Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9.

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

Crystal structure and solution scattering of Geobacillus stearothermophilus S9 peptidase reveal structural adaptations for carboxypeptidase activity.

Acylaminoacyl peptidases (AAPs) play a pivotal role in various pathological conditions and are recognized as potential therapeutic targets. AAPs exhibit a wide range of activities, such as acylated amino acid-dependent aminopeptidase, endopeptidase, and less studied carboxypeptidase activity. We have determined the crystal structure of an AAP from Geobacillus stearothermophilus (S9gs) at 2.0 Å resolution. Despite being annotated as an aminopeptidase in the NCBI database, our enzymatic characterization proved S9gs to be a carboxypeptidase. Solution-scattering studies showed that S9gs exists as a tetramer in solution, and crystal structure analysis revealed adaptations responsible for the carboxypeptidase activity of S9gs. The findings present a hypothesis for substrate selection, substrate entry, and product exit from the active site, enriching our understanding of this rare carboxypeptidase.

Improved thermostability of type I pullulanase from Bacillus thermoliquefaciens by error-prone PCR.

Pullulanase (PulB) is a starch-debranching enzyme. In order to improve its catalytic performance, random mutagenesis was performed on the pullulanase gene derived from Bacillus thermoliquefaciens. Two rounds of error-prone PCR were carried out. Mutant T252S was screened in the first round of error-prone library, which had the highest catalytic activity. During the second round of mutations, mutant enzyme G250P/T252S/G253T/N255K was screened, which had further improved catalytic activity and the best thermostability. Compared with the parent enzyme, the specific activity of mutant enzyme G250P/T252S/G253T/N255K increased by 1.9 times, Km decreased by 22.7 %, kcat increased by 28.7 %, and kcat/Km increased by 68.4 %. The thermostability of the mutant enzyme improved significantly, showing that the half-life at 60 °C was extended to 7.5 h, which was 87.5 % higher than that of the parent enzyme. The mutation sites in these two rounds were concentrated in the 250-255 regions, indicating that this region was an important region affecting the catalytic activity and Thermostability. The reasons for the change of enzymtic properties was also preliminarily analyzed through three-dimensional simulation.

Thermostable homologues of the periplasmic siderophore-binding protein CeuE from Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius.

Siderophore-binding proteins from two thermophilic bacteria, Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius, were identified from a search of sequence databases, cloned and overexpressed. They are homologues of the well characterized protein CjCeuE from Campylobacter jejuni. The iron-binding histidine and tyrosine residues are conserved in both thermophiles. Crystal structures were determined of the apo proteins and of their complexes with iron(III)-azotochelin and its analogue iron(III)-5-LICAM. The thermostability of both homologues was shown to be about 20°C higher than that of CjCeuE. Similarly, the tolerance of the homologues to the organic solvent dimethylformamide (DMF) was enhanced, as reflected by the respective binding constants for these ligands measured in aqueous buffer at pH 7.5 in the absence and presence of 10% and 20% DMF. Consequently, these thermophilic homologues offer advantages in the development of artificial metalloenzymes using the CeuE family.

Polyhydroxyalkanoate biosynthesis and optimisation of thermophilic Geobacillus stearothermophilus strain K4E3_SPR_NPP.

Polyhydroxyalkanoates (PHA) can be used to combat the challenges associated with plastic because it is biodegradable and can be produced from renewable resources. Extremophiles are considered to be potential PHA producers. An initial screening for the PHA synthesizing ability of a thermophilic bacteria Geobacillus stearothermophilus strain K4E3_SPR_NPP was carried out using Sudan black B staining. Nile red viable colony staining was used to further verify that the isolates produced PHA. Crotonic acid assays were used to determine the concentrations of PHA. The bacteria showed 31% PHA accumulation per dry cell weight (PHA/DCW) when glucose was used as a carbon source for growth. The molecule was identified to be medium chain length PHA, A copolymer of PHA containing poly(3-hydroxybutyrate)-poly(3-hydroxyvalerate)-poly(3-hydroxyhexanoate) (PHB-PHV-PHHX) using 1H-NMR. Six carbon sources and four nitrogen sources were screened for the synthesis of maximum PHA content, of which lactose and ammonium nitrate showed 45% and 53% PHA/DCW respectively. The important factors in the experiment are identified using the Plackett-Burman design, and optimization is performed using the response surface method. Response surface methodology was used to optimize the three important factors, and the maximum biomass and PHA productions were discovered. Optimal concentrations yielded a maximum of 0.48 g/l biomass and 0.32 g/l PHA, measuring 66.66% PHA accumulation. Dairy industry effluent was employed for the synthesis of PHA, yielding 0.73 g/l biomass and 0.33 g/l PHA, measuring 45% PHA accumulation. These findings add credibility to the possibility of adopting thermophilic isolates for PHA production using low-cost substrates.

Reducing substrate inhibition of malate dehydrogenase from Geobacillus stearothermophilus by C-terminal truncation.

Malate dehydrogenase (MDH) catalyzes the reduction of oxaloacetate to L-malate. Geobacillus stearothermophilus MDH (gs-MDH) is used as a diagnostic reagent; however, gs-MDH is robustly inhibited at high substrate concentrations, which limits its reaction rate. Here, we reduced substrate inhibition of gs-MDH by deleting its C-terminal residues. Computational analysis showed that C-terminal residues regulate the position of the active site loop. C-terminal deletions of gs-MDH successfully increased Ki values by 5- to 8-fold with maintained thermal stability (>90% of the wild-type enzyme), although kcat/Km values were decreased by <2-fold. The structure of the mutant showed a shift in the location of the active site loop and a decrease in its volume, suggesting that substrate inhibition was reduced by eliminating the putative substrate binding site causing inhibition. Our results provide an effective method to reduce substrate inhibition of the enzyme without loss of other parameters, including binding and stability constants.

Effect of solid-state fermentation by three different Bacillus species on composition and protein structure of soybean meal.

BACKGROUND: Fermentation efficiency of thermophiles of Bacillus licheniformis YYC4 and Geobacillus stearothermophilus A75, and mesophilic Bacillus subtilis 10 160 on soybean meal (SBM), was evaluated by examining the nutritional and protein structural changes. RESULTS: SBM fermentation by B. licheniformis YYC4, B. subtilis 10 160 and G. stearothemophilus A75 increased significantly the crude and soluble protein from 442.4 to 524.8, 516.1 and 499.9 g kg-1 , and from 53.9 to 203.3, 291.3 and 74.6 g kg-1 , and decreased trypsin inhibitor from 8.19 to 3.19, 2.14 and 5.10 mg g-1 , respectively. Bacillus licheniformis YYC4 and B. subtilis 10 160 significantly increased phenol and pyrazine content. Furthermore, B. licheniformis YYC4 fermentation could produce abundant alcohols, ketones, esters and acids. Surface hydrophobicity, sulfhydryl groups and disulfide bond contents of SBM protein were increased significantly from 98.27 to 166.13, 173.27 and 150.71, from 3.26 to 4.88, 5.03 and 4.21 µmol g-1 , and from 20.77 to 27.95, 29.53 and 25.5 µmol g-1 after their fermentation. Fermentation induced red shifts of the maximum absorption wavelength (λmax ) of fluorescence spectra from 353 to 362, 376 and 361 nm, while significantly reducing the fluorescence intensity of protein, especially when B. subtilis 10 160 was used. Moreover, fermentation markedly changed the secondary structure composition of SBM protein. Analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and atomic force microscopy showed that macromolecule protein was degraded into small-sized protein or peptide during fermentation of SBM. CONCLUSION: Bacillus licheniformis YYC4 fermentation (without sterilization) improved nutrition and protein structure of SBM as B. subtilis 10 160, suggesting its potential application in the SBM fermentation industry. © 2021 Society of Chemical Industry.

Increasing loop flexibility affords low-temperature adaptation of a moderate thermophilic malate dehydrogenase from Geobacillus stearothermophilus.

Malate dehydrogenase (MDH) catalyzes the reversible reduction of nicotinamide adenine dinucleotide from oxaloacetate to L-malate. MDH from moderate thermophilic Geobacillus stearothermophilus (gs-MDH) has high thermal stability and substrate specificity and is used as a diagnostic reagent. In this study, gs-MDH was engineered to increase its catalytic activity at low temperatures. Based on sequential and structural comparison with lactate dehydrogenase from G. stearothermophilus, we selected G218 as a mutation site to increase the loop flexibility pivotal for MDH catalysis. The G218 mutants showed significantly higher specific activities than the wild type at low temperatures and maintained thermal stability. The crystal structure of the G218Y mutant, which had the highest catalytic efficiency among all the G218 mutants, suggested that the flexibility of the mobile loop was successfully increased by the bulky side chain. Therefore, this study demonstrated the low-temperature adaptation of MDH by facilitating conformational changes during catalysis.

Extracellular expression in Bacillus subtilis of a thermostable Geobacillus stearothermophilus lipase

BACKGROUND The extracellular expression of enzymes in a secretion host such as Bacillus subtilis is a useful strategy in reducing the cost of downstream processing of industrial enzymes. Here, we present the first report of the successful extracellular expression in Bacillus subtilis WB800 of Geobacillus stearothermophilus lipase (T1.2RQ), a novel industriallydesirable thermostable lipolytic enzyme which has an excellent hydrolytic and transesterification activity. Signal peptides of a-amylase, extracellular protease, and lipase A, as well as two different promoters, were used in the secretion and expression of lipase T1.2RQ. RESULTS Lipase activity assay using p-nitrophenyl laurate showed that all three signal peptides directed the secretion of lipase T1.2RQ into the extracellular medium. The signal peptide of lipase A, resulted in the highest extracellular yield of 5.6 U/ml, which corresponds to a 6-fold increase over the parent Bacillus subtilis WB800 strain. SDS-PAGE and zymogram analysis confirmed that lipase T1.2RQ was correctly processed and secreted in its original size of 44 kDa. A comparison of the expression levels of lipase T1.2RQ in rich medium and minimal media showed that the enzyme was better expressed in rich media, with up to an 8-fold higher yield over minimal media. An attempt to further increase the lipase expression level by promoter optimization showed that, contrary to expectation, the optimized promoter exhibited similar expression levels as the original one, suggesting the need for the optimization of downstream factors. CONCLUSIONS The successful extracellular secretion of lipase T1.2RQ in Bacillus subtilis represents a remarkable feat in the industrial-scale production of this enzyme

Incidence of Bacillus cereus, Bacillus sporothermodurans and Geobacillus stearothermophilus in ultra-high temperature milk and biofilm formation capacity of isolates.

The presence of mesophilic and thermophilic spore-forming bacteria in UHT milk, as well as biofilm formation in dairy plants, are concerning. The current study explored the spore-forming bacilli diversity in 100 samples of UHT milk (skimmed and whole). Through this work, a total of 239 isolates from UHT milk samples were obtained. B. cereus s.s. was isolated from 7 samples, B. sporothermodurans from 19 and, G. stearothermophilus from 25 samples. Genes encoding hemolysin (HBL), and non-hemolytic (NHE) enterotoxins were detected in B. cereus s.s. isolates. All isolates of B. cereus s.s. (12) B. sporothermodurans (38), and G. stearothermophilus (47) were selected to verify the ability of biofilm formation in microtiter plates. The results showed all isolates could form biofilms. The OD595 values of biofilm formation varied between 0.14 and 1.04 for B. cereus, 0.20 to 1.87 for B. sporothermodurans, and 0.49 to 2.77 for G. stearothermophilus. The data highlights that the dairy industry needs to reinforce control in the initial quality of the raw material and in CIP cleaning procedures; avoiding biofilm formation and consequently a persistent microbiota in processing plants, which can shelter pathogenic species such as B. cereus s.s.

Producing molecular biology reagents without purification.

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.

Ribosome-Amplified Metabolism, RAMBO, Measured by NMR Spectroscopy.

NMR spectroscopy was used to investigate the phenomenon of ribosome-amplified metabolism or RAMBO between pyruvate kinase and ribosomes. Because the concentration of ribosomes increases as the cell grows, ribosome binding interactions may regulate metabolic fluxes by altering the distribution of bound and free enzymes. Pyruvate kinase (PK) catalyzes the last step of glycolysis and represents a major drug target for controlling bacterial infections. The binding of metabolic enzymes to ribosomes creates protein quinary structures with altered catalytic activities. NMR spectroscopy and chemical cross-linking combined with high-resolution mass spectrometry were used to establish that PK binds to ribosome at three independent sites, the L1 stalk, the A site, and the mRNA entry pore. The bioanalytical methodology described characterizes the altered kinetics and confirms the specificity of pyruvate kinase-ribosome interaction, affording an opportunity to investigate the ribosome dependence of metabolic reactions under solution conditions that closely mimic the cytosol. Expanding on the concept of ribosomal heterogeneity, which describes variations in ribosomal constituents that contribute to the specificity of cellular processes, this work firmly establishes the reciprocal process by which ribosome-dependent quinary interactions affect metabolic activity.

Comparison of conventional and extractive fermentation using aqueous two-phase system to extract fibrinolytic proteases produced by Bacillus stearothermophilus DPUA 1729.

Fibrinolytic enzymes have been considered promising for treatment and protection of healthy circulation due its ability to dissolve the fibrin in blood clots. Extractive fermentation is a not explored and efficient downstream process which segregates the desired product simultaneously in a fermentation process fast and economically. Extraction of fibrinolytic enzymes by Bacillus stearothermophilus DPUA 1729 employing conventional aqueous two-phase systems (ATPS) and extractive fermentation with ATPS was evaluated. The results of both systems were compared using a factorial design with PEG molar mass, PEG and salt concentrations as independent variables and extraction parameters as a response. In all conditions evaluated it was observed a similar partitioning of fibrinolytic enzymes through the phases, both in conventional ATPS and extractive fermentation. Salt concentration and interaction among PEG and salt concentration influenced in the partition coefficient. The fibrinolytic activity was determined by hydrolysis of fibrin in plate using the extract of one condition from extractive fermentation. The zone degradation presented a diameter of 7.03 ± 0.94 mm. In conclusion, there was no significant difference among the results obtained using conventional ATPS and extractive fermentation, however, the second one presents more advantages and can integrate production and extraction in one single step, reducing the costs.

Cross-utilization of ß-galactosides and cellobiose in Geobacillus stearothermophilus.

Strains of the Gram-positive, thermophilic bacterium Geobacillus stearothermophilus possess elaborate systems for the utilization of hemicellulolytic polysaccharides, including xylan, arabinan, and galactan. These systems have been studied extensively in strains T-1 and T-6, representing microbial models for the utilization of soil polysaccharides, and many of their components have been characterized both biochemically and structurally. Here, we characterized routes by which G. stearothermophilus utilizes mono- and disaccharides such as galactose, cellobiose, lactose, and galactosyl-glycerol. The G. stearothermophilus genome encodes a phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) for cellobiose. We found that the cellobiose-PTS system is induced by cellobiose and characterized the corresponding GH1 6-phospho-ß-glucosidase, Cel1A. The bacterium also possesses two transport systems for galactose, a galactose-PTS system and an ABC galactose transporter. The ABC galactose transport system is regulated by a three-component sensing system. We observed that both systems, the sensor and the transporter, utilize galactose-binding proteins that also bind glucose with the same affinity. We hypothesize that this allows the cell to control the flux of galactose into the cell in the presence of glucose. Unexpectedly, we discovered that G. stearothermophilus T-1 can also utilize lactose and galactosyl-glycerol via the cellobiose-PTS system together with a bifunctional 6-phospho-ß-gal/glucosidase, Gan1D. Growth curves of strain T-1 growing in the presence of cellobiose, with either lactose or galactosyl-glycerol, revealed initially logarithmic growth on cellobiose and then linear growth supported by the additional sugars. We conclude that Gan1D allows the cell to utilize residual galactose-containing disaccharides, taking advantage of the promiscuity of the cellobiose-PTS system.

Aspartic acid racemization constrains long-term viability and longevity of endospores.

Certain microorganisms survive long periods of time as endospores to cope with adverse conditions. Since endospores are metabolically inactive, the extent of aspartic acid (Asp) racemization will increase over time and might kill the spores by preventing their germination. Therefore, understanding the relationship between endospore survivability and Asp racemization is important for constraining the long-term survivability and global dispersion of spore-forming bacteria in nature. Geobacillus stearothermophilus was selected as a model organism to investigate racemization kinetics and survivability of its endospores at 65°C, 75°C and 98°C. This study found that the Asp racemization rates of spores and autoclaved spores were similar at all temperatures. The Asp racemization rate of spores was not significantly different from that of vegetative cells at 65°C. The Asp racemization rate of G. stearothermophilus spores was not significantly different from that of Bacillus subtilis spores at 98°C. The viability of spores and vegetative cells decreased dramatically over time, and the mortality of spores correlated exponentially with the degree of racemization (R2 = 0.9). This latter correlation predicts spore half-lives on the order of hundreds of years for temperatures typical of shallow marine sediments, a result consistent with studies about the survivability of thermophilic spores found in these environments.

Effect of Different Proportions of Three Microbial Agents on Ammonia Mitigation during the Composting of Layer Manure.

Odor emissions represent one of the important issues of aerobic composting. The addition of microbial agents to compost is an important method for solving this problem, but this process is often unstable when a single microbial agent is added to the compost. Therefore, in this study, five treatments comprising different proportions of Bacillus stearothermophilus, Candida utilis, and Bacillus subtilis were tested to determine the best combination of the three microbial agents for ammonia reduction, as follows: control group (CK), 2:1:1 (A), 1:1:2 (B), 1:2:1 (C), and 1:1:1 (D). Compared with the CK group, the A, B, C, and D groups reduced ammonia emissions by 17.02, 9.68, 53.11, and 46.23%, respectively. The total ammonia emissions were significantly lower in C and D than in CK (p < 0.05). These two treatment groups had significantly increased nitrate nitrogen concentrations and decreased pH values and ammonium nitrogen concentrations (p < 0.05). Throughout the composting process, the total bacterial number was significantly higher in C and D than in CK (p < 0.05). Therefore, it is likely that B. stearothermophilus, C. utilis, and B. subtilis compounded from 1:2:1 (C) to 1:1:1 (D) reduced the ammonia emissions due to (1) a reduction in the pH and (2) the promotion of the growth of ammonia-oxidizing bacteria and the conversion of ammonium nitrogen to nitrate nitrogen. This study provides a theoretical basis and technical support for the odor problem of layer manure compost and promotes the development of composting technology.

A Monostyryl Boradiazaindacene (BODIPY)-based lanthanide-free colorimetric and fluorogenic probe for sequential sensing of copper (II) ions and dipicolinic acid as a biomarker of bacterial endospores.

A new catechol-substituted monostyryl boradiazaindacene (BODIPY)-based lanthanide-free colorimetric and fluorogenic probe was developed for the sequential detection of Cu2+ ions and dipicolinic acid (DPA), a distinctive biomarker of bacterial endospores, with high sensitivity and selectivity. In the presence of Cu2+ ions, the blue solution of the probe changes to cyan and the fluorescence is quenched, however, the cyan color changes to blue immediately and the fluorescence is restored on contact with DPA, resulting from competitive binding of DPA that interact with Cu2+ ions. A practical application by using Geobacillus stearothermophilus spores was further studied and as low as 1.0 x 105 spores were detected.

Feasibility study on direct fermentation of soybean meal by Bacillus stearothermophilus under non-sterile conditions.

BACKGROUND: To evaluate the feasibility of high-temperature solid-state fermentation (SSF) using soybean meal (SBM) during the non-sterile process, Bacillus stearothermophilus was employed to assess the nutritional quality and bioactivity of SBM after fermentation. RESULTS: The fermented SBM (FSBM) without autoclaving showed significant improvements in nutritional quality and bioactivity. The contents of peptides and crude and soluble proteins increased by 131.21%, 5.3% and 15.52%, respectively. Meanwhile, DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging ability, reducing ability and hydroxyl free radical-scavenging activity rose by 57.07%, 238.92% and 368.26%, respectively. The inhibitory activity of angiotensin I-converting enzyme increased from 1.43 ± 0.83% to 26.89 ± 1.03%, while the trypsin inhibitor activity decreased by 74.05%. The contents of neutral and alkaline proteases and the growth of microorganisms in FSBM without autoclaving were higher and better than in steam-treated FSBM. After steam treatment, the water-holding capacity of SBM decreased, and a high crosslink density was observed on the surface of SBM particles. CONCLUSIONS: It is feasible to ferment SBM by high-temperature SSF using B. stearothermophilus under non-sterile conditions. Adverse effects of SSF using sterile SBM might be owing to the low water-holding capacity caused by autoclaving. © 2018 Society of Chemical Industry.

Fe-S Clusters and MutY Base Excision Repair Glycosylases: Purification, Kinetics, and DNA Affinity Measurements.

A growing number of iron-sulfur (Fe-S) cluster cofactors have been identified in DNA repair proteins. MutY and its homologs are base excision repair (BER) glycosylases that prevent mutations associated with the common oxidation product of guanine (G), 8-oxo-7,8-dihydroguanine (OG) by catalyzing adenine (A) base excision from inappropriately formed OG:A mispairs. The finding of an [4Fe-4S]2+ cluster cofactor in MutY, Endonuclease III, and structurally similar BER enzymes was surprising and initially thought to represent an example of a purely structural role for the cofactor. However, in the two decades subsequent to the initial discovery, purification and in vitro analysis of bacterial MutYs and mammalian homologs, such as human MUTYH and mouse Mutyh, have demonstrated that proper Fe-S cluster coordination is required for OG:A substrate recognition and adenine excision. In addition, the Fe-S cluster in MutY has been shown to be capable of redox chemistry in the presence of DNA. The work in our laboratory aimed at addressing the importance of the MutY Fe-S cluster has involved a battery of approaches, with the overarching hypothesis that understanding the role(s) of the Fe-S cluster is intimately associated with understanding the biological and chemical properties of MutY and its unique damaged DNA substrate as a whole. In this chapter, we focus on methods of enzyme expression and purification, detailed enzyme kinetics, and DNA affinity assays. The methods described herein have not only been leveraged to provide insight into the roles of the MutY Fe-S cluster but have also been provided crucial information needed to delineate the impact of inherited variants of the human homolog MUTYH associated with a colorectal cancer syndrome known as MUTYH-associated polyposis or MAP. Notably, many MAP-associated variants have been found adjacent to the Fe-S cluster further underscoring the intimate relationship between the cofactor, MUTYH-mediated DNA repair, and disease.

One Intact Transmembrane Substrate Binding Site Is Sufficient for the Function of the Homodimeric Type I ATP-Binding Cassette Importer for Positively Charged Amino Acids Art(MP)2 of Geobacillus stearothermophilus.

ATP-binding cassette (ABC) transport systems comprise two transmembrane domains/subunits that form a translocation path and two nucleotide-binding domains/subunits that bind and hydrolyze ATP. Prokaryotic canonical ABC import systems require an extracellular substrate-binding protein for function. Knowledge of substrate-binding sites within the transmembrane subunits is scarce. Recent crystal structures of the ABC importer Art(QN)2 for positively charged amino acids of Thermoanerobacter tengcongensis revealed the presence of one substrate molecule in a defined binding pocket in each of the transmembrane subunits, ArtQ (J. Yu, J. Ge, J. Heuveling, E. Schneider, and M. Yang, Proc Natl Acad Sci U S A 112:5243-5248, 2015, https://doi.org/10.1073/pnas.1415037112). This finding raised the question of whether both sites must be loaded with substrate prior to initiation of the transport cycle. To address this matter, we first explored the role of key residues that form the binding pocket in the closely related Art(MP)2 transporter of Geobacillus stearothermophilus, by monitoring consequences of mutations in ArtM on ATPase and transport activity at the level of purified proteins embedded in liposomes. Our results emphasize that two negatively charged residues (E153 and D160) are crucial for wild-type function. Furthermore, the variant Art[M(L67D)P]2 exhibited strongly impaired activities, which is why it was considered for construction of a hybrid complex containing one intact and one impaired substrate-binding site. Activity assays clearly revealed that one intact binding site was sufficient for function. To our knowledge, our study provides the first biochemical evidence on transmembrane substrate-binding sites of an ABC importer.IMPORTANCE Canonical prokaryotic ATP-binding cassette importers mediate the uptake of a large variety of chemicals, including nutrients, osmoprotectants, growth factors, and trace elements. Some also play a role in bacterial pathogenesis, which is why full understanding of their mode of action is of the utmost importance. One of the unsolved problems refers to the chemical nature and number of substrate binding sites formed by the transmembrane subunits. Here, we report that a hybrid amino acid transporter of G. stearothermophilus, encompassing one intact and one impaired transmembrane binding site, is fully competent in transport, suggesting that the binding of one substrate molecule is sufficient to trigger the translocation process.