Loss of YhcB results in dysregulation of coordinated peptidoglycan, LPS and phospholipid synthesis during Escherichia coli cell growth

Abstract

The cell envelope is essential for viability in all domains of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the best studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole-genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies, we report the comprehensive genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a ΔyhcB background. Subsequent analyses suggest that YhcB functions at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.

A New Factor LapD Is Required for the Regulation of LpxC Amounts and Lipopolysaccharide Trafficking

“…However, the molecular basis of such a lethality remained unknown. Although LapD has recently been implicated in cell division or the maintenance of cell envelope homeostasis, its function has remained unknown [ 36 , 37 ].…”

“…Our studies do not rule out a direct link between LapD and cell division machinery; however, our suppressor approach clearly shows suppressors that either increase LpxC amounts (mutations in lpxC , ftsH and lapB ) or enhance LPS translocation ( msbA suppressor mutations) support direct participation of LapD in LPS assembly. Another study using whole genome transposon mutagenesis approaches also proposes that LapD (YhcB) functions at the junction of several envelope biosynthetic pathways including peptidoglycan biogenesis [ 36 ]. Some phenotypes, such as defects in biofilm formation of Δ lapD mutant bacteria [ 72 ], can be explained as an indirect consequence of the alteration in LPS amounts.…”

“…We have previously shown that the lapD gene is required for the growth of E. coli at critical high temperatures [ 34 ]. LapD (YhcB) is an inner membrane protein and has recently been implicated in either the cell division process or the cell envelope homeostasis; although, molecular mechanisms in either of these functions remain unknown [ 35 , 36 , 37 , 38 ]. In this work, we show that in the absence of LapD, LpxL and LpxM acyl transferases become essential and the synthetic lethality of either Δ( lpxL lapD ) or Δ( lpxM lapD ) can be overcome by extragenic suppressor mutations mapping to the essential msbA gene ( Figure 2 B).…”

TraDIS-validate: a method for curating ordered gene-replacement libraries

“…A limitation of this method is that it only informs whether a mutant is present within the total pool of mutants, the nature of the method cannot distinguish whether the mutant of a given well is correct; further validation checks are required to test this. One caveat to consider is that the presence of a correctly positioned kanamycin resistance cassette is not sufficient evidence for the correct construction of a mutant, as is seen with yciM, yhcB, hda and holD mutants (Mahalakshmi et al 2014; Goodall et al 2021). In each of these examples, the kanamycin cassette is in the correct location, but secondary mutations have been reported elsewhere in the genome.…”

“…One caveat to consider is that the presence of a correctly positioned kanamycin resistance cassette is not sufficient evidence for the correct construction of a mutant, as is seen with yciM, yhcB, hda and holD mutants Goodall et al 2021). In each of these examples, the kanamycin cassette is in the correct location, but secondary mutations have been reported elsewhere in the genome.…”