Programmed genome editing by a miniature CRISPR-Cas12f nuclease.

The RNA-guided CRISPR-associated (Cas) nucleases are versatile tools for genome editing in various organisms. The large sizes of the commonly used Cas9 and Cas12a nucleases restrict their flexibility in therapeutic applications that use the cargo-size-limited adeno-associated virus delivery vehicle. More compact systems would thus offer more therapeutic options and functionality for this field. Here, we report a miniature class 2 type V-F CRISPR-Cas genome-editing system from Acidibacillus sulfuroxidans (AsCas12f1, 422 amino acids). AsCas12f1 is an RNA-guided endonuclease that recognizes 5' T-rich protospacer adjacent motifs and creates staggered double-stranded breaks to target DNA. We show that AsCas12f1 functions as an effective genome-editing tool in both bacteria and human cells using various delivery methods, including plasmid, ribonucleoprotein and adeno-associated virus. The small size of AsCas12f1 offers advantages for cellular delivery, and characterizations of AsCas12f1 may facilitate engineering more compact genome-manipulation technologies.

Cohnella pontilimi sp. nov., isolated from tidal-flat mud.

A Gram-positive, aerobic, endospore-forming, rod-shaped bacterial strain, CAU 1483 T, was isolated from tidal-flat mud in the Republic of Korea. It grew optimally at 30 °C, in a pH 7.0 medium with 2% (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain CAU 1483 T formed a separate clade within Paenibacillaceae together with members of the genus Cohnella. Strain CAU 1483 T exhibited the highest 16S rRNA gene sequence similarity (97.1%) to C. candidum 18JY8-7 T. Whole genome of strain CAU 1483 T was 4.29 Mb in size with a 53.7 mol% G + C content, and included 4046 coding sequences and included 4046 coding sequences, some of which associated with stress response. The average nucleotide identity and digital DNA-DNA hybridization similarity between strain CAU 1483 T and related members of the genus Cohnella were 71.8-74.9% and 22.6-33.9%, respectively. The major respiratory quinone present in this strain was menaquinone-7. Strain CAU 1483 T contained anteiso-C15:0 and iso-C16:0 as the major fatty acids, while its polar lipids consisted of phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, lysyl-phosphatidylglycerol, phosphatidylcholine, three unidentified aminophospholipids, two unidentified lipids and an unidentified phospholipid. Peptidoglycan type was A1γ meso-Dpm. On the basis of taxonomic characterization, strain CAU 1483 T constitutes a novel species, for which the name Cohnella pontilimi sp. nov. is proposed. The type strain of this novel species is CAU 1483 T (= KCTC 43047 T = NBRC 113953 T).

Fructuronate-tagaturonate epimerase UxaE from Cohnella laeviribosi has a versatile TIM-barrel scaffold suitable for a sugar metabolizing biocatalyst.

Xylan and pectin are major structural components of plant cell walls. There are two independent catabolic pathways for xylan and pectin. UxaE bridges these two pathways by reversibly epimerizing D-fructuronate and D-tagaturonate. The crystal structure of UxaE from Cohnella laeviribosi (ClUxaE) shows a core scaffold of TIM-barrel with a position-changing divalent metal cofactor. ClUxaE has the flexible metal-coordination loop to allow the metal shift and the extra domains to bind a phosphate ion in the active site, which are important for catalysis and substrate specificity. Elucidation of the structure and mechanism of ClUxaE will assist in understanding the catalytic mechanism of UxaE family members, which are useful for processing both xylan and pectin-derived carbohydrates for practical and industrial purposes, including the transformation of agricultural wastes into numerous valuable products.

Modulation of innate immunity, expression of cytokine genes and disease resistance against Aeromonas hydrophila infection in goldfish (Carassius auratus) by dietary supplementation with Exiguobacterium acetylicum S01.

Probiotic strains play an increasing role in the production of healthy animals used as a food source. Elucidating the mechanisms of action that allow probiotic-driven immunomodulation may facilitate different applications such as the prevention of infectious diseases in food organisms. This study elucidates the probiotic effects of Exiguobacterium acetylicum S01 on the growth, haematological profile, innate immune capacity, expression of cytokine genes, and resistance to diseases of Carassius auratus caused by Aeromonas hydrophila infection. Three fish groups were fed with the following diets containing different doses of E. acetylicum S01 (CFU g-1): basal diet 0 (BD, without probiotic), 2.5 × 107 (DI) and 2.7 × 109 (DII)-CFU g-1 for 4 weeks. After 4 weeks, the fish were injected intraperitoneally with A. hydrophila and the percentage of survival was recorded over 21 days of post-challenge. Results revealed that dietary supplementation of E. acetylicum S01 significantly (P < 0.05) enhanced the growth, haematological profile and cellular immune responses including respiratory burst, phagocytic activities and antimicrobial enzymes (myeloperoxidase and lysozyme) and total immunoglobulin levels were improved by probiotic feeding at both occasions. Comparatively, expression of c- and g-type lysozyme followed by pro- and anti-inflammatory cytokines (IL-1ß, IL-10 and TGFß) was up-regulated in kidney, head-kidney and spleen. Moreover, fish fed with diet DII had a significantly higher (P < 0.05) survival rate (73.2%) after challenging. The survival rate was only 33.2% of the BD group against A. hydrophila infection. Our results revealed that E. acetylicum S01 delivered probiotic in feed exerts its influence on growth performance and provides disease resistance by stimulating the immune system at the cellular and molecular levels in C. auratus.

Improved biotransformation of arsenic by arsenite oxidase - Chitosan nanoparticle conjugates.

Recent developments in the potential use of nanoparticles as carriers of enzyme have attracted great attention. In the present study, arsenite oxidase (AOase) enzyme capable of transforming the more toxic arsenite [As(III)] to the less toxic arsenate [As(V)] was extracted from an arsenic resistant bacterium (Exiguobacterium sp. As-9) and partially purified. Chitosan nanoparticles were prepared on the basis of ionic gelation of chitosan with tripolyphosphate (TPP) anions. The purified AOase was immobilized efficiently by physical adsorption on to chitosan nanoparticles and were characterized for particle size, morphology, zeta potential, AOase loading efficiency and in vitro transformation assay. The chitosan nanoparticles were spherical in shape with the average diameter of 100nm which increased to 294nm upon successful loading of AOase. Under optimized conditions, the loading capacity of the chitosan nanoparticle was determined to be 71% for AOase. Further, immobilization also increased the stability of AOase at varying temperature (4-37°C) and pH (5-10) for a period of 30days with the increased enzymatic activity (159.57Uml-1). It also facilitated increased biotransformation (89%) of As(III) to As(V). A conceptual understanding of biological responses to AOase loaded chitosan nanoparticles is needed for the development of novel methods of drug delivery.

Cold adaptation of tRNA nucleotidyltransferases: A tradeoff in activity, stability and fidelity.

Cold adaptation is an evolutionary process that has dramatic impact on enzymatic activity. Increased flexibility of the protein structure represents the main evolutionary strategy for efficient catalysis and reaction rates in the cold, but is achieved at the expense of structural stability. This results in a significant activity-stability tradeoff, as it was observed for several metabolic enzymes. In polymerases, however, not only reaction rates, but also fidelity plays an important role, as these enzymes have to synthesize copies of DNA and RNA as exact as possible. Here, we investigate the effects of cold adaptation on the highly accurate CCA-adding enzyme, an RNA polymerase that uses an internal amino acid motif within the flexible catalytic core as a template to synthesize the CCA triplet at tRNA 3'-ends. As the relative orientation of these residues determines nucleotide selection, we characterized how cold adaptation impacts template reading and fidelity. In a comparative analysis of closely related psychro-, meso-, and thermophilic enzymes, the cold-adapted polymerase shows a remarkable error rate during CCA synthesis in vitro as well as in vivo. Accordingly, CCA-adding activity at low temperatures is not only achieved at the expense of structural stability, but also results in a reduced polymerization fidelity.

Bacterial Inoculant Treatment of Bermudagrass Alters Ovipositional Behavior, Larval and Pupal Weights of the Fall Armyworm (Lepidoptera: Noctuidae).

Nonpathogenic soil bacteria can colonize the rhizosphere and induce unique plant phenotypes that may influence plant-insect interactions. However, few studies have considered the influences of bacteria-plant interactions on insect feeding and oviposition. The objective of this study was to determine how rhizobacterial inoculation of bermudagrass affects larval development and ovipositional behaviors of the fall armyworm (Spodoptera frugiperda J.E. Smith). Eight blends of rhizobacteria known to induce root or shoot growth in grasses were applied weekly to hybrid bermudagrass for 5 wk. Oviposition was evaluated in two no-choice trials with bacteria-treated, fertilized, or nontreated grass. Grass blades from these treatments were extracted in polar and nonpolar solvents and assayed for oviposition responses. Another experiment compared the development of fall armyworm larvae on bermudagrass treated with each of the eight rhizobacterial blends for 5 wk to larvae fed nontreated bermudagrass. Females deposited more eggs on nontreated and fertilized grass and ≤34% of eggs on grass treated with rhizobacterial blends. Moths exposed to polar and nonpolar extracts were unable to reproduce these results. Larval and pupal weights at days 10 and 12 and the number of adults to eclose were lower for larvae fed some, but not all, bacteria-treated bermudagrass relative to controls. This is one of the few studies to investigate plant-microbe-insect interactions in an economically important system. Although the effects noted with fall armyworm are limited, induced changes in roots also reported for these bacteria may have greater utility than foliar changes for mediating interactions with biotic or abiotic stresses.

Detection and quantification of Alicyclobacillus acidoterrestris by electrical impedance in apple juice.

Alicyclobacillus acidoterrestris is a thermotolerant bacterium able to grow in fruit juices and drinks, as the spoilage by Alicyclobacillus in the final product does not product any gas, but leads to a "medicine flavor" due to the formation of guaicol. Also, its detection is a challenge for the quality control departments, because it takes several days to get the results of traditional microbiology methods. This study aimed at developing a more accurate electrical impedance technique for the detection of A. acidoterrestris in concentrated apple juice. Samples of apple juice were inoculated with A. acidoterrestris spores isolated from a peach and grape juice. For the spore germination, several heat-shock treatments were tested (80 °C/10 min, 70 °C/20 min and 60 °C/30 min). Direct and indirect electrical impedance was applied to detect and quantify the microorganism in the inoculated apple juice, using BAT broth and Bimedia 002A (pH 4). The 80 °C/10 min treatment was selected for spore activation. The valid electrical impedance technique was the indirect method in BAT broth, which measured the changes in the impedance through the formation of CO2. In addition, a positive correlation (r = 0.98, R2 = 0.97) was observed between the classical microbiology (BAM agar) and the indirect impedance method.

Antifungal peptidic compound from the deep-sea bacterium Aneurinibacillus sp. YR247.

Aneurinibacillus: sp. YR247 was newly isolated from the deep-sea sediment inside the Calyptogena community at a depth of 1171 m in Sagami Bay. The strain exhibited antifungal activity against the filamentous fungus Aspergillus brasiliensis NBRC9455. A crude extract prepared from the YR247 cells by ethanol extraction exhibited broad antimicrobial activities. The antifungal compound is stable at 4-70 °C and pH 2.0-12.0. After treatment with proteinase K, the antifungal activity was not detected, indicating that the antifungal compound of strain YR247 is a peptidic compound. Electrospray ionization mass spectrometry of the purified antifungal compound indicated that the peptidic compound has an average molecular weight of 1167.9. The molecular weight of the antifungal compound from strain YR247 is different from those of antimicrobial peptides produced by the related Aneurinibacillus and Bacillus bacteria. The antifungal peptidic compound from the deep-sea bacterium Aneurinibacillus sp. YR247 may be useful as a biocontrol agent.

Aneurinifactin, a new lipopeptide biosurfactant produced by a marine Aneurinibacillus aneurinilyticus SBP-11 isolated from Gulf of Mannar: Purification, characterization and its biological evaluation.

Biosurfactants are microbial-derived amphiphilic molecules having hydrophobic and hydrophilic moieties produced by bacteria, fungi, yeasts and algae and are extracellular or cell wall-associated compounds. In an ongoing survey for bioactive microbial metabolites from microbes isolated from diverse ecological niches, a new lipopeptide biosurfactant was identified from a marine bacterium; Aneurinibacillus aneurinilyticus strain SBP-11, which was isolated from a marine diversity hotspot, Gulf of Mannar, India. A new lipopeptide biosurfactant was purified and characterized based on TLC, FT-IR, NMR, GC-MS, HPLC, MALDI-TOF-MS and tandem MS analysis as Stearic acid-Thr-Tyr-Val-Ser-Tyr-Thr (named as Aneurinifactin). The critical micelle concentration of Aneurinifactin was 26mgL-1 at a surface tension of 26mNm-1. Further, the biosurfactant showed stable emulsification at a wide range of pH (2-9) and temperature up to 80°C. Aneurinifactin showed promising antimicrobial activity and concentration dependent efficient oil recovery. This is the first report on Aneurinifactin, a lipopeptide biosurfactant produced by a marine A. aneurinilyticus SBP-11, which could be explored as a promising candidate for use in various biomedical and industrial applications.

Comparison of the effects of dietary single and multi-probiotics on growth, non-specific immune responses and disease resistance in starry flounder, Platichthys stellatus.

An 8-week feeding trial was conducted to evaluate the effects of dietary probiotics on growth performance and non-specific immune responses in starry flounder, Platichthys stellatus. Fish averaging 46.5 ± 0.65 g (mean ± SD) were fed one of the six experimental diets; one control (Cont), and five other diets were prepared by supplementing single-probiotics 1 (Bacillus subtilis; SP1, 2 × 109 CFU kg-1 diet), single-probiotics 2 (Bacillus licheniformis; SP2, 2 × 109 CFU kg-1 diet), multi-probiotics 1 (Bacillus subtilis + Bacillus licheniformis; MP1, 2 × 109 CFU kg-1 diet), multi-probiotics 2 (commercial probiotics; Bacillus subtills + Bacillus licheniformis + Paenibacillus polymyxa + Aspergillus oryzae + Saccharomyces cerevisiae; MP2, 2 × 109 CFU kg-1 diet) and oxytetracycline (OTC) at 5 g OTC kg-1 diet. At the end of 8 weeks feeding trial, weight gain (WG) and specific growth rate (SGR) of fish fed SP1, MP1 and MP2 diets were significantly higher than those of fish fed control diet (P < 0.05). Superoxide dismutase (SOD) activity of fish fed MP2 diet was significantly higher than those of fish fed OTC diet (P < 0.05). Nitro blue tetrazolium (NBT) activity and lysozyme activity of fish fed SP1, MP1 and MP2 diets were significantly higher than those of fish fed OTC diet (P < 0.05). However, there was no significant difference among fish fed SP1, SP2, MP1 and MP2 diets. During the Edwardsiella tarda challenge test, the first mortality occurred on day 2. After the 14 days challenge test, cumulative survival rate of fish fed MP1 and MP2 diets were significantly higher than those of fish fed control diet (P < 0.05). However, there was no significant difference among fish fed SP1, SP2, MP1, MP2 and OTC diets in survival rate at the termination of the challenge test. Although there was little advantage in immunological parameters with fish fed MP diets, single and multi-probiotics were equally effective statistically. These results demonstrated that single or multi-probiotics had equal beneficial effect as an antibiotic replacer in terms of growth performance, non-specific immune responses and disease resistance in starry flounder.

The Spore Coat.

Spores of Clostridiales and Bacillales are encased in a complex series of concentric shells that provide protection, facilitate germination, and mediate interactions with the environment. Analysis of diverse spore-forming species by thin-section transmission electron microscopy reveals that the number and morphology of these encasing shells vary greatly. In some species, they appear to be composed of a small number of discrete layers. In other species, they can comprise multiple, morphologically complex layers. In addition, spore surfaces can possess elaborate appendages. For all their variability, there is a consistent architecture to the layers encasing the spore. A hallmark of all Clostridiales and Bacillales spores is the cortex, a layer made of peptidoglycan. In close association with the cortex, all species examined possess, at a minimum, a series of proteinaceous layers, called the coat. In some species, including Bacillus subtilis, only the coat is present. In other species, including Bacillus anthracis, an additional layer, called the exosporium, surrounds the coat. Our goals here are to review the present understanding of the structure, composition, assembly, and functions of the coat, primarily in the model organism B. subtilis, but also in the small but growing number of other spore-forming species where new data are showing that there is much to be learned beyond the relatively well-developed basis of knowledge in B. subtilis. To help summarize this large field and define future directions for research, we will focus on key findings in recent years.

Caenibacillus caldisaponilyticus gen. nov., sp. nov., a thermophilic, spore-forming and phospholipid-degrading bacterium isolated from acidulocompost.

A thermophilic and phospholipid-degrading bacterium, designated strain B157T, was isolated from acidulocompost, a garbage compost processed under acidic conditions at moderately high temperature. The organism was Gram-stain-positive, aerobic, spore-forming and rod-shaped. Growth was observed to occur at 40-65 °C and pH 4.8-8.1 (optimum growth: 50-60 °C, pH 6.2). The strain was catalase- and oxidase-positive. The cell wall contained meso-diaminopimelic acid, alanine, glutamic acid and galactose. The predominant respiratory quinone was menaquinone-7 (MK-7) and the major fatty acids were anteiso-C17 : 0 and iso-C17 : 0. Comparative 16S rRNA gene sequence analysis showed that strain B157T was related most closely to Tuberibacillus calidus 607T (94.8 % identity), and the phylogenetic analysis revealed that it belonged to the family Sporolactobacillaceae. The DNA G+C content was determined as 51.8 mol%. In spite of many similarities with the type strains of members of the family Sporolactobacillaceae, genotypic analyses suggest that strain B157T represents a novel species of a new genus, Caenibacilluscaldisaponilyticus gen. nov., sp. nov. The type strain of Caenibacilluscaldisaponilyticus is B157T (=NBRC 111400T=DSM 101100T).

Affinity Purification Method for the Identification of Nonribosomal Peptide Biosynthetic Enzymes Using a Synthetic Probe for Adenylation Domains.

A series of inhibitors have been designed based on 5'-O-sulfamoyl adenosine (AMS) that display tight binding characteristics towards the inhibition of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs). We recently developed an affinity probe for A domains that could be used to facilitate the specific isolation and identification of NRPS modules. Our synthetic probe, which is a biotinylated variant of L-Phe-AMS (L-Phe-AMS-biotin), selectively targets the A domains in NRPS modules that recognize and convert L-Phe to an aminoacyl adenylate in whole proteomes. In this chapter, we describe the design and synthesis of L-Phe-AMS-biotin and provide a summary of our work towards the development of a series of protocols for the specific enrichment of NRPS modules using this probe.

The Exiguobacterium sibiricum 255-15 GtfC Enzyme Represents a Novel Glycoside Hydrolase 70 Subfamily of 4,6-α-Glucanotransferase Enzymes.

The glycoside hydrolase 70 (GH70) family originally was established for glucansucrase enzymes found solely in lactic acid bacteria synthesizing α-glucan polysaccharides from sucrose (e.g., GtfA). In recent years, we have characterized GtfB and related Lactobacillus enzymes as 4,6-α-glucanotransferase enzymes. These GtfB-type enzymes constitute the first GH70 subfamily of enzymes that are unable to act on sucrose as a substrate but are active with maltodextrins and starch, cleave α1→4 linkages, and synthesize linear α1→6-glucan chains. The GtfB disproportionating type of activity results in the conversion of malto-oligosaccharides into isomalto/malto-polysaccharides with a relatively high percentage of α1→6 linkages. This paper reports the identification of the members of a second GH70 subfamily (designated GtfC enzymes) and the characterization of the Exiguobacterium sibiricum 255-15 GtfC enzyme, which is also inactive with sucrose and displays 4,6-α-glucanotransferase activity with malto-oligosaccharides. GtfC differs from GtfB in synthesizing isomalto/malto-oligosaccharides. Biochemically, the GtfB- and GtfC-type enzymes are related, but phylogenetically, they clearly constitute different GH70 subfamilies, displaying only 30% sequence identity. Whereas the GtfB-type enzyme largely has the same domain order as glucansucrases (with α-amylase domains A, B, and C plus domains IV and V), this GtfC-type enzyme differs in the order of these domains and completely lacks domain V. In GtfC, the sequence of conserved regions I to IV of clan GH-H is identical to that in GH13 (I-II-III-IV) but different from that in GH70 (II-III-IV-I because of a circular permutation of the (ß/α)8 barrel. The GtfC 4,6-α-glucanotransferase enzymes thus represent structurally and functionally very interesting evolutionary intermediates between α-amylase and glucansucrase enzymes.

[Characterization of D-lactate dehydrogenase isozymes from a D-lactic acid producing bacterium Sporolactobacillus inulinus].

Objective: Sporolactobacillus inulinus, a typical homofermentative lactic acid bacterium, is an efficient D-lactic acid producer. Various environment factors affect the productivity of S. inulinus. Glucokinase, phosphofructokinase, pyruvate kinase and lactic dehydrogenase are the key enzymes of D-lactic acid production from glucose by S. inulinus. The characteristics of these enzymes are important in controlling and regulating the fermentation process. According to the genome bioinformatics analysis of S. inulinus CASD, three putative D-lactate dehydrogenases were identified, among which the bifunctional protein had been reported. In this study, we provided insights into the characteristics of the other two D-lactate dehydrogenase isozymes. Methods: S. inulinus Y2-8 genome was used as the template to amplify D-lactate dehydrogenase gene (dldh) and D-isomer specific 2-hydroxyacid dehydrogenase gene (dhdh). The two recombinant strains E-pET-28a/dldh and E-pET-28a/dhdh were constructed for enzyme expression. Both recombinants DLDH and DHDH could convert pyruvic acid into D-lactic acid. Results: Enzymes expressed by recombinant strains were purified by Ni-NTA chromatography. The apparent molecular mass of DLDH was approximately 37 kDa by SDS-PAGE analysis, and DLDH showed a high affinity to pyruvate with the Km value of (0.58±0.04) mmol/L. The optimal reaction temperature and pH for DLDH was 35℃ and 6.5, respectively. The apparent molecular mass of DHDH was approximately 39 kDa, and the Km of DHDH toward pyruvate was (1.70±0.08) mmol/L. The optimum catalysis temperature and pH of DHDH were 30℃ and 7.5, respectively. Conclusion: According to the Km and optimal reaction pH, DLDH was suggested as the main catalyst in formation D-lactic acid from pyruvate during the fermentation. The enzymatic properties would contribute to the regulation of the fermentation of S. inulinus.

Broad spectrum antimicrobial compounds from the bacterium Exiguobacterium mexicanum MSSRFS9.

Clinical bacterial pathogens front a major challenge for the clinical researchers and physicians. In particular microbial pathogens like Escherichia coli, Shigella flexneri, Klebsiella pneumonia and Salmonella enterica are apparelled with systemic machineries to bring down the human immune system as well as proliferate dramatically in a short period which in turn cause a pronounced ailment to the human health. In vitro evaluation of four purified compounds isolated from rhizosphere bacterium Exiguobacterium mexicanum tested against clinical pathogens mentioned above by disc diffusion method showed the two compounds viz., 3,6,18-trione, 9,10-dihydro-12'-hydroxyl-2methyl-5-(phenyl methyl) (5'-alpha, 10-alpha)-dihydroergotamine (C3) and dipropyl - S-propyl ester (C4) exhibit antibacterial property against all the tested pathogens. Among the four clinical pathogens tested, compound C3 has shown higher zone of inhibition against S. enterica with 17±0 mm, followed by S. flexneri with 16.5±0.7 mm, E. coli with 15±0 mm and K. pneumoniae with 14±0 mm, respectively. The compound C4 has shown higher antimicrobial activity against S. enterica with 21.5±0.7 mm zone of inhibition, followed by S. flexneri with 19.5±0.7 mm, E. coli with 17±0 mm and K. pneumoniae with 16±0 mm, these two compounds were found to be safer when subjected to rat haematological and enzymatic analysis.

Thermophilic Coenzyme B12-Dependent Acyl Coenzyme A (CoA) Mutase from Kyrpidia tusciae DSM 2912 Preferentially Catalyzes Isomerization of (R)-3-Hydroxybutyryl-CoA and 2-Hydroxyisobutyryl-CoA.

The recent discovery of a coenzyme B12-dependent acyl-coenzyme A (acyl-CoA) mutase isomerizing 3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in the mesophilic bacterium Aquincola tertiaricarbonis L108 (N. Yaneva, J. Schuster, F. Schäfer, V. Lede, D. Przybylski, T. Paproth, H. Harms, R. H. Müller, and T. Rohwerder, J Biol Chem 287:15502-15511, 2012, http://dx.doi.org/10.1074/jbc.M111.314690) could pave the way for a complete biosynthesis route to the building block chemical 2-hydroxyisobutyric acid from renewable carbon. However, the enzyme catalyzes only the conversion of the stereoisomer (S)-3-hydroxybutyryl-CoA at reasonable rates, which seriously hampers an efficient combination of mutase and well-established bacterial poly-(R)-3-hydroxybutyrate (PHB) overflow metabolism. Here, we characterize a new 2-hydroxyisobutyryl-CoA mutase found in the thermophilic knallgas bacterium Kyrpidia tusciae DSM 2912. Reconstituted mutase subunits revealed highest activity at 55°C. Surprisingly, already at 30°C, isomerization of (R)-3-hydroxybutyryl-CoA was about 7,000 times more efficient than with the mutase from strain L108. The most striking structural difference between the two mutases, likely determining stereospecificity, is a replacement of active-site residue Asp found in strain L108 at position 117 with Val in the enzyme from strain DSM 2912, resulting in a reversed polarity at this binding site. Overall sequence comparison indicates that both enzymes descended from different prokaryotic thermophilic methylmalonyl-CoA mutases. Concomitant expression of PHB enzymes delivering (R)-3-hydroxybutyryl-CoA (beta-ketothiolase PhaA and acetoacetyl-CoA reductase PhaB from Cupriavidus necator) with the new mutase in Escherichia coli JM109 and BL21 strains incubated on gluconic acid at 37°C led to the production of 2-hydroxyisobutyric acid at maximal titers of 0.7 mM. Measures to improve production in E. coli, such as coexpression of the chaperone MeaH and repression of thioesterase II, are discussed.

First report of a lipopeptide biosurfactant from thermophilic bacterium Aneurinibacillus thermoaerophilus MK01 newly isolated from municipal landfill site.

A biosurfactant-producing thermophile was isolated from the Kahrizak landfill of Tehran and identified as a bacterium belonging to the genus Aneurinibacillus. A thermostable lipopeptide-type biosurfactant was purified from the culture medium of this bacterium and showed stability in the temperature range of 20-90 °C and pH range of 5-10. The produced biosurfactant could reduce the surface tension of water from 72 to 43 mN/m with a CMC of 1.21 mg/mL. The strain growing at a temperature of 45 °C produces a substantial amount of 5 g/L of biosurfactant in the medium supplemented with sunflower oil as the sole carbon source. Response surface methodology was employed to optimize the biosurfactant production using sunflower oil, sodium nitrate, and yeast extract as variables. The optimization resulted in 6.75 g/L biosurfactant production, i.e., 35% improved as compared to the unoptimized condition. Thin-layer chromatography, FTIR spectroscopy, 1H-NMR spectroscopy, and biochemical composition analysis confirmed the lipopeptide structure of the biosurfactant.

Photocycle of Exiguobacterium sibiricum rhodopsin characterized by low-temperature trapping in the IR and time-resolved studies in the visible.

The photocycle of the retinal protein from Exiguobacterium sibiricum, which differs from bacteriorhodopsin in both its primary donor and acceptor, is characterized by visible and infrared spectroscopy. At pH above pKa ~6.5, we find a bacteriorhodopsin-like photocycle, which originates from excitation of the all-trans retinal chromophore with K-, L-, M-, and N-like intermediates. At pH below pKa ~6.5, the M state, which reflects Schiff base deprotonation during proton pumping, is not accumulated. However, using the infrared band at ~1760 cm(-1) as a marker for transient protonation of the primary acceptor, we find that Schiff base deprotonation must have occurred at pH not only above but also below the pKa ~6.5. Thus, the M state is formed but not accumulated for kinetic reasons. Further, chromophore reisomerization from the 13-cis to the all-trans conformation occurs very late in the photocycle. The strongly red-shifted states that dominate the second half of the cycle are produced before the reisomerization step, and by this criterion, they are not O-like but rather N-like states. The assignment of photocycle intermediates enables reevaluation of the photocycle; its specific features are discussed in relation to the general mechanism of proton transport in retinal proteins.