PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease.

C2c1 is a newly identified guide RNA-mediated type V-B CRISPR-Cas endonuclease that site-specifically targets and cleaves both strands of target DNA. We have determined crystal structures of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Moreover, C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA. crRNA adopts a pre-ordered five-nucleotide A-form seed sequence in the binary complex, with release of an inserted tryptophan, facilitating zippering up of 20-bp guide RNA:target DNA heteroduplex on ternary complex formation. Notably, the PAM-interacting cleft adopts a "locked" conformation on ternary complex formation. Structural comparison of C2c1 ternary complexes with their Cas9 and Cpf1 counterparts highlights the diverse mechanisms adopted by these distinct CRISPR-Cas systems, thereby broadening and enhancing their applicability as genome editing tools.

C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism.

C2c1 is a type V-B CRISPR-Cas system dual-RNA-guided DNA endonuclease. Here, we report the crystal structure of Alicyclobacillus acidoterrestris C2c1 in complex with a chimeric single-molecule guide RNA (sgRNA). AacC2c1 exhibits a bi-lobed architecture consisting of a REC and NUC lobe. The sgRNA scaffold forms a tetra-helical structure, distinct from previous predictions. The crRNA is located in the central channel of C2c1, and the tracrRNA resides in an external surface groove. Although AacC2c1 lacks a PAM-interacting domain, our analysis revealed that the PAM duplex has a similar binding position found in Cpf1. Importantly, C2c1-sgRNA system is highly sensitive to single-nucleotide mismatches between guide RNA and target DNA. The resulting reduction in off-target cleavage renders C2c1 a valuable addition to the current arsenal of genome-editing tools. Together, our findings indicate that sgRNA assembly is achieved through a mechanism distinct from that reported previously for Cas9 or Cpf1 endonucleases.

Pyrococcus furiosus argonaute based Alicyclobacillus acidoterrestrsis detection in fruit juice.

Alicyclobacillus acidoterrestris is the major threat to fruit juice for its off-odor producing characteristic. In this study, Pyrococcus furiosus Argonaute (PfAgo), a novel endonuclease with precise DNA cleavage activity, was used for A. acidoterrestrisdetection, termed as PAD. The partially amplified 16 S rRNA gene of A. acidoterrestris can be cleaved by PfAgo activated by a short 5'-phosphorylated single strand DNA, producing a new guide DNA (gDNA). Then, PfAgo was activated by the new gDNA to cut a molecular beacon (MB) with fluorophore-quencher reporter, resulting in the recovery of fluorescence. The fluorescent intensity is positively related with the concentration of A. acidoterrestris. The PAD assay showed excellent specificity and sensitivity as low as 101 CFU/mL, which can be a powerful tool for on-site detection of A. acidoterrestris in fruit juice industry in the future, reducing the economic loss.

Exploration of Alicyclobacillus spp. Genome in Search of Antibiotic Resistance.

The study investigates the antibiotic resistance (AR) profiles and genetic determinants in three strains of guaiacol-producing Alicyclobacillus spp. isolated from orchard soil and pears. Their phenotypic characteristics, such as spore formation; resistance to different factors, including drugs or disinfectants; or production of off-flavor compounds, can affect the taste and aroma of spoiled products. Food and beverages are potential vectors for the transfer of antibiotic resistance genes, which is a growing health concern; thus, microorganisms in food and beverages should not be a potential source of drug resistance to consumers. Whole-genome sequencing (WGS) was utilized to identify antibiotic resistance genes, metabolic pathways, and elements associated with guaiacol and halophenol production. Minimum inhibitory concentration (MIC) testing revealed that all strains were susceptible to eight out of nine tested antibiotics (ampicillin, gentamycin, kanamycin, streptomycin, clindamycin, tetracycline, chloramphenicol, and vancomycin) but exhibited high resistance to erythromycin. Analysis indicated that the erythromycin resistance gene, ribosomal RNA small subunit methyltransferase A (RsmA), was intrinsic and likely acquired through horizontal gene transfer (HGT). The comprehensive genomic analysis provides insights into the molecular mechanisms of antibiotic resistance in Alicyclobacillus spp., highlighting the potential risk of these bacteria as vectors for antibiotic resistance genes in the food chain. This study expands the understanding of the genetic makeup of these spoilage bacteria and their role in antimicrobial resistance dissemination.

Transcriptomic analysis of the antibacterial mechanism of ε-polylysine-functionalized magnetic composites against Alicyclobacillus acidoterrestris and its application in apple juice.

BACKGROUND: Alicyclobacillus acidoterrestris is a common microorganism in fruit juice. It can produce off-odor metabolites and has been considered to be an important factor in juice contamination. Thus, the development of new strategy for the control of A. acidoterrestris has important practical significance. The primary objective of this work was to assess the antibacterial performance of ε-polylysine-functionalized magnetic composites (Fe3O4@MoS2@PAA-EPL) in apple juice and its effect on juice quality. Moreover, the molecular mechanism of Fe3O4@MoS2@PAA-EPL against A. acidoterrestris was explored by RNA sequencing (RNA-Seq). RESULTS: Experimental results indicated that the synthesized composites possessed the ability to inhibit the viability of A. acidoterrestris vegetative cells and spores. Besides, investigation on the quality of apple juice incubated with Fe3O4@MoS2@PAA-EPL implied that the fabricated composites displayed negligible adverse effects on juice quality. In addition, the results of RNA-Seq demonstrated that 833 differentially expressed genes (DEGs) were identified in Fe3O4@MoS2@PAA-EPL-treated A. acidoterrestris, which were associated with translation, energy metabolism, amino acid metabolism, membrane transport and cell integrity. CONCLUSION: These results suggested that the treatment of Fe3O4@MoS2@PAA-EPL disrupted energy metabolism, repressed cell wall synthesis and caused membrane transport disorder of bacterial cells. This work provides novel insights into the molecular antibacterial mechanism for ε-polylysine-functionalized magnetic composites against A. acidoterrestris. © 2024 Society of Chemical Industry.

Contribution of amino acids to Alicyclobacillus acidoterrestris DSM 3922T resistance towards acid stress.

Spoilage of juice and beverages by a thermo-acidophilic bacterium, Alicyclobacillus acidoterrestris, has been considered to be a major and widespread concern for juice industry. Acid-resistant property of A. acidoterrestris supports its survival and multiplication in acidic juice and challenges the development of corresponding control measures. In this study, intracellular amino acid differences caused by acid stress (pH 3.0, 1 h) were determined by targeted metabolomics. The effect of exogenous amino acids on acid resistance of A. acidoterrestris and the related mechanisms were also investigated. The results showed that acid stress affected the amino acid metabolism of A. acidoterrestris, and the selected glutamate, arginine, and lysine contributed to its survival under acid stress. Exogenous glutamate, arginine, and lysine significantly increased the intracellular pH and ATP level, alleviated cell membrane damage, reduced surface roughness, and suppressed deformation caused by acid stress. Additionally, the up-regulated gadA and speA genes and the enhanced enzymatic activity confirmed that glutamate and arginine decarboxylase systems played a crucial role in maintaining pH homeostasis of A. acidoterrestris under acid stress. Our research reveals an important factor contributing to acid resistance of A. acidoterrestris, which provides an alternative target for effectively controlling this contaminant in fruit juices.

Computational analysis of carboxylesterase genes and proteins in non-pathogenic food bacterium Alicyclobacillus acidocaldarius: insights from proteogenomics.

Over recent years, Alicyclobacillus acidocaldarius, a Gram-positive nonpathogenic rod-shaped thermo-acid-tolerant bacterium, has posed numerous challenges for the fruit juice industry. However, the bacterium's unique characteristics, particularly its nonpathogenic and thermophilic capabilities, offer significant opportunities for genetic exploration by biotechnologists. This study presents the computational proteogenomics report on the carboxylesterase (CE) enzyme in A. acidocaldarius, shedding light on structural and evolutional of CEs from this bacterium. Our analysis revealed that the average molecular weight of CEs in A. acidocaldarius was 41 kDa, with an isoelectric point around 5. The amino acid composition favored negative amino acids over positive ones. The aliphatic index and hydropathicity were approximately 88 and - 0.15, respectively. While the protein sequence showed no disulfide bonds in the CEs' structure, the presence of Cys amino acids was observed in the structure of CEs. Phylogenetic analysis presented more than 99% similarity between CEs, indicating their close evolutionary relationship. By applying homology modeling, the 3-dimensional structural models of the carboxylesterase were constructed, which with the help of structural conservation and solvent accessibility analysis highlighted key residues and regions responsible for enzyme stability and conformation. The specific patterns presented the total solvent accessibility of less than 25 (Å2) was in considerable position as well as Gly residues were noticeably have high accessibility to solvent in all structures. Ala was the more frequent amino acids in the conserved-SASA of carboxylesterases. Furthermore, unsupervised agglomerative hierarchical clustering based on solvent accessibility feature successfully clustered and even distinguished this enzyme from proteases from the same genome. These findings contribute to a deeper understanding of the nonpathogenic A. acidocaldarius carboxylesterase and its potential applications in biotechnology. Additionally, structural analysis of CEs would help to address potential solutions in fruit juice industry with utilization of computational structural biology.

Insight into CAZymes of Alicyclobacillus mali FL18: Characterization of a New Multifunctional GH9 Enzyme.

In the bio-based era, cellulolytic and hemicellulolytic enzymes are biocatalysts used in many industrial processes, playing a key role in the conversion of recalcitrant lignocellulosic waste biomasses. In this context, many thermophilic microorganisms are considered as convenient sources of carbohydrate-active enzymes (CAZymes). In this work, a functional genomic annotation of Alicyclobacillus mali FL18, a recently discovered thermo-acidophilic microorganism, showed a wide reservoir of putative CAZymes. Among them, a novel enzyme belonging to the family 9 of glycosyl hydrolases (GHs), named AmCel9, was identified; in-depth in silico analyses highlighted that AmCel9 shares general features with other GH9 members. The synthetic gene was expressed in Escherichia coli and the recombinant protein was purified and characterized. The monomeric enzyme has an optimal catalytic activity at pH 6.0 and has comparable activity at temperatures ranging from 40 °C to 70 °C. It also has a broad substrate specificity, a typical behavior of multifunctional cellulases; the best activity is displayed on ß-1,4 linked glucans. Very interestingly, AmCel9 also hydrolyses filter paper and microcrystalline cellulose. This work gives new insights into the properties of a new thermophilic multifunctional GH9 enzyme, that looks a promising biocatalyst for the deconstruction of lignocellulose.

Mobile genetic elements mediate the mixotrophic evolution of novel Alicyclobacillus species for acid mine drainage adaptation.

Alicyclobacillus species inhabit diverse environments and have adapted to broad ranges of pH and temperature. However, their adaptive evolutions remain elusive, especially regarding the role of mobile genetic elements (MGEs). Here, we characterized the distributions and functions of MGEs in Alicyclobacillus species across five environments, including acid mine drainage (AMD), beverages, hot springs, sediments, and soils. Nine Alicyclobacillus strains were isolated from AMD and possessed larger genome sizes and more genes than those from other environments. Four AMD strains evolved to be mixotrophic and fell into distinctive clusters in phylogenetic tree. Four types of MGEs including genomic island (GI), insertion sequence (IS), prophage, and integrative and conjugative element (ICE) were widely distributed in Alicyclobacillus species. Further, AMD strains did not possess CRISPR-Cas systems, but had more GI, IS, and ICE, as well as more MGE-associated genes involved in the oxidation of iron and sulfide and the resistance of heavy metal and low temperature. These findings highlight the differences in phenotypes and genotypes between strains isolated from AMD and other environments and the important role of MGEs in rapid environment niche expansions.

Evaluation of temperature, pH and nutrient conditions in bacterial growth and extracellular hydrolytic activities of two Alicyclobacillus spp. strains.

Extremophile bacteria have developed the metabolic machinery for living in extreme temperatures, pH, and high-salt content. Two novel bacterium strains Alicyclobacillus sp. PA1 and Alicyclobacillus sp. PA2, were isolated from crater lake El Chichon in Chiapas, Mexico. Phylogenetic tree analysis based on the 16SrRNA gene sequence revealed that the strain Alicyclobacillus sp. PA1 and Alicyclobacillus sp. PA2 were closely related to Alicyclobacillus species (98% identity and 94.73% identity, respectively). Both strains were Gram variable, and colonies were circular, smooth and creamy. Electron microscopy showed than Alicyclobacillus sp. PA1 has a daisy-like form and Alicyclobacillus sp. PA2 is a regular rod. Both strains can use diverse carbohydrates and triglycerides as carbon source and they also can use organic and inorganic nitrogen source. But, the two strains can grow without any carbon or nitrogen sources in the culture medium. Temperature, pH and nutrition condition affect bacterial growth. Maximum growth was produced at 65 °C for Alicyclobacillus sp. PA1 (0.732 DO600) at pH 3 and Alicyclobacillus sp. PA2 (0.725 DO600) at pH 5. Inducible extracellular extremozyme activities were determined for ß-galactosidase (Alicyclobacillus sp. PA1: 88.07 ± 0.252 U/mg, Alicyclobacillus sp. PA2: 51.57 ± 0.308 U/mg), cellulose (Alicyclobacillus sp. PA1: 141.20 ± 0.585 U/mg, Alicyclobacillus sp. PA2: 51.57 ± 0.308 U/mg), lipase (Alicyclobacillus sp. PA1: 138.25 ± 0.600 U/mg, Alicyclobacillus sp. PA2: 175.75 ± 1.387 U/mg), xylanase (Alicyclobacillus sp. PA1: 174.72 ± 1.746 U/mg, Alicyclobacillus sp. PA2: 172.69 ± 0.855U/mg), and protease (Alicyclobacillus sp. PA1: 15.12 ± 0.121 U/mg, Alicyclobacillus sp. PA2: 15.33 ± 0.284 U/mg). These results provide new insights on extreme enzymatic production on Alicyclobacillus species.

Alicyclobacillus mali sp. nov., Alicyclobacillus suci sp. nov. and Alicyclobacillus fructus sp. nov., thermoacidophilic sporeforming bacteria isolated from fruit beverages.

Six thermo-acidophilic, spore-forming strains were isolated from a variety of juice products and were characterized genetically and phenotypically. According to 16S rRNA and rpoB gene phylogenetic analyses and average nucleotide identity comparisons against the species demarcation cutoff at <95 %, these six strains were determined to represent three novel species of Alicyclobacillus. The isolates were designated FSL-W10-0018T, FSL-W10-0037, FSL-W10-0048, VF-FSL-W10-0049T, FSL-W10-0057 and FSL-W10-0059T. All six isolates were Gram-positive, motile, rod shaped, contained menaquinone 7 as the major respiratory quinone and had ω-cyclohexane C17 : 0 as a major fatty acid. They were all able to grow aerobically in a range of acidic and moderate thermal conditions. Only isolates FSL-W10-0048 and VF-FSL-W10-0049T were able to produce guaiacol. The following names are proposed for the three new species: Alicyclobacillus mali sp. nov. (type strain FSL-W10-0018T =DSM 112016T=NCIMB 15266T); Alicyclobacillus suci sp. nov (VF-FSL-W10-0049T=DSM 112017T=NCIMB 15265T); and Alicyclobacillus fructus sp. nov. (FSL-W10-0059T=DSM 112018T=NCIMB 15264T).

New insights into thermo-acidophilic properties of Alicyclobacillus acidoterrestris after acid adaptation.

Alicyclobacillus acidoterrestris has unique thermo-acidophilic properties and is the main cause of fruit juice deterioration. Given the acidic environment and thermal treatment during juice processing, the effects of acid adaptation (pH 3.5, 3.2, and 3.0) on the resistance of A. acidoterrestris to heat (65 °C, 5 min) and acid (pH = 2.2, 1 h) stresses were investigated for the first time. The results showed that acid adaptation induced cross-protection against heat stress of A. acidoterrestris and acid tolerance response, and the extent of induced tolerance was increased with the decrease of adaptive pH values. Acid adaptation treatments did not disrupt the membrane potential stability and intracellular pH homeostasis, but reduced intracellular ATP concentration, increased cyclic fatty acids content, and changed the acquired Fourier transform infrared spectra. Transcription levels of stress-inducible (dnaK, grpE, clpP, ctsR) genes and genes related to spore formation (spo0A, ctoX) were up-regulated after acid adaptation, and spore formation was observed by scanning electron microscopy. This study revealed that the intracellular microenvironment homeostasis, expression of chaperones and proteases, and spore formation played a coordinated role in acid stress adaptive responses, with implications for applications in fruit juice processing.

Heat resistance and genomics of spoilage Alicyclobacillus spp. Isolated from fruit juice and fruit-based beverages.

Alicyclobacillus acidoterrestris is a spore-forming bacterium of importance to the fruit juice industry due to its remarkable heat resistance and production of guaiacol taint. Whole genome sequencing analysis reveals species demarcation corresponds to the two major genotypic groups to which A. acidoterrestris isolates belong. Heat resistance was significantly different between genotypic groups 1 and 2 with D90 values of 15.5 and 9.3 min, respectively (p < 0.01). Comparison of squalene-hopene cyclase (shc) encoding sequences reveals non-synonymous changes and the alteration of glutamine residues. Glutamine absence may link to the stability reinforcement of the enzyme structure against thermal denaturation. Genomic islands harbouring heavy metal resistance genes are found in the majority of genotypic group 1 genomes (63%) but occurs in only one genome (5%) of genotypic group 2. Distribution of the genomic islands in the genotypic groups 1 and 2 is also consistent with phylogenetic trees and ANI and dDDH values. Subsequently, we propose genotypic group 1 as a new species closely related to A. acidoterrestris that possesses enhanced heat resistance.

Analysis of differential expression proteins reveals the key pathway in response to heat stress in Alicyclobacillus acidoterrestris DSM 3922T.

For the purpose of investigating the heat resistance mechanism of Alicyclobacillus acidoterrestris, label-free quantification was used to reveal some cellular changes in A. acidoterrestris during heat stress. Totally, 545 differential expression proteins were respectively identified at heat stress of 65 °C for 5 min, of which 258 proteins were up-regulated and 287 proteins were down-regulated. These significantly changed proteins were mapped to 100 pathways and some of them were mostly related to protection or repair of macromolecules such as proteins and DNA, cell wall formation, which indicated that these proteins might play crucial roles in response to heat stress. The KEGG pathway analysis combined with protein functional analysis and further validation at mRNA level suggested that A. acidoterrestris sensed the temperature rise in environment through alterations in the secondary structure of DNA and RNA molecules. The biosynthesis of antibiotics pathway and the ribosomes might be involved in signal transduction in heat stress and further trigger a large number of proteins playing a critical role in the regulation of heat stress in A. acidoterrestris. The study firstly demonstrated the global physiological response to heat stress and the results provided a better understanding of thermal adaption mechanism of A. acidoterrestris.

Linear enzyme cascade for the production of (-)-iso-isopulegol.

Biocatalysis has developed enormously in the last decade and now offers solutions for the sustainable production of chiral and highly functionalised asset molecules. Products generated by enzymatic transformations are already being used in the food, feed, chemical, pharmaceutical and cosmetic industry, and the accessible compound panoply is expected to expand even further. In particular, the combination of stereo-selective enzymes in linear cascade reactions is an elegant strategy toward enantiomeric pure compounds, as it reduces the number of isolation and purification steps and avoids accumulation of potentially unstable intermediates. Here, we present the set-up of an enzyme cascade to selectively convert citral to (-)-iso-isopulegol by combining an ene reductase and a squalene hopene cyclase. In the initial reaction step, the ene reductase YqjM from Bacillus subtilis selectively transforms citral to (S)-citronellal, which is subsequently cyclised exclusively to (-)-iso-isopulegol by a mutant of the squalene hopene cyclase from Alicyclobacillus acidocaldarius (AacSHC). With this approach, we can convert citral to an enantiopure precursor for isomenthol derivatives.

Comparison of Alicyclobacillus acidocaldarius o-Succinylbenzoate Synthase to Its Promiscuous N-Succinylamino Acid Racemase/ o-Succinylbenzoate Synthase Relatives.

Studying the evolution of catalytically promiscuous enzymes like those from the N-succinylamino acid racemase/ o-succinylbenzoate synthase (NSAR/OSBS) subfamily can reveal mechanisms by which new functions evolve. Some enzymes in this subfamily have only OSBS activity, while others catalyze OSBS and NSAR reactions. We characterized several NSAR/OSBS subfamily enzymes as a step toward determining the structural basis for evolving NSAR activity. Three enzymes were promiscuous, like most other characterized NSAR/OSBS subfamily enzymes. However, Alicyclobacillus acidocaldarius OSBS (AaOSBS) efficiently catalyzes OSBS activity but lacks detectable NSAR activity. Competitive inhibition and molecular modeling show that AaOSBS binds N-succinylphenylglycine with moderate affinity in a site that overlaps its normal substrate. On the basis of possible steric conflicts identified by molecular modeling and sequence conservation within the NSAR/OSBS subfamily, we identified one mutation, Y299I, that increased NSAR activity from undetectable to 1.2 × 102 M-1 s-1 without affecting OSBS activity. This mutation does not appear to affect binding affinity but instead affects kcat, by reorienting the substrate or modifying conformational changes to allow both catalytic lysines to access the proton that is moved during the reaction. This is the first site known to affect reaction specificity in the NSAR/OSBS subfamily. However, this gain of activity was obliterated by a second mutation, M18F. Epistatic interference by M18F was unexpected because a phenylalanine at this position is important in another NSAR/OSBS enzyme. Together, modest NSAR activity of Y299I AaOSBS and epistasis between sites 18 and 299 indicate that additional sites influenced the evolution of NSAR reaction specificity in the NSAR/OSBS subfamily.

Description of Alicyclobacillus montanus sp. nov., a mixotrophic bacterium isolated from acidic hot springs.

Three morphologically similar thermo-acidophilic strains, USBA-GBX-501, USBA-GBX-502 and USBA-GBX-503T, were isolated from acidic thermal springs at the National Natural Park Los Nevados (Colombia). All isolates were spore-forming, Gram-stain-positive and motile, growing aerobically at 25-55 °C (optimum ~45 °C) and at pH 1.5-4.5 (optimum pH ~3.0). Phylogenetic analysis of the 16S rRNA gene sequences of these isolates showed an almost identical sequence (99.0 % similarity) and they formed a robust cluster with the closest relative Alicyclobacillus tolerans DSM 16297T with a sequence similarity of 99.0 %. Average similarity to other species of the genus Alicyclobacillus was 93.0 % and average similarity to species of the genus Effusibacillus was 90 %. In addition, the level of DNA-DNA hybridization between strain USBA-GBX-503T and Alicyclobacillus tolerans DSM 16297T was 31.7 %. The genomic DNA G+C content of strain USBA-GBX-503T was 44.6 mol%. The only menaquinone was MK-7 (100.0 %). No ω-alicyclic fatty acids were detected in strain USBA-GBX-503T, and the major cellular fatty acids were C18 : 1ω7c, anteiso-C17 : 0 and iso-C17 : 0. Based on phenotypic and chemotaxonomic characteristics, phylogenetic analysis and DNA-DNA relatedness values, along with low levels of identity at the whole genome level (ANIb and ANIm values of <67.0 and <91.0 %, respectively), it can be concluded that strain USBA-GBX-503T represents a novel species of the genus Alicyclobacillus, for which the name Alicyclobacillus montanus sp. nov. is proposed. The type strain is USBA-GBX-503T (=CMPUJ UGB U503T=CBMAI1927T).

Introducing transgalactosylation activity into a family 42 ß-galactosidase.

Chemo-enzymatic synthesis of oligosaccharides exploits the diversity of glycosidases and their ability to promote transglycosylation reactions in parallel with hydrolysis. Methods to increase the transglycosylation/hydrolysis ratio include site-directed mutagenesis and medium modification. The former approach was successful in several cases and has provided the best synthetic yields with glycosynthases-mutants at the catalytic nucleophile position that promote transglycosylation with high efficiency, but do not hydrolyze the oligosaccharide products. Several glycosidases have proven recalcitrant to this conversion, thus alternative methods to increase the transglycosylation/hydrolysis ratio by mutation would be very useful. Here we show that a mutant of a ß-galactosidase from Alicyclobacillus acidocaldarius in an invariant residue in the active site of the enzymes of this family (glutamic acid 361) carries out efficient transglycosylation reactions on different acceptors only in the presence of external ions with yields up to 177-fold higher than that of the wild type. This is the first case in which sodium azide and sodium formate in combination with site-directed mutagenesis have been used to introduce transglycosylation activity into a glycosidase. These observations will hopefully guide further efforts to generate useful synthases.

Concurrent metabolism of pentose and hexose sugars by the polyextremophile Alicyclobacillus acidocaldarius.

Alicyclobacillus acidocaldarius is a thermoacidophilic bacterium capable of growth on sugars from plant biomass. Carbon catabolite repression (CCR) allows bacteria to focus cellular resources on a sugar that provides efficient growth, but also allows sequential, rather than simultaneous use when more than one sugar is present. The A. acidocaldarius genome encodes all components of CCR, but transporters encoded are multifacilitator superfamily and ATP-binding cassette-type transporters, uncommon for CCR. Therefore, global transcriptome analysis of A. acidocaldarius grown on xylose or fructose was performed in chemostats, followed by attempted induction of CCR with glucose or arabinose. Alicyclobacillus acidocaldarius grew while simultaneously metabolizing xylose and glucose, xylose and arabinose, and fructose and glucose, indicating that CCR did not control carbon metabolism. Microarrays showed down-regulation of genes during growth on one sugar compared to two, and occurred primarily in genes encoding: (1) regulators; (2) enzymes for cell wall synthesis; and (3) sugar transporters.

Identification and Characterization of a New 7-Aminocephalosporanic Acid Deacetylase from Thermophilic Bacterium Alicyclobacillus tengchongensis.

UNLABELLED: Deacetylation of 7-aminocephalosporanic acid (7-ACA) at position C-3 provides valuable starting material for producing semisynthetic ß-lactam antibiotics. However, few enzymes have been characterized in this process before now. Comparative analysis of the genome of the thermophilic bacterium Alicyclobacillus tengchongensis revealed a hypothetical protein (EstD1) with typical esterase features. The EstD1 protein was functionally cloned, expressed, and purified from Escherichia coli BL21(DE3). It indeed displayed esterase activity, with optimal activity at around 65°C and pH 8.5, with a preference for esters with short-chain acyl esters (C2 to C4). Sequence alignment revealed that EstD1 is an SGNH hydrolase with the putative catalytic triad Ser15, Asp191, and His194, which belongs to carbohydrate esterase family 12. EstD1 can hydrolyze acetate at the C-3 position of 7-aminocephalosporanic acid (7-ACA) to form deacetyl-7-ACA, which is an important starting material for producing semisynthetic ß-lactam antibiotics. EstD1 retained more than 50% of its initial activity when incubated at pH values ranging from 4 to 11 at 65°C for 1 h. To the best of our knowledge, this enzyme is a new SGNH hydrolase identified from thermophiles that is able to hydrolyze 7-ACA. IMPORTANCE: Deacetyl cephalosporins are highly valuable building blocks for the industrial production of various kinds of semisynthetic ß-lactam antibiotics. These compounds are derived mainly from 7-ACA, which is obtained by chemical or enzymatic processes from cephalosporin C. Enzymatic transformation of 7-ACA is the main method because of the adverse effects chemical deacylation brought to the environment. SGNH hydrolases are widely distributed in plants. However, the tools for identifying and characterizing SGNH hydrolases from bacteria, especially from thermophiles, are rather limited. Here, our work demonstrates that EstD1 belongs to the SGNH family and can hydrolyze acetate at the C-3 position of 7-ACA. Moreover, this study can enrich our understanding of the functions of these enzymes from this family.