Biology subjects

Pullela, K.

Publications and source records attributed to Pullela, K..

3 recordsLinked to original sources

bioRxiv2021

YhcB coordinates peptidoglycan and LPS biogenesis with phospholipid synthesis during Escherichia coli cell growth.

The cell envelope is essential for viability in all kingdoms 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 most well 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 complete 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 {Delta}yhcB background. Subsequent analyses suggest that YhcB sits at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.

microbiology↗

bioRxiv2021

DpaA detaches Braun's lipoprotein from peptidoglycan

Gram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Brauns lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB and LdtC. LdtD and LdtE are members of the same family of LD-transpeptidases but they catalyse a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF remains unclear, although it has been shown to become essential in cells with inhibited LPS export to the outer membrane. We now show that LdtF hydrolyses the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product down-regulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated. IMPORTANCEGram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Brauns lipoprotein (Lpp) is the most abundant protein in E. coli and about one third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from PG and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.

microbiology↗

bioRxiv2020

Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

Protein acylation is critical for many cellular functions including signal transduction, cell division and development. In bacteria, such lipoproteins have important roles in virulence and are therefore potential targets for the development of novel antimicrobials and vaccines. To date, all known bacterial lipoproteins are secreted from the cytosol via the Sec pathway, acylated on an N-terminal cysteine residue through the action of Lgt, Lsp and Lnt, and then targeted to the appropriate cellular location. In the case of Gram-negative bacteria, the lipoprotein trafficking Lol pathway transports the lipoproteins to the outer membrane where most substrate molecules are retained within the cell. Here we identify a new secretion pathway that displays the substrate lipoprotein on the cell surface. We demonstrate that the previously identified E. coli Aat secretion system is a composite system that shares similarity with type I secretion systems and elements of the Lol pathway. Remarkably, during secretion by the Aat system, the AatD subunit acylates the substrate CexE on a highly conserved N-terminal glycine residue (rather than the canonical cysteine). Mutations in AatD or CexE that disrupt glycine acylation interfere with membrane incorporation and trafficking. Our data suggest that CexE is the first member of a new class of glycine-acylated bacterial lipoprotein, while Aat represents a new secretion system that we propose be defined as a lipoprotein secretion system (LSS).

microbiology↗