Biology subjects

Mitchell, A. M.

Publications and source records attributed to Mitchell, A. M..

5 recordsLinked to original sources

bioRxiv2026

Stress-response regulators tune enterobacterial common antigen synthesis to support acid and reactive nitrogen species stress resistance

Enteric bacteria must survive envelope-damaging environmental conditions in both host and environmental niches (e.g., acidic environments, reactive oxygen and nitrogen species). The Gram-negative outer membrane (OM) is central to the cells stress resistance, acting as a permeability barrier to antimicrobials and as an adaptable structure remodeled in response to environmental changes. Enterobacterales (e.g., Escherichia coli, Salmonella enterica, Klebsiella pneumoniae) produce an invariant glycan, enterobacterial common antigen (ECA), which is localized in the OM and periplasm and contributes to the OM permeability barrier. Yet, how ECA biosynthesis is controlled and the conditions under which ECA is important remain poorly defined. Therefore, we investigated transcriptional regulation of the ECA biosynthesis wec operon in E. coli K-12. We assayed wec operon promoter activity across deletions of 36 bioinformatically identified potential regulator genes and identified four for which deletion significantly altered wec promoter activity. Of these, two repressors, GadX, a regulator of the glutamate-dependent acid stress response system, and MetJ, the master regulator of methionine biosynthesis and a reactive nitrogen species (RNS) stress regulator, bound to the wec promoter in vitro, consistent with direct transcriptional regulation. In accordance with a link between ECA and these regulators, wec promoter activity increased under acidic pH and under RNS stress, and loss of ECA impaired growth under both conditions and fitness in acidic pH. Together, these findings reveal that biosynthesis of a conserved OM glycan is embedded within dedicated environmental stress regulons and identify previously unrecognized roles for ECA in bacterial stress adaptation.

microbiology↗

bioRxiv2025

Loss of DNA mismatch repair genes leads to acquisition of antibiotic resistance independent of secondary mutations

Antibiotic resistant bacteria have been a rising clinical concern for decades. Beyond acquisition of alleles conferring resistance, bacteria under stress (e.g., from changing environmental conditions or mutations) can have higher intrinsic resistance to antibiotics than unstressed cells. This concern is expanded for gram-negative bacteria which have a protective outer membrane serving as an additional barrier against harmful molecules such as antibiotics. Here, we report a pathway which increases antibiotic resistance (i.e., minimum inhibitory concentration) in response to inactivation of the DNA Mismatch Repair pathway (MMR). This pathway led to increased intrinsic resistance and was independent of secondary mutations. Specifically, deletion of the DNA mismatch repair genes mutL or mutS caused resistance to various antibiotics spanning different classes, molecular sizes, and mechanisms of action in several different E. coli K-12 MG1655 strains, and in Salmonella enterica serovar Typhimurium LT2. This pathway was independent of the SOS response (severe DNA damage response). However, the patterns of resistance correlated with previously reported increases in MMR mutants in rates of homoeologous recombination, homologous recombination between non-identical DNA strands. Mutations expected to lower rates of recombination in MMR mutants also decreased the resistance to most antibiotics. Finally, we found lysis occurs in MMR mutants and may contribute to resistance to other antibiotics. Our results have demonstrated a novel mechanism that increases antibiotic resistance in direct response to loss of MMR genes, and we propose this resistance involves increased rates of homoeologous recombination and cell lysis. The increased antibiotic resistance of MMR mutants provides a path for these cells to survive in antibiotics long enough to develop more specific resistance mutations and so may contribute to the development of new clinical resistance alleles. Author SummaryAntibiotic resistance has become a worldwide clinical threat and understanding resistance mechanisms is essential for continued treatment of bacterial infections. Here, we investigate a novel mechanism acting in E. coli and Salmonella when DNA mismatch repair (MMR) is lost that increases the concentration of many antibiotics needed to kill cells and does not require secondary mutations. Our data suggest increased rates of homoeologous recombination (between non-identical DNA strands) and the lysis of some cells within a population are involved in this resistance. This pathway provides a mechanism through which cells with an increased mutation rate due to loss of MMR could survive long enough in the presence of antibiotics to develop new resistance mutations, leading to the spread of antibiotic resistance.

microbiology↗

bioRxiv2023

Differentiation of gram-negative intermembrane phospholipid transporter function by fatty acid saturation preference

The outer membrane of Gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. We investigated these functions using synthetic cold sensitivity (at 30 {degrees}C) caused by deletion of yhdP and fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, but not by {Delta}tamB {Delta}fadR or {Delta}ydbH {Delta}fadR. Deletion of tamB suppresses the {Delta}yhdP {Delta}fadR cold sensitivity suggesting this phenotype is related to phospholipid transport. The {Delta}yhdP {Delta}fadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not despite lower cardiolipin levels. In addition to increased cardiolipin, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the {Delta}yhdP {Delta}fadR strain. Although indirect effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential substrate transport preferences, most likely with YhdP preferentially transporting more saturated phospholipids and TamB preferentially transporting more unsaturated phospholipids. We envision cardiolipin contributing to this transport preference by sterically clogging TamB-mediated transport of saturated phospholipids. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions. Author SummaryGram-negative bacteria possess a highly impermeable outer membrane, which protects against environmental stress and antibiotics. Outer membrane phospholipid transport remained mysterious until YhdP, TamB, and YdbH were recently implicated in phospholipid transport between the inner and outer membranes of E. coli. Similar roles for YhdP and/or TamB have been suggested in both closely and distantly related gram-negative bacteria. Here, given the transporters apparent partial redundancy, we investigated functional differentiation between YhdP and TamB. Our data suggest YhdP and TamB have differential involvement with fatty acid and phospholipid metabolism. In fact, transport of higher than normal levels of cardiolipin and saturated phospholipids in the absence of YhdP and presence of TamB at a non-permissive temperature is lethal. We suggest a model where the functions of YhdP and TamB are distinguished by phospholipid transport preference with YhdP preferentially transporting more saturated phospholipids and TamB more unsaturated phospholipids. Cardiolipin headgroup specificity may contribute transport inhibition due to its bulky nature inhibiting the passage of other phospholipids. Diversification of function between YhdP and TamB provides a mechanism for regulation of phospholipid composition, and possibly the mechanical strength and permeability of the outer membrane, and so the cells intrinsic antibiotic resistance, in changing environmental conditions.

microbiology↗

bioRxiv2022

Phosphatidylglycerol is the lipid donor for synthesis of phospholipid-linked enterobacterial common antigen

The gram-negative outer membrane (OM) is an asymmetric bilayer with phospholipids in its inner leaflet and mainly lipopolysaccharide (LPS) in its outer leaflet and is largely impermeable to many antibiotics. In Enterobacterales (e.g., Escherichia, Salmonella, Klebsiella, Yersinia), the outer leaflet of the OM also contains phosphoglyceride-linked enterobacterial common antigen (ECAPG). This molecule consists of the conserved ECA carbohydrate linked to diacylglycerol-phosphate (DAG-P) through a phosphodiester bond. ECAPG contributes to the OM permeability barrier and modeling suggests that it may alter the packing of LPS molecules in the OM. Here, we investigate, in Escherichia coli K-12, the reaction synthesizing ECAPG from ECA precursor linked to an isoprenoid carrier to identify the lipid donor that provides the DAG-P moiety to ECAPG. Through overexpression of phospholipid biosynthesis genes, we observed alterations expected to increase levels of phosphatidylglycerol (PG) increased synthesis of ECAPG, whereas alterations expected to decrease levels of PG decreased synthesis of ECAPG. We discovered depletion of PG levels in strains that could synthesize ECAPG, but not other forms of ECA, causes additional growth defects, likely due to the buildup of ECA precursor on the isoprenoid carrier inhibiting peptidoglycan biosynthesis. Our results demonstrate ECAPG can be synthesized in the absence of the other major phospholipids (phosphatidylethanolamine and cardiolipin). Overall, these results conclusively demonstrate PG is the lipid donor for the synthesis of ECAPG and provide a key insight into the reaction producing ECAPG. In addition, these results provide an interesting parallel to lipoprotein acylation, which also uses PG as its DAG donor. IMPORTANCEThe outer membrane of gram-negative bacteria is a permeability barrier that prevents the entry of many antibiotics into the cell. However, the pathways responsible for outer membrane biogenesis are potential targets for small molecule development. Here, we investigate the synthesis of a form of enterobacterial common antigen (ECA), ECAPG, found in the outer membrane of Enterobacterales such as Escherichia, Salmonella, Klebsiella, and Yersinia. ECAPG consists of the conserved ECA carbohydrate unit linked to diacylglycerol-phosphate--ECA is the headgroup of a phospholipid. The details of the reaction forming this molecule from ECA linked to an isoprenoid carrier are unknown. We determined that the lipid donor that provides the phospholipid moiety to ECAPG is phosphatidylglycerol. Understanding the synthesis of outer membrane constituents such as ECAPG provides the opportunity for the development of molecules to increase outer membrane permeability, expanding the antibiotics available to treat gram-negative infections.

microbiology↗

bioRxiv2020

The inner membrane protein YhdP modulates the rate of anterograde phospholipid flow in Escherichia coli

The outer membrane (OM) of Gram-negative bacteria is a selective permeability barrier that allows uptake of nutrients while simultaneously protecting the cell from harmful compounds. The basic pathways and molecular machinery responsible for transporting lipopolysaccharides (LPS), lipoproteins, and {beta}-barrel proteins to the OM have been identified, but very little is known about phospholipid (PL) transport. To identify genes capable of affecting PL transport, we screened for genetic interactions with mlaA*, a mutant in which anterograde PL transport causes the inner membrane (IM) to shrink and eventually rupture; characterization of mlaA*-mediated lysis suggested that PL transport can occur via a high-flux, diffusive flow mechanism. We found that YhdP, an IM protein involved in maintaining the OM permeability barrier, modulates the rate of PL transport during mlaA*-mediated lysis. Deletion of yhdP from mlaA* reduced the rate of IM transport to the OM by 50%, slowing shrinkage of the IM and delaying lysis. As a result, the weakened OM of {Delta}ydhP cells was further compromised and ruptured before the IM during mlaA*-mediated death. These findings demonstrate the existence of a high-flux, diffusive pathway for PL flow in Escherichia coli that is modulated by YhdP. Significance StatementThe outer membrane (OM) of Gram-negative bacteria serves as a barrier that protects cells from harmful chemical compounds, including many antibiotics. Understanding how bacteria build this barrier is an important step in engineering strategies to circumvent it. A long-standing mystery in the field is how phospholipids (PLs) are transported from the inner membrane (IM) to the OM. We previously discovered that a mutation in the gene mlaA causes rapid flow of PLs to the OM, eventually resulting in IM rupture. Here, we found that deletion of the gene yhdP delayed cell death in the mlaA mutant by slowing flow of PLs to the OM. These findings reveal a high-flux, diffusive pathway for PL transport in Gram-negative bacteria modulated by YhdP.

molecular biology↗