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Insights into the function of YciM, a heat shock membrane protein required to maintain envelope integrity in Escherichia coli.

ABSTRACT: The cell envelope of Gram-negative bacteria is an essential organelle that is important for cell shape and protection from toxic compounds. Proteins involved in envelope biogenesis are therefore attractive targets for the design of new antibacterial agents. In a search for new envelope assembly factors, we screened a collection of Escherichia coli deletion mutants for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of defects in the cell envelope. Strains lacking yciM were among the most sensitive strains of the mutant collection. Further characterization of yciM mutants revealed that they display a thermosensitive growth defect on low-osmolarity medium and that they have a significantly altered cell morphology. At elevated temperatures, yciM mutants form bulges containing cytoplasmic material and subsequently lyse. We also discovered that yciM genetically interacts with envC, a gene encoding a regulator of the activity of peptidoglycan amidases. Altogether, these results indicate that YciM is required for envelope integrity. Biochemical characterization of the protein showed that YciM is anchored to the inner membrane via its N terminus, the rest of the protein being exposed to the cytoplasm. Two CXXC motifs are present at the C terminus of YciM and serve to coordinate a redox-sensitive iron center of the rubredoxin type. Both the N-terminal membrane anchor and the C-terminal iron center of YciM are important for function.

SUBMITTER: Nicolaes V

PROVIDER: S-EPMC3911248 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Insights into the function of YciM, a heat shock membrane protein required to maintain envelope integrity in Escherichia coli.

Nicolaes Valérie V El Hajjaji Hayat H Davis Rebecca M RM Van der Henst Charles C Depuydt Matthieu M Leverrier Pauline P Aertsen Abram A Haufroid Vincent V Ollagnier de Choudens Sandrine S De Bolle Xavier X Ruiz Natividad N Collet Jean-Francois JF

Journal of bacteriology 20131101 2

The cell envelope of Gram-negative bacteria is an essential organelle that is important for cell shape and protection from toxic compounds. Proteins involved in envelope biogenesis are therefore attractive targets for the design of new antibacterial agents. In a search for new envelope assembly factors, we screened a collection of Escherichia coli deletion mutants for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of defects in the cell envelope. Strains lacking yc ...[more]

PMID: 24187084

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Project description:The alternative sigma factor ?E is a key component of the Escherichia coli response to cell envelope stress and is required for viability even in the absence of stress. The activity of ?E increases during entry into stationary phase, suggesting an important role for ?E when nutrients are limiting. Elevated ?E activity has been proposed to activate a pathway leading to the lysis of nonculturable cells that accumulate during early stationary phase. To better understand ?E-directed cell lysis and the role of ?E in stationary phase, we investigated the effects of elevated ?E activity in cultures grown for 10 days. We demonstrate that high ?E activity is lethal for all cells in stationary phase, not only those that are nonculturable. Spontaneous mutants with reduced ?E activity, due primarily to point mutations in the region of ?E that binds the -35 promoter motif, arise and take over cultures within 5 to 6 days after entry into stationary phase. High ?E activity leads to large reductions in the levels of outer membrane porins and increased membrane permeability, indicating membrane defects. These defects can be counteracted and stationary-phase lethality delayed significantly by stabilizing membranes with Mg2+ and buffering the growth medium or by deleting the ?E-dependent small RNAs (sRNAs) MicA, RybB, and MicL, which inhibit the expression of porins and Lpp. Expression of these sRNAs also reverses the loss of viability following depletion of ?E activity. Our results demonstrate that appropriate regulation of ?E activity, ensuring that it is neither too high nor too low, is critical for envelope integrity and cell viability.IMPORTANCE The Gram-negative cell envelope and cytoplasm differ significantly, and separate responses have evolved to combat stress in each compartment. An array of cell envelope stress responses exist, each of which is focused on different parts of the envelope. The ?E response is conserved in many enterobacteria and is tuned to monitor pathways for the maturation and delivery of outer membrane porins, lipoproteins, and lipopolysaccharide to the outer membrane. The activity of ?E is tightly regulated to match the production of ?E regulon members to the needs of the cell. In E. coli, loss of ?E results in lethality. Here we demonstrate that excessive ?E activity is also lethal and results in decreased membrane integrity, the very phenotype the system is designed to prevent.

Project description:The envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measure Escherichia coli envelope permeability to a ?-galactosidase chromogenic substrate. The signal produced by cytoplasmic ?-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds and E. coli gene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinct E. coli strains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 ?M of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R > 0.5 ?g/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

Dataset Information

Insights into the function of YciM, a heat shock membrane protein required to maintain envelope integrity in Escherichia coli.

Publications

Insights into the function of YciM, a heat shock membrane protein required to maintain envelope integrity in Escherichia coli.

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