Reduced peptidoglycan synthesis capacity impairs growth of E. coli at high salt concentration.

Gram-negative bacteria have a thin peptidoglycan layer between the cytoplasmic and outer membranes protecting the cell from osmotic challenges. Hydrolases of this structure are needed to cleave bonds to allow the newly synthesized peptidoglycan strands to be inserted by synthases. These enzymes need to be tightly regulated and their activities coordinated to prevent cell lysis. To better understand this process in Escherichia coli, we probed the genetic interactions of mrcA (encodes PBP1A) and mrcB (encodes PBP1B) with genes encoding peptidoglycan amidases and endopeptidases in envelope stress conditions. Our extensive genetic interaction network analysis revealed relatively few combinations of hydrolase gene deletions with reduced fitness in the absence of PBP1A or PBP1B, showing that none of the amidases or endopeptidases is strictly required for the functioning of one of the class A PBPs. This illustrates the robustness of the peptidoglycan growth mechanism. However, we discovered that the fitness of ∆mrcB cells is significantly reduced under high salt stress and in vitro activity assays suggest that this phenotype is caused by a reduced peptidoglycan synthesis activity of PBP1A at high salt concentration.IMPORTANCEEscherichia coli and many other bacteria have a surprisingly high number of peptidoglycan hydrolases. These enzymes function in concert with synthases to facilitate the expansion of the peptidoglycan sacculus under a range of growth and stress conditions. The synthases PBP1A and PBP1B both contribute to peptidoglycan expansion during cell division and growth. Our genetic interaction analysis revealed that these two penicillin-binding proteins (PBPs) do not need specific amidases, endopeptidases, or lytic transglycosylases for function. We show that PBP1A and PBP1B do not work equally well when cells encounter high salt stress and demonstrate that PBP1A alone cannot provide sufficient PG synthesis activity under this condition. These results show how the two class A PBPs and peptidoglycan hydrolases govern cell envelope integrity in E. coli in response to environmental challenges and particularly highlight the importance of PBP1B in maintaining cell fitness under high salt conditions.

In vitro cytocompatibility and antibacterial studies on biodegradable Zn alloys supplemented by a critical assessment of direct contact cytotoxicity assay.

In vitro cytotoxicity assessment is indispensable in developing new biodegradable implant materials. Zn, which demonstrates an ideal corrosion rate between Mg- and Fe-based alloys, has been reported to have excellent in vivo biocompatibility. Therefore, modifications aimed at improving Zn's mechanical properties should not degrade its biological response. As sufficient strength, ductility and corrosion behavior required of load-bearing implants has been obtained in plastically deformed Zn-3Ag-0.5Mg, the effect of simultaneous Ag and Mg additions on in vitro cytocompatibility and antibacterial properties was studied, in relation to Zn and Zn-3Ag. Direct cell culture on samples and indirect extract-based tests showed almost no significant differences between the tested Zn-based materials. The diluted extracts of Zn, Zn-3Ag, and Zn-3Ag-0.5Mg showed no cytotoxicity toward MG-63 cells at a concentration of ≤12.5%. The cytotoxic effect was observed only at high Zn2+ ion concentrations and when in direct contact with metallic samples. The highest LD50 (lethal dose killing 50% of cells) of 13.4 mg/L of Zn2+ ions were determined for the Zn-3Ag-0.5Mg. Similar antibacterial activity against Escherichia coli and Staphylococcus aureus was observed for Zn and Zn alloys, so the effect is attributed mainly to the released Zn2+ ions exhibiting bactericidal properties. Most importantly, our experiments indicated the limitations of water-soluble tetrazolium salt-based cytotoxicity assays for direct tests on Zn-based materials. The discrepancies between the WST-8 assay and SEM observations are attributed to the interference of Zn2+ ions with tetrazolium salt, therefore favoring its transformation into formazan, giving false cell viability quantitative results.

LI-Detector: a Method for Curating Ordered Gene-Replacement Libraries.

In recent years the availability of genome sequence information has grown logarithmically resulting in the identification of a plethora of uncharacterized genes. To address this gap in functional annotation, many high-throughput screens have been devised to uncover novel gene functions. Gene-replacement libraries are one such tool that can be screened in a high-throughput way to link genotype and phenotype and are key community resources. However, for a phenotype to be attributed to a specific gene, there needs to be confidence in the genotype. Construction of large libraries can be laborious and occasionally errors will arise. Here, we present a rapid and accurate method for the validation of any ordered library where a gene has been replaced or disrupted by a uniform linear insertion (LI). We applied our method (LI-detector) to the well-known Keio library of Escherichia coli gene-deletion mutants. Our method identified 3,718 constructed mutants out of a total of 3,728 confirmed isolates, with a success rate of 99.7% for identifying the correct kanamycin cassette position. This data set provides a benchmark for the purity of the Keio mutants and a screening method for mapping the position of any linear insertion, such as an antibiotic resistance cassette in any ordered library. IMPORTANCE The construction of ordered gene replacement libraries requires significant investment of time and resources to create a valuable community resource. During construction, technical errors may result in a limited number of incorrect mutants being made. Such mutants may confound the output of subsequent experiments. Here, using the remarkable E. coli Keio knockout library, we describe a method to rapidly validate the construction of every mutant.

The lipoprotein DolP affects cell separation in Escherichia coli, but not as an upstream regulator of NlpD.

Bacterial amidases are essential to split the shared envelope of adjunct daughter cells to allow cell separation. Their activity needs to be precisely controlled to prevent cell lysis. In Escherichia coli, amidase activity is controlled by three regulatory proteins NlpD, EnvC and ActS. However, recent studies linked the outer membrane lipoprotein DolP (formerly YraP) as a potential upstream regulator of NlpD. In this study we explored this link in further detail. To our surprise DolP did not modulate amidase activity in vitro and was unable to interact with NlpD in pull-down and MST (MicroScale Thermophoresis) assays. Next, we excluded the hypothesis that ΔdolP phenocopied ΔnlpD in a range of envelope stresses. However, morphological analysis of double deletion mutants of amidases (AmiA, AmiB AmiC) and amidase regulators with dolP revealed that ΔamiAΔdolP and ΔenvCΔdolP mutants display longer chain length compared to their parental strains indicating a role for DolP in cell division. Overall, we present evidence that DolP does not affect NlpD function in vitro, implying that DolP is not an upstream regulator of NlpD. However, DolP may impact daughter cell separation by interacting directly with AmiA or AmiC, or by a yet undiscovered mechanism.

Early midcell localization of Escherichia coli PBP4 supports the function of peptidoglycan amidases.

Insertion of new material into the Escherichia coli peptidoglycan (PG) sacculus between the cytoplasmic membrane and the outer membrane requires a well-organized balance between synthetic and hydrolytic activities to maintain cell shape and avoid lysis. Since most bacteria carry multiple enzymes carrying the same type of PG hydrolytic activity, we know little about the specific function of given enzymes. Here we show that the DD-carboxy/endopeptidase PBP4 localizes in a PBP1A/LpoA and FtsEX dependent fashion at midcell during septal PG synthesis. Midcell localization of PBP4 requires its non-catalytic domain 3 of unknown function, but not the activity of PBP4 or FtsE. Microscale thermophoresis with isolated proteins shows that PBP4 interacts with NlpI and the FtsEX-interacting protein EnvC, an activator of amidases AmiA and AmiB, which are needed to generate denuded glycan strands to recruit the initiator of septal PG synthesis, FtsN. The domain 3 of PBP4 is needed for the interaction with NlpI and EnvC, but not PBP1A or LpoA. In vivo crosslinking experiments confirm the interaction of PBP4 with PBP1A and LpoA. We propose that the interaction of PBP4 with EnvC, whilst not absolutely necessary for mid-cell recruitment of either protein, coordinates the activities of PBP4 and the amidases, which affects the formation of denuded glycan strands that attract FtsN. Consistent with this model, we found that the divisome assembly at midcell was premature in cells lacking PBP4, illustrating how the complexity of interactions affect the timing of cell division initiation.

Loss of YhcB results in dysregulation of coordinated peptidoglycan, LPS and phospholipid synthesis during Escherichia coli cell growth.

The cell envelope is essential for viability in all domains 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 best 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 comprehensive 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 ΔyhcB background. Subsequent analyses suggest that YhcB functions at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.

Structure of dual BON-domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation.

The Gram-negative outer-membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents, and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here, we reveal DolP is a lipoprotein functionally conserved amongst Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for Escherichia coli DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds anionic phospholipids through an extensive membrane:protein interface. This interaction is essential for DolP function and is required for sub-cellular localisation of the protein to the cell division site, providing evidence of subcellular localisation of these phospholipids within the outer membrane. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.

Structure-Function Characterization of the Conserved Regulatory Mechanism of the Escherichia coli M48 Metalloprotease BepA.

The asymmetric Gram-negative outer membrane (OM) is the first line of defense for bacteria against environmental insults and attack by antimicrobials. The key component of the OM is lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system component LptD is regulated by a periplasmic metalloprotease, BepA. Here, we present the crystal structure of BepA from Escherichia coli, solved to a resolution of 2.18 Å, in which the M48 protease active site is occluded by an active-site plug. Informed by our structure, we demonstrate that free movement of the active-site plug is essential for BepA function, suggesting that the protein is autoregulated by the active-site plug, which is conserved throughout the M48 metalloprotease family. Targeted mutagenesis of conserved residues reveals that the negative pocket and the tetratricopeptide repeat (TPR) cavity are required for function and degradation of the BAM complex component BamA under conditions of stress. Last, we show that loss of BepA causes disruption of OM lipid asymmetry, leading to surface exposed phospholipid.IMPORTANCE M48 metalloproteases are widely distributed in all domains of life. E. coli possesses four members of this family located in multiple cellular compartments. The functions of these proteases are not well understood. Recent investigations revealed that one family member, BepA, has an important role in the maturation of a central component of the lipopolysaccharide (LPS) biogenesis machinery. Here, we present the structure of BepA and the results of a structure-guided mutagenesis strategy, which reveal the key residues required for activity that inform how all M48 metalloproteases function.

Biofilm Inhibitor Taurolithocholic Acid Alters Colony Morphology, Specialized Metabolism, and Virulence of Pseudomonas aeruginosa.

Biofilm inhibition by exogenous molecules has been an attractive strategy for the development of novel therapeutics. We investigated the biofilm inhibitor taurolithocholic acid (TLCA) and its effects on the specialized metabolism, virulence, and biofilm formation of the clinically relevant bacterium Pseudomonas aeruginosa strain PA14. Our study shows that TLCA alters the specialized metabolism, thereby affecting P. aeruginosa colony biofilm physiology. We observed an upregulation of metabolites correlated to virulence such as the siderophore pyochelin. A wax moth virulence assay confirmed that treatment with TLCA increases the virulence of P. aeruginosa. On the basis of our results, we believe that future endeavors to identify biofilm inhibitors must consider how a putative lead alters the specialized metabolism of a bacterial community to prevent pathogens from entering a highly virulent state.

Pseudallescheria boydii and Meyerozyma guilliermondii: behavior of deteriogenic fungi during simulated storage of diesel, biodiesel, and B10 blend in Brazil.

Due to their renewable and sustainable nature, biodiesel blends boost studies predicting their stability during storage. Besides chemical degradation, biodiesel is more susceptible to biodegradation due to its raw composition. The aim of this work was to evaluate the deteriogenic potential (growth and degradation) of Pseudallescheria boydii and Meyerozyma guilliermondii in degrading pure diesel (B0), pure biodiesel (B100), and a B10 blend in mineral medium during storage. The biodeterioration susceptibility at different fuel ratios and in BH minimal mineral medium were evaluated. The biomass measurements of P. boydii during 45 days indicated higher biomass production in the B10 blend. The growth curve of M. guilliermondii showed similar growth in B10 and B100. Although there was no significant production of biosurfactant, lipase production was detected in the tributyrin agar medium of both microorganisms. The main compounds identified in the aqueous phase by GC-MS were alcohols, esters, acids, sulfur, ketones, and phenols. The results showed that P. boydii grew at the expense of fuels, degrading biodiesel esters, and diesel hydrocarbons. M. guilliermondii grew in B100 and B10; however, degradation was not detected.

UCP2 Expression Is Increased in Pancreas From Brain-Dead Donors and Involved in Cytokine-Induced ß Cells Apoptosis.

BACKGROUND: Systemic inflammation associated with brain death (BD) decreases islet yield and quality, negatively affecting outcomes of human islet transplantation. A recent study from our group showed an increased expression of uncoupling protein-2 (UCP2) in pancreas from rats with BD as compared with controls. UCP2 is located in the mitochondrial inner membrane and regulates production of reactive oxygen species and glucose-stimulated insulin secretion. It has been suggested that UCP2 also plays a role in ß cell apoptosis, but these findings remain controversial. METHODS: We have presently performed a case-control study to assess UCP2 expression in pancreas from BD donors (cases) and subjects who underwent pancreatectomy (controls). We next investigated the role of Ucp2 in cytokine-induced apoptosis of rat insulin-producing INS-1E cells. RESULTS: UCP2 gene expression was higher in pancreas from BD donors compared with controls (1.73 ± 0.93 vs 0.75 ± 0.66; fold change, P < 0.05). Ucp2 knockdown (80% at the protein and messenger RNA levels) reduced by 30% cytokine-induced apoptosis and nitric oxide production in INS-1E cells. This protection was associated with decreased expression of cleaved (activated) caspases 9 and 3, suggesting that Ucp2 knockdown interferes with cytokine triggering of the intrinsic apoptotic pathway. Moreover, both messenger RNA and protein concentrations of the antiapoptotic protein Bcl-2 were increased after Ucp2 knockdown in INS-1E cells. CONCLUSIONS: These data suggest that UCP2 has an apoptotic effect in ß cells via regulation of the intrinsic pathway of apoptosis.

Irisin-encoding gene (FNDC5) variant is associated with changes in blood pressure and lipid profile in type 2 diabetic women but not in men.

INTRODUCTION: Irisin has recently been described as a novel myokine, which reduces visceral obesity and improves glucose metabolism in mice. Thus, polymorphisms in the gene encoding irisin, fibronectin type III domain containing 5 (FNDC5), may be associated with type 2 diabetes mellitus (T2DM) and related disorders. However, to date, no study has investigated the association between FNDC5 polymorphisms and susceptibility to T2DM. OBJECTIVE: To investigate the association of FNDC5 rs3480 (A/G) and rs1746661 (G/T) polymorphisms, alone or in combination, with T2DM and its clinical features. METHODS: We analyzed 1006 T2DM patients and 434 nondiabetic subjects. Polymorphisms were genotyped by real-time PCR using TaqMan MGB probes. Haplotypes constructed from the combination of rs1746661 and rs3480 polymorphisms were inferred using the Phase 2.1 program. RESULTS: Genotype, allele and haplotype frequencies of rs1746661 and rs3480 polymorphisms did not differ significantly between nondiabetic subjects and T2DM patients. Women with T2DM carrying the G allele of rs3480 showed increased HbA1c levels compared with A/A carriers, adjusted for age. The T allele of rs1746661 was associated with increased systolic blood pressure, total cholesterol and LDL-cholesterol and decreased HDL-cholesterol in women with T2DM, adjusted for covariates. Moreover, prevalence of hypercholesterolemia was higher in women carrying the T allele of rs1746661 than in G/G carriers (72.4% vs. 58.7%, OR=2.010, 95% CI=1.210-3.390), but it was not significantly different in men. CONCLUSIONS: These results indicate that, although not associated with T2DM, the G allele of rs3480 appears to be associated with increased HbA1c, while the T allele of rs1746661 appears to be associated with higher systolic blood pressure and dyslipidemia in women with T2DM.