Maltose accumulation-induced cell death in Saccharomyces cerevisiae.

Pretreatment of lignocellulose yields a complex sugar mixture that potentially can be converted into bioethanol and other chemicals by engineered yeast. One approach to overcome competition between sugars for uptake and metabolism is the use of a consortium of specialist strains capable of efficient conversion of single sugars. Here, we show that maltose inhibits cell growth of a xylose-fermenting specialist strain IMX730.1 that is unable to utilize glucose because of the deletion of all hexokinase genes. The growth inhibition cannot be attributed to a competition between maltose and xylose for uptake. The inhibition is enhanced in a strain lacking maltase enzymes (dMalX2) and completely eliminated when all maltose transporters are deleted. High-level accumulation of maltose in the dMalX2 strain is accompanied by a hypotonic-like transcriptional response, while cells are rescued from maltose-induced cell death by the inclusion of an extracellular osmolyte such as sorbitol. These data suggest that maltose-induced cell death is due to high levels of maltose uptake causing hypotonic-like stress conditions and can be prevented through engineering of the maltose transporters. Transporter engineering should be included in the development of stable microbial consortia for the efficient conversion of lignocellulosic feedstocks.

Unraveling the multiplicity of geranylgeranyl reductases in Archaea: potential roles in saturation of terpenoids.

The enzymology of the key steps in the archaeal phospholipid biosynthetic pathway has been elucidated in recent years. In contrast, the complete biosynthetic pathways for proposed membrane regulators consisting of polyterpenes, such as carotenoids, respiratory quinones, and polyprenols remain unknown. Notably, the multiplicity of geranylgeranyl reductases (GGRs) in archaeal genomes has been correlated with the saturation of polyterpenes. Although GGRs, which are responsible for saturation of the isoprene chains of phospholipids, have been identified and studied in detail, there is little information regarding the structure and function of the paralogs. Here, we discuss the diversity of archaeal membrane-associated polyterpenes which is correlated with the genomic loci, structural and sequence-based analyses of GGR paralogs.

Membrane Adaptations and Cellular Responses of Sulfolobus acidocaldarius to the Allylamine Terbinafine.

Cellular membranes are essential for compartmentalization, maintenance of permeability, and fluidity in all three domains of life. Archaea belong to the third domain of life and have a distinct phospholipid composition. Membrane lipids of archaea are ether-linked molecules, specifically bilayer-forming dialkyl glycerol diethers (DGDs) and monolayer-forming glycerol dialkyl glycerol tetraethers (GDGTs). The antifungal allylamine terbinafine has been proposed as an inhibitor of GDGT biosynthesis in archaea based on radiolabel incorporation studies. The exact target(s) and mechanism of action of terbinafine in archaea remain elusive. Sulfolobus acidocaldarius is a strictly aerobic crenarchaeon thriving in a thermoacidophilic environment, and its membrane is dominated by GDGTs. Here, we comprehensively analyzed the lipidome and transcriptome of S. acidocaldarius in the presence of terbinafine. Depletion of GDGTs and the accompanying accumulation of DGDs upon treatment with terbinafine were growth phase-dependent. Additionally, a major shift in the saturation of caldariellaquinones was observed, which resulted in the accumulation of unsaturated molecules. Transcriptomic data indicated that terbinafine has a multitude of effects, including significant differential expression of genes in the respiratory complex, motility, cell envelope, fatty acid metabolism, and GDGT cyclization. Combined, these findings suggest that the response of S. acidocaldarius to terbinafine inhibition involves respiratory stress and the differential expression of genes involved in isoprenoid biosynthesis and saturation.

Integrative Analysis of the Ethanol Tolerance of Saccharomyces cerevisiae.

Ethanol (EtOH) alters many cellular processes in yeast. An integrated view of different EtOH-tolerant phenotypes and their long noncoding RNAs (lncRNAs) is not yet available. Here, large-scale data integration showed the core EtOH-responsive pathways, lncRNAs, and triggers of higher (HT) and lower (LT) EtOH-tolerant phenotypes. LncRNAs act in a strain-specific manner in the EtOH stress response. Network and omics analyses revealed that cells prepare for stress relief by favoring activation of life-essential systems. Therefore, longevity, peroxisomal, energy, lipid, and RNA/protein metabolisms are the core processes that drive EtOH tolerance. By integrating omics, network analysis, and several other experiments, we showed how the HT and LT phenotypes may arise: (1) the divergence occurs after cell signaling reaches the longevity and peroxisomal pathways, with CTA1 and ROS playing key roles; (2) signals reaching essential ribosomal and RNA pathways via SUI2 enhance the divergence; (3) specific lipid metabolism pathways also act on phenotype-specific profiles; (4) HTs take greater advantage of degradation and membraneless structures to cope with EtOH stress; and (5) our EtOH stress-buffering model suggests that diauxic shift drives EtOH buffering through an energy burst, mainly in HTs. Finally, critical genes, pathways, and the first models including lncRNAs to describe nuances of EtOH tolerance are reported here.

A promiscuous archaeal cardiolipin synthase enables construction of diverse natural and unnatural phospholipids.

Cardiolipins (CL) are a class of lipids involved in the structural organization of membranes, enzyme functioning, and osmoregulation. Biosynthesis of CLs has been studied in eukaryotes and bacteria, but has been barely explored in archaea. Unlike the common fatty acyl chain-based ester phospholipids, archaeal membranes are made up of the structurally different isoprenoid-based ether phospholipids, possibly involving a different cardiolipin biosynthesis mechanism. Here, we identified a phospholipase D motif-containing cardiolipin synthase (MhCls) from the methanogen Methanospirillum hungatei. The enzyme was overexpressed in Escherichia coli, purified, and its activity was characterized by LC-MS analysis of substrates/products. MhCls utilizes two archaetidylglycerol (AG) molecules in a transesterification reaction to synthesize glycerol-di-archaetidyl-cardiolipin (Gro-DACL) and glycerol. The enzyme is nonselective to the stereochemistry of the glycerol backbone and the nature of the lipid tail, as it also accepts phosphatidylglycerol (PG) to generate glycerol-di-phosphatidyl-cardiolipin (Gro-DPCL). Remarkably, in the presence of AG and PG, MhCls formed glycerol-archaetidyl-phosphatidyl-cardiolipin (Gro-APCL), an archaeal-bacterial hybrid cardiolipin species that so far has not been observed in nature. Due to the reversibility of the transesterification, in the presence of glycerol, Gro-DPCL can be converted back into two PG molecules. In the presence of other compounds that contain primary hydroxyl groups (e.g., alcohols, water, sugars), various natural and unique unnatural phospholipid species could be synthesized, including multiple di-phosphatidyl-cardiolipin species. Moreover, MhCls can utilize a glycolipid in the presence of phosphatidylglycerol to form a glycosyl-mono-phosphatidyl-cardiolipin species, emphasizing the promiscuity of this cardiolipin synthase that could be of interest for bio-catalytic purposes.

The catalytic and structural basis of archaeal glycerophospholipid biosynthesis.

Archaeal glycerophospholipids are the main constituents of the cytoplasmic membrane in the archaeal domain of life and fundamentally differ in chemical composition compared to bacterial phospholipids. They consist of isoprenyl chains ether-bonded to glycerol-1-phosphate. In contrast, bacterial glycerophospholipids are composed of fatty acyl chains ester-bonded to glycerol-3-phosphate. This largely domain-distinguishing feature has been termed the "lipid-divide". The chemical composition of archaeal membranes contributes to the ability of archaea to survive and thrive in extreme environments. However, ether-bonded glycerophospholipids are not only limited to extremophiles and found also in mesophilic archaea. Resolving the structural basis of glycerophospholipid biosynthesis is a key objective to provide insights in the early evolution of membrane formation and to deepen our understanding of the molecular basis of extremophilicity. Many of the glycerophospholipid enzymes are either integral membrane proteins or membrane-associated, and hence are intrinsically difficult to study structurally. However, in recent years, the crystal structures of several key enzymes have been solved, while unresolved enzymatic steps in the archaeal glycerophospholipid biosynthetic pathway have been clarified providing further insights in the lipid-divide and the evolution of early life.

D-glucose overflow metabolism in an evolutionary engineered high-performance D-xylose consuming Saccharomyces cerevisiae strain.

Co-consumption of D-xylose and D-glucose by Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. There is a need for improved sugar conversion rates to minimize fermentation times. Previously, we have employed evolutionary engineering to enhance D-xylose transport and metabolism in the presence of D-glucose in a xylose-fermenting S. cerevisiae strain devoid of hexokinases. Re-introduction of Hxk2 in the high performance xylose-consuming strains restored D-glucose utilization during D-xylose/D-glucose co-metabolism, but at rates lower than the non-evolved strain. In the absence of D-xylose, D-glucose consumption was similar to the parental strain. The evolved strains accumulated trehalose-6-phosphate during sugar co-metabolism, and showed an increased expression of trehalose pathway genes. Upon the deletion of TSL1, trehalose-6-phosphate levels were decreased and D-glucose consumption and growth on mixed sugars was improved. The data suggest that D-glucose/D-xylose co-consumption in high-performance D-xylose consuming strains causes the glycolytic flux to saturate. Excess D-glucose is phosphorylated enters the trehalose pathway resulting in glucose recycling and energy dissipation, accumulation of trehalose-6-phosphate which inhibits the hexokinase activity, and release of trehalose into the medium.

Nonribosomal peptide synthetases and their biotechnological potential in Penicillium rubens.

Nonribosomal peptide synthetases (NRPS) are large multimodular enzymes that synthesize a diverse variety of peptides. Many of these are currently used as pharmaceuticals, thanks to their activity as antimicrobials (penicillin, vancomycin, daptomycin, echinocandin), immunosuppressant (cyclosporin) and anticancer compounds (bleomycin). Because of their biotechnological potential, NRPSs have been extensively studied in the past decades. In this review, we provide an overview of the main structural and functional features of these enzymes, and we consider the challenges and prospects of engineering NRPSs for the synthesis of novel compounds. Furthermore, we discuss secondary metabolism and NRP synthesis in the filamentous fungus Penicillium rubens and examine its potential for the production of novel and modified ß-lactam antibiotics.

Converting Escherichia coli into an archaebacterium with a hybrid heterochiral membrane.

One of the main differences between bacteria and archaea concerns their membrane composition. Whereas bacterial membranes are made up of glycerol-3-phosphate ester lipids, archaeal membranes are composed of glycerol-1-phosphate ether lipids. Here, we report the construction of a stable hybrid heterochiral membrane through lipid engineering of the bacterium Escherichia coli By boosting isoprenoid biosynthesis and heterologous expression of archaeal ether lipid biosynthesis genes, we obtained a viable E. coli strain of which the membranes contain archaeal lipids with the expected stereochemistry. It has been found that the archaeal lipid biosynthesis enzymes are relatively promiscuous with respect to their glycerol phosphate backbone and that E. coli has the unexpected potential to generate glycerol-1-phosphate. The unprecedented level of 20-30% archaeal lipids in a bacterial cell has allowed for analyzing the effect on the mixed-membrane cell's phenotype. Interestingly, growth rates are unchanged, whereas the robustness of cells with a hybrid heterochiral membrane appeared slightly increased. The implications of these findings for evolutionary scenarios are discussed.

A Unified Approach for the Total Synthesis of cyclo-Archaeol, iso-Caldarchaeol, Caldarchaeol, and Mycoketide.

Ir-catalyzed asymmetric alkene hydrogenation is presented as the strategy par excellence to prepare saturated isoprenoids and mycoketides. This highly stereoselective synthesis approach is combined with an established 13 C-NMR method to determine the enantioselectivity of each methyl-branched stereocenter. It is shown that this analysis is fit for purpose and the combination allows the synthesis of the title compounds with a significant increase in efficiency.

Impact of Classical Strain Improvement of Penicillium rubens on Amino Acid Metabolism during ß-Lactam Production.

To produce high levels of ß-lactams, the filamentous fungus Penicillium rubens (previously named Penicillium chrysogenum) has been subjected to an extensive classical strain improvement (CSI) program during the last few decades. This has led to the accumulation of many mutations that were spread over the genome. Detailed analysis reveals that several mutations targeted genes that encode enzymes involved in amino acid metabolism, in particular biosynthesis of l-cysteine, one of the amino acids used for ß-lactam production. To examine the impact of the mutations on enzyme function, the respective genes with and without the mutations were cloned and expressed in Escherichia coli, purified, and enzymatically analyzed. Mutations severely impaired the activities of a threonine and serine deaminase, and this inactivates metabolic pathways that compete for l-cysteine biosynthesis. Tryptophan synthase, which converts l-serine into l-tryptophan, was inactivated by a mutation, whereas a mutation in 5-aminolevulinate synthase, which utilizes glycine, was without an effect. Importantly, CSI caused increased expression levels of a set of genes directly involved in cysteine biosynthesis. These results suggest that CSI has resulted in improved cysteine biosynthesis by the inactivation of the enzymatic conversions that directly compete for resources with the cysteine biosynthetic pathway, consistent with the notion that cysteine is a key component during penicillin production.IMPORTANCEPenicillium rubens is an important industrial producer of ß-lactam antibiotics. High levels of penicillin production were enforced through extensive mutagenesis during a classical strain improvement (CSI) program over 70 years. Several mutations targeted amino acid metabolism and resulted in enhanced l-cysteine biosynthesis. This work provides a molecular explanation for the interrelation between secondary metabolite production and amino acid metabolism and how classical strain improvement has resulted in improved production strains.

Efficient, D-glucose insensitive, growth on D-xylose by an evolutionary engineered Saccharomyces cerevisiae strain.

Optimizing D-xylose consumption in Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. An evolutionary engineering approach was used to elevate D-xylose consumption in a xylose-fermenting S. cerevisiae strain carrying the D-xylose-specific N367I mutation in the endogenous chimeric Hxt36 hexose transporter. This strain carries a quadruple hexokinase deletion that prevents glucose utilization, and allows for selection of improved growth rates on D-xylose in the presence of high D-glucose concentrations. Evolutionary engineering resulted in D-glucose-insensitive growth and consumption of D-xylose, which could be attributed to glucose insensitive D-xylose uptake via a novel chimeric Hxt37 N367I transporter that emerged from a fusion of the HXT36 and HXT7 genes, and a down regulation of a set of Hxt transporters that mediate glucose sensitive xylose transport. RNA sequencing revealed the downregulation of HXT1 and HXT2 which, together with the deletion of HXT7, resulted in a 21% reduction of the expression of all plasma membrane transporters genes. Morphological analysis showed an increased cell size and corresponding increased cell surface area of the evolved strain, which could be attributed to genome duplication. Mixed strain fermentation of the D-xylose-consuming strain DS71054-evo6 with the D-glucose consuming CEN.PK113-7D strain resulted in decreased residual sugar concentrations and improved ethanol production yields compared to a strain which sequentially consumes D-glucose and D-xylose.

Synthetic control devices for gene regulation in Penicillium chrysogenum.

BACKGROUND: Orthogonal, synthetic control devices were developed for Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. In the synthetic transcription factor, the QF DNA-binding domain of the transcription factor of the quinic acid gene cluster of Neurospora crassa is fused to the VP16 activation domain. This synthetic transcription factor controls the expression of genes under a synthetic promoter containing quinic acid upstream activating sequence (QUAS) elements, where it binds. A gene cluster may demand an expression tuned individually for each gene, which is a great advantage provided by this system. RESULTS: The control devices were characterized with respect to three of their main components: expression of the synthetic transcription factors, upstream activating sequences, and the affinity of the DNA binding domain of the transcription factor to the upstream activating domain. This resulted in synthetic expression devices, with an expression ranging from hardly detectable to a level similar to that of highest expressed native genes. The versatility of the control device was demonstrated by fluorescent reporters and its application was confirmed by synthetically controlling the production of penicillin. CONCLUSIONS: The characterization of the control devices in microbioreactors, proved to give excellent indications for how the devices function in production strains and conditions. We anticipate that these well-characterized and robustly performing control devices can be widely applied for the production of secondary metabolites and other compounds in filamentous fungi.

Reshaping of the conformational search of a protein by the chaperone trigger factor.

Protein folding is often described as a search process, in which polypeptides explore different conformations to find their native structure. Molecular chaperones are known to improve folding yields by suppressing aggregation between polypeptides before this conformational search starts, as well as by rescuing misfolds after it ends. Although chaperones have long been speculated to also affect the conformational search itself--by reshaping the underlying folding landscape along the folding trajectory--direct experimental evidence has been scarce so far. In Escherichia coli, the general chaperone trigger factor (TF) could play such a role. TF has been shown to interact with nascent chains at the ribosome, with polypeptides released from the ribosome into the cytosol, and with fully folded proteins before their assembly into larger complexes. To investigate the effect of TF from E. coli on the conformational search of polypeptides to their native state, we investigated individual maltose binding protein (MBP) molecules using optical tweezers. Here we show that TF binds folded structures smaller than one domain, which are then stable for seconds and ultimately convert to the native state. Moreover, TF stimulates native folding in constructs of repeated MBP domains. The results indicate that TF promotes correct folding by protecting partially folded states from distant interactions that produce stable misfolded states. As TF interacts with most newly synthesized proteins in E. coli, we expect these findings to be of general importance in understanding protein folding pathways.

Pathway for the Biosynthesis of the Pigment Chrysogine by Penicillium chrysogenum.

Chrysogine is a yellow pigment produced by Penicillium chrysogenum and other filamentous fungi. Although the pigment was first isolated in 1973, its biosynthetic pathway has so far not been resolved. Here, we show that deletion of the highly expressed nonribosomal peptide synthetase (NRPS) gene Pc21g12630 (chyA) resulted in a decrease in the production of chrysogine and 13 related compounds in the culture broth of P. chrysogenum Each of the genes of the chyA-containing gene cluster was individually deleted, and corresponding mutants were examined by metabolic profiling in order to elucidate their function. The data suggest that the NRPS ChyA mediates the condensation of anthranilic acid and alanine into the intermediate 2-(2-aminopropanamido)benzoic acid, which was verified by feeding experiments of a ΔchyA strain with the chemically synthesized product. The remainder of the pathway is highly branched, yielding at least 13 chrysogine-related compounds.IMPORTANCEPenicillium chrysogenum is used in industry for the production of ß-lactams, but also produces several other secondary metabolites. The yellow pigment chrysogine is one of the most abundant metabolites in the culture broth, next to ß-lactams. Here, we have characterized the biosynthetic gene cluster involved in chrysogine production and elucidated a complex and highly branched biosynthetic pathway, assigning each of the chrysogine cluster genes to biosynthetic steps and metabolic intermediates. The work further unlocks the metabolic potential of filamentous fungi and the complexity of secondary metabolite pathways.

The Canonical and Accessory Sec System of Gram-positive Bacteria.

The Sec system is present in all bacteria and responsible for the translocation of the majority of proteins across the cytoplasmic membrane. The system consists of two principal components: the ATPase motor protein, SecA, and the protein-conducting channel, SecYEG. In addition to this canonical Sec system, several Gram-positive bacteria also possess a so-called accessory Sec system. This is a specialized translocation system that is responsible for the export of a subset of secretory proteins, including virulence factors. The accessory Sec system consists of a second SecA paralog, termed SecA2, with or without a second SecY paralog, termed SecY2. In some bacteria, the accessory Sec system is dependent on the canonical Sec system for functionality, while in other bacteria, they can function independently. In this review, we provide an overview of the current knowledge of the canonical and accessory Sec system of Gram-positive bacteria with a focus on the primary component of the Sec translocase, SecA and SecYEG.

Laboratory evolution of a glucose-phosphorylation-deficient, arabinose-fermenting S. cerevisiae strain reveals mutations in GAL2 that enable glucose-insensitive l-arabinose uptake.

Cas9-assisted genome editing was used to construct an engineered glucose-phosphorylation-negative S. cerevisiae strain, expressing the Lactobacillus plantaruml-arabinose pathway and the Penicillium chrysogenum transporter PcAraT. This strain, which showed a growth rate of 0.26 h-1 on l-arabinose in aerobic batch cultures, was subsequently evolved for anaerobic growth on l-arabinose in the presence of d-glucose and d-xylose. In four strains isolated from two independent evolution experiments the galactose-transporter gene GAL2 had been duplicated, with all alleles encoding Gal2N376T or Gal2N376I substitutions. In one strain, a single GAL2 allele additionally encoded a Gal2T89I substitution, which was subsequently also detected in the independently evolved strain IMS0010. In 14C-sugar-transport assays, Gal2N376S, Gal2N376T and Gal2N376I substitutions showed a much lower glucose sensitivity of l-arabinose transport and a much higher Km for d-glucose transport than wild-type Gal2. Introduction of the Gal2N376I substitution in a non-evolved strain enabled growth on l-arabinose in the presence of d-glucose. Gal2N376T, T89I and Gal2T89I variants showed a lower Km for l-arabinose and a higher Km for d-glucose than wild-type Gal2, while reverting Gal2N376T, T89I to Gal2N376 in an evolved strain negatively affected anaerobic growth on l-arabinose. This study indicates that optimal conversion of mixed-sugar feedstocks may require complex 'transporter landscapes', consisting of sugar transporters with complementary kinetic and regulatory properties.

Lipids Activate SecA for High Affinity Binding to the SecYEG Complex.

Protein translocation across the bacterial cytoplasmic membrane is an essential process catalyzed predominantly by the Sec translocase. This system consists of the membrane-embedded protein-conducting channel SecYEG, the motor ATPase SecA, and the heterotrimeric SecDFyajC membrane protein complex. Previous studies suggest that anionic lipids are essential for SecA activity and that the N terminus of SecA is capable of penetrating the lipid bilayer. The role of lipid binding, however, has remained elusive. By employing differently sized nanodiscs reconstituted with single SecYEG complexes and comprising varying amounts of lipids, we establish that SecA gains access to the SecYEG complex via a lipid-bound intermediate state, whereas acidic phospholipids allosterically activate SecA for ATP-dependent protein translocation.

Photocontrol of Antibacterial Activity: Shifting from UV to Red Light Activation.

The field of photopharmacology aims to introduce smart drugs that, through the incorporation of molecular photoswitches, allow for the remote spatial and temporal control of bioactivity by light. This concept could be particularly beneficial in the treatment of bacterial infections, by reducing the systemic and environmental side effects of antibiotics. A major concern in the realization of such light-responsive drugs is the wavelength of the light that is applied. Studies on the photocontrol of biologically active agents mostly rely on UV light, which is cytotoxic and poorly suited for tissue penetration. In our efforts to develop photoswitchable antibiotics, we introduce here antibacterial agents whose activity can be controlled by visible light, while getting into the therapeutic window. For that purpose, a UV-light-responsive core structure based on diaminopyrimidines with suitable antibacterial properties was identified. Subsequent modification of an azobenzene photoswitch moiety led to structures that allowed us to control their activity against Escherichia coli in both directions with light in the visible region. For the first time, full in situ photocontrol of antibacterial activity in the presence of bacteria was attained with green and violet light. Most remarkably, one of the diaminopyrimidines revealed an at least 8-fold difference in activity before and after irradiation with red light at 652 nm, showcasing the effective "activation" of a biological agent otherwise inactive within the investigated concentration range, and doing so with red light in the therapeutic window.

Archaeal phospholipids: Structural properties and biosynthesis.

Phospholipids are major components of the cellular membranes present in all living organisms. They typically form a lipid bilayer that embroiders the cell or cellular organelles, constitute a barrier for ions and small solutes and form a matrix that supports the function of membrane proteins. The chemical composition of the membrane phospholipids present in the two prokaryotic domains Archaea and Bacteria are vastly different. Archaeal lipids are composed of highly-methylated isoprenoid chains that are ether-linked to a glycerol-1-phosphate backbone while bacterial phospholipids consist of straight fatty acids bound by ester bonds to the enantiomeric glycerol-3-phosphate backbone. The chemical structure of the archaeal lipids and their compositional diversity ensures the required stability at extreme environmental conditions as many archaea thrive at such conditions including high or low temperature, high salinity and extreme acidic or alkaline pH values. However, not all archaea are extremophiles, and the presence of ether-linked phospholipids is a phylogenetic marker that distinguishes Archaea from other life forms. During the past decade, our understanding of the biosynthesis of archaeal lipids has progressed resulting in the characterization of the main biosynthetic steps of the pathway including the reconstitution of lipid biosynthesis in vitro. Here we describe the chemical and physical properties of archaeal lipids and membranes derived thereof, summarize the existing knowledge about the enzymology of the archaeal lipid biosynthetic pathway and discuss evolutionary theories associated with the "Lipid Divide" that resulted in the differentiation of bacterial and archaeal organisms. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.