Biology subjects

Morona, R.

Publications and source records attributed to Morona, R..

5 recordsLinked to original sources

bioRxiv2026

Genome-wide screen for genes required for smooth lipopolysaccharide production in Escherichia coli K-12

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria, contributing to membrane integrity and environmental interactions. Genome-wide studies defining bacterial gene functions have been extensively performed in the model strain Escherichia coli K-12 which lacks O-antigen (OAg), and therefore does not produce smooth LPS (S-LPS). Consequently, the genetic requirements for S-LPS production in this model system remain incompletely defined. Here, a functional wbbL gene was introduced into the E. coli K-12 KEIO single-gene deletion mutant library to restore OAg synthesis, enabling genome-wide analysis of S-LPS production by screening with colicin E2 (ColE2) and validated with LPS silver staining. This identified 319 mutants with increased sensitivity to ColE2 in the presence of OAg, suggesting broader envelope-associated effects during screening. In addition, 27 mutants showed defects in S-LPS production, corresponding to genes involved in OAg biosynthesis, LPS core and sugar precursor synthesis, OAg ligation and regulation, and enterobacterial common antigen biosynthesis. A further 18 mutants initially appeared defective in S-LPS production but could not be validated upon reconstruction, and whole-genome sequencing revealed secondary mutations responsible for the observed phenotypes. This study provides a validated genetic framework for S-LPS production in E. coli K-12 and highlights the importance of rigorous validation in genome-wide screening approaches.

microbiology↗

bioRxiv2025

Disruptions in Outer Membrane-Peptidoglycan Interactions Enhance Bile Salt Resistance in O-antigen-Producing E. coli

Bile salts (BS) are derived from cholesterol in the liver and act as antimicrobial agents in the intestines by disrupting bacterial cell membranes and inducing oxidative stress. The gut bacterium E. coli is naturally resistant to BS, including the model strain K12 that produces a truncated LPS without O-antigen (OAg). Paradoxically, restoring a wild-type like LPS with OAg sensitises E. coli K12 to exogenous BS. In this study, we investigate this phenomenon. We show that mutations causing truncation of the LPS core oligosaccharide render these strains even more susceptible to BS, similar to the mutant strain MG1655-S{Delta}waaL defective in OAg ligase, primarily due to the accumulation of the lipid-linked intermediate UndPP-OAg. Through the characterisation of BS-resistant suppressor mutants of MG1655-S{Delta}waaL, we identify key genetic disruptions involved in resistance. Notably, we observed the highest BS resistance in strains with a weaker connection between the outer membrane (OM) and peptidoglycan (PG), including strains lacking the major OM-anchored, PG-binding proteins OmpA or Lpp, or expressing versions of these that lack PG-binding. Our data suggest that BS-induced stress in OAg-producing E. coli is due to the spatial constraints between OM and PG, and that mutations disrupting OM-PG interactions alleviate this stress, enhancing BS resistance. These findings provide new insights into a major challenge E. coli faces in the gut environment where it needs to produce OAg for stable colonisation and resists BS. E. coli can only survive BS exposure by fine-tuning the connectivity between its cell envelope layers, which highlights a potential target for modulating bacterial responses to BS in the gut. Author summaryEnteric bacteria residing in the human gut must withstand the host-derived antimicrobial agents, bile salts (BS), but the underlying resistance mechanisms are not fully elucidated. This study investigates the BS resistance mechanisms in O-antigen (OAg)-producing Escherichia coli K-12. We show that truncation of lipopolysaccharide (LPS) core oligosaccharides or restoration of OAg production increases BS sensitivity due to the accumulation of UndPP-OAg intermediates. By analysing suppressor mutants, we identify key genetic disruptions, particularly affecting the level of outer membrane-peptidoglycan (OM-PG) interactions involving OmpA and Lpp, which confer heightened BS resistance. Our findings highlight how BS-induced stress is linked to spatial constraints between the OM and PG layer, offering new insights into bacterial adaptation to BS stress. This research may provide new targets for therapeutic interventions to modulate gut microbial responses to BS.

microbiology↗

bioRxiv2024

Tolerance Mechanisms in Polysaccharide Biosynthesis: Implications for Undecaprenol Phosphate Recycling in Escherichia coli and Shigella flexneri

Bacterial polysaccharide synthesis is catalysed on the universal lipid carrier, undecaprenol phosphate (UndP). The cellular UndP pool is shared by other polysaccharide synthesis pathways and in peptidoglycan (PG) biogenesis. Disruptions in cytosolic polysaccharide synthesis steps are detrimental to bacterial survival due to affecting UndP recycling. In contrast, bacteria can survive disruptions in the periplasmic steps, suggesting a tolerance mechanism to mitigate UndP sequestration. Here we investigated tolerance mechanisms to disruptions of polymerases that are involved in UndP-releasing steps in two related polysaccharide synthesis pathways: the enterobacterial common antigen (ECA) and the O antigen (OAg), in Escherichia coli and Shigella flexneri. Our study reveals that polysaccharide polymerisation is crucial for efficient UndP recycling. In E. coli K-12, cell survival upon disruptions in OAg polymerase is dependent on a functional ECA synthesis pathway and vice versa. This is because disruptions in OAg synthesis leads to the redirection of the shared lipid-linked sugar substrate UndPP-GlcNAc towards increased ECA production. Conversely, in S. flexneri, the OAg polymerase is essential due to its limited ECA production, which inadequately redirects UndP flow to support cell survival. We propose a model whereby sharing the initial sugar intermediate UndPP-GlcNAc between the ECA and OAg synthesis pathways allows UndP to be redirected towards ECA production, mitigating sequestration issues caused by disruptions in the OAg pathway. These findings suggest an evolutionary buffering mechanism that enhances bacterial survival when UndP sequestration occurs due to stalled polysaccharide biosynthesis, which may allow polysaccharide diversity in the species to increase over time. ImportanceEnzymes involved in bacterial polysaccharide biosynthesis have substrate specificity to ensure the correct polysaccharide is produced at the appropriate place and time. However, this specificity poses a challenge for the diversification of polysaccharide structure and hence function, as the acquisition of a novel oligosaccharide RU would likely disrupt the synthesis pathways, leading to sequestration of the essential universal lipid carrier UndP and ultimately cause cell death. We investigated how cells tolerate disruptions in polysaccharide synthesis pathways and provide evidence that suggests that sharing a common substrate between the synthesis pathways of two common enteric bacterial surface polysaccharides (ECA and OAg), can redirect the flow of UndP. Our study provides insights into the mechanism of how bacteria alleviate sequestration issues, thereby enhancing cell survival which may allow them additional capacity for polysaccharide diversification.

microbiology↗

bioRxiv2023

O antigen biogenesis sensitises Escherichia coli K-12 to bile salts, a likely cause for how it lost its O antigen

Escherichia coli K-12 is a model organism for bacteriology and has served as a workhorse for molecular biology and biochemistry for over a century since its first isolation in 1922. However, Escherichia coli K-12 strains are phenotypically devoid of an O antigen (OAg) since early reports in the scientific literature. Recent studies reported the presence of independent mutations that abolish OAg biogenesis in E. coli K-12 strains from the same original source, suggesting unknown evolutionary forces have selected for loss of OAg during the early propagation of K-12. Here, we show for the first time that restoration of OAg in E. coli K-12 strain MG1655 synergistically sensitises bacteria to vancomycin with bile salts (VBS). Suppressor mutants surviving lethal doses of VBS mostly contained disruptions in OAg biogenesis. We present data supporting a model where the transient presence and accumulation of lipid-carried OAg intermediates in the bacterial periplasm interfere with peptidoglycan synthesis, causing growth defects that are synergistically enhanced by bile salts. Lastly, we demonstrate that continuous bile salt exposure of OAg-producing MG1655 in the laboratory, can recreate a scenario where OAg disruption is selected for. Hence our work provides a likely explanation for the long-held mystery of how E. coli K-12 lost its OAg production and opens new avenues for exploring long-standing questions on the intricate network coordinating the synthesis of different cell envelope components in Gram-negative bacteria. Significance statementEscherichia coli K-12 is the most studied microorganism, widely used in laboratories for studying bacteriology and as a tool for molecular biology. The reason why it is devoid of O antigen remains a long-standing question. Our work has uncovered a previously unknown selection pressure of bile salts on bacterial O antigen biogenesis, which provides a plausible scenario for how the early propagation of E. coli K-12 strains in bile salt containing media could have led to loss of O antigen in K-12. Our results also suggest that the accumulation of O antigen intermediates in the bacterial periplasm may interfere with bacterial cell wall synthesis, which paves a new research direction into the interplay of different cell envelope component synthesis pathways.

microbiology↗

bioRxiv2022

Cysteine dependent conformation heterogeneity of Shigella flexneri autotransporter IcsA and implications in its function

Shigella IcsA is a versatile surface virulence factor required for both early and late pathogenesis stages, extracellularly to intracellularly. Despite IcsA serving as a model Type V secretion system (T5SS) autotransporter to study host pathogen interactions, its detailed molecular architecture is poorly understood. Recently, IcsA was found to switch to a different conformation for its adhesin activity upon sensing of the host stimuli by Shigella Type III secretion system (T3SS). Here, we report that the single cysteine residue (C130) near the N-terminus of IcsA passenger has a role in IcsA adhesin activity. We also show that the IcsA passenger (IcsAp) exists in multiple conformations, and the conformation populations are influenced by a central pair of cysteine residues (C375 and C379), which is not previously reported for any Type V autotransporter passengers. Disruption of either or both central cysteine residues alters the exposure of IcsA epitopes to polyclonal anti-IcsA antibodies previously shown to block Shigella adherence, yet without loss of IcsA intracellular functions in actin-based motility (ABM). Anti-IcsA antibody reactivity was restored when the IcsA paired cysteine substitution mutants were expressed in a{triangleup} ipaD background with a constitutively active T3SS, highlighting an interplay between T3SS and T5SS. The work here uncovers a novel molecular switch empowered by a centrally localised, short-spaced cysteine pair in the Type V autotransporter IcsA that ensures conformational heterogeneity to aid IcsA evasion of host immunity. ImportanceShigella species are the leading cause of diarrheal related death globally by causing bacillary dysentery. The surface virulence factor IcsA which is essential for Shigella pathogenesis is a unique multi-functional autotransporter that is responsible for cell adhesion, and actin-based motility, yet detailed mechanistic understanding is lacking. Here, we show that the three cysteine residues in IcsA contribute to the proteins distinct functions. The N terminus cysteine residue within the IcsA passenger domain plays a role in adhesin function, while a centrally localised cysteine pair provides conformational heterogeneity resulting in IcsA molecules with different reactivity to adhesion-blocking anti-IcsA antibodies. In synergy with the Type III secretion system, this molecular switch preserves biological function in distinct IcsA conformations for cell adhesion, actin-based motility and autophagy escape, providing a potential strategy by which Shigella evade host immunity targeting of this essential virulence factor.

microbiology↗