Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics, and a synthetic lethal screen, we show that lengthening Lpp to the upper limit does not change the spatial constraint but is accommodated by other factors which thereby become essential for viability. Our findings demonstrate E. coli expressing elongated Lpp does not simply enlarge the periplasm in response, but the bacteria accommodate by a combination of tilting Lpp and reducing the amount of the covalent bridge. By genetic screening, we identified all of the genes in E. coli that become essential in order to enact this adaptation, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to accommodate the longer Lpp. We observed increased levels of factors determining cell stiffness, a decrease in membrane integrity, an increased membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling, and protein translocation.

Polymyxin causes cell envelope remodelling and stress responses in mcr-1-harbouring Escherichia coli.

Polymyxins remain important last-line antibiotics against multidrug-resistant Gram-negative bacteria. Unfortunately, polymyxin resistance is emerging and the mobile polymyxin resistance gene, mcr, is contributing to the wide dissemination of polymyxin resistance, especially among Escherichia coli, with mcr-1 being the most commonly found variant. The objective of this study was to provide mechanistic insights into concentration-dependent transcriptomic responses of mcr-harbouring E. coli following polymyxin treatment. An mcr-1-carrying clinical isolate of E. coli (LH30) was treated with polymyxin B at 2 and 8 mg/L. Bacterial cultures were collected before and 1 h following treatment for viable counting and transcriptomic analysis. Growth of E. coli LH30 was unaffected by 2 mg/L polymyxin B, whereas killing of approximately 2 log10 colony-forming units/mL occurred with 8 mg/L at 1 h. All four phosphoethanolamine (pEtN) transferase genes (mcr-1, eptA, eptB and eptC) were upregulated (fold change 2.4-4.0) by 8 mg/L polymyxin B, indicating that pEtN modifications were the preferred polymyxin resistance mechanism. The higher polymyxin B concentration also affected the expression of genes involved in fatty acid, lipopolysaccharide, lipid A, phospholipid biosynthesis, iron homeostasis and oxidative stress pathways. This transcriptomic analysis revealed that cell envelope remodelling, pEtN modification, iron acquisition and oxidative stress protective mechanisms play a key role in the survival of mcr-carrying E. coli treated with polymyxin. These findings provide new mechanistic information at the gene expression level to counter polymyxin resistance.

Investigation of LuxS-mediated quorum sensing in Klebsiella pneumoniae.

Introduction. Autoinducer-2 (AI-2) quorum sensing is a bacterial communication system that responds to cell density. The system requires luxS activity to produce AI-2, which can regulate gene expression and processes such as biofilm formation.Aim. To investigate the role of luxS in biofilm formation and gene expression in the nosocomial pathogen Klebsiella pneumoniae.Methodology. A ΔluxS gene deletion was made in K. pneumoniae KP563, an extensively drug-resistant isolate. AI-2 production was assessed in wild-type and ΔluxS strains grown in media supplemented with different carbohydrates. Potential roles of luxS in biofilm formation were investigated using a microtiter plate biofilm assay and scanning electron microscopy. Quantitative RT-PCR evaluated the expression of lipopolysaccharide (wzm and wbbM), polysaccharide (pgaA), and type 3 fimbriae (mrkA) synthesis genes in wild-type and ΔluxS mutant biofilm extracts.Results. AI-2 production was dependent on the presence of luxS. AI-2 accumulation was highest during early stationary phase in media supplemented with glucose, sucrose or glycerol. Changes in biofilm architecture were observed in the ΔluxS mutant, with less surface coverage and reduced macrocolony formation; however, no differences in biofilm formation between the wild-type and ΔluxS mutant using a microtiter plate assay were observed. In ΔluxS mutant biofilm extracts, the expression of wzm was down-regulated, and the expression of pgaA, which encodes a porin for poly-ß-1,6-N-acetyl-d-glucosamine (PNAG) polysaccharide secretion, was upregulated.Conclusion. Relationships among AI-2-mediated quorum sensing, biofilm formation and gene expression of outer-membrane components were identified in K. pneumoniae. These inter-connected processes could be important for bacterial group behaviour and persistence.

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase.

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.