations with altered buoyancy as a surrogate measure of CPS production in two hvKp strains, NTUH-K2044 and ATCC 43816 [23]. Transposon insertions were identified that increase buoyancy in NTUH-K2044 or decrease buoyancy in NTUH-K2044 and/or ATCC 43816, then the hmv and CPS production of ten targeted deletion mutants were quantified to validate the density-TraDISort.…”

“…To validate the use of the library and more broadly examine the relationship between hmv and CPS overproduction, forward and reverse genetic screens were performed to systematically quantify both CPS production and hmv exhibited by transposon mutants. The use of a forward screen allowed for the unbiased identification of mutants that impact hmv and/or CPS production, while the reverse screen enabled methodical screening of CPS production and hmv for genes previously ascribed to affect K. pneumoniae buoyancy [23]. Global analyses of the CPS production and hmv of 100 transposon mutants and 27 targeted mutants revealed a significant correlation between the two biological features, although several mutants displayed discordant regulation of hmv and CPS biosynthesis.…”