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Background: The objective of this work was to engineer Deinococcus radiodurans R1 as a microbial cell factory for the production of pinene, a monoterpene molecule prominently used for the production of fragrances, pharmaceutical products, and jet engine biofuels. Our objective was to produce pinene from glycerol, an abundant by-product of various industries. Results: To enable pinene production in D. radiodurans, we expressed the pinene synthase from Abies grandis, the geranyl pyrophosphate (GPP) synthase from Escherichia coli, and overexpressed the native 1-deoxy-d-xylulose 5-phosphate synthase. Further, we disrupted the deinoxanthin pathway competing for the substrate GPP by either inactivating the gene dr0862, encoding phytoene synthase, or substituting the native GPP synthase with that of E. coli. These manipulations resulted in a D. radiodurans strain capable of producing 3.2 ± 0.2 mg/L pinene in a minimal medium supplemented with glycerol, with a yield of 0.13 ± 0.04 mg/g glycerol in shake flask cultures. Additionally, our results indicated a higher tolerance of D. radiodurans towards pinene as compared to E. coli. Conclusions: In this study, we successfully engineered the extremophile bacterium D. radiodurans to produce pinene. This is the first study demonstrating the use of D. radiodurans as a cell factory for the production of terpenoid molecules. Besides, its high resistance to pinene makes D. radiodurans a suitable host for further engineering efforts to increase pinene titer as well as a candidate for the production of the other terpenoid molecules. [ABSTRACT FROM AUTHOR]

Background: The objective of this work was to engineer Deinococcus radiodurans R1 as a microbial cell factory for the production of pinene, a monoterpene molecule prominently used for the production of fragrances, pharmaceutical products, and jet engine biofuels. Our objective was to produce pinene from glycerol, an abundant by-product of various industries. Results: To enable pinene production in D. radiodurans, we expressed the pinene synthase from Abies grandis, the geranyl pyrophosphate (GPP) synthase from Escherichia coli, and overexpressed the native 1-deoxy-d-xylulose 5-phosphate synthase. Further, we disrupted the deinoxanthin pathway competing for the substrate GPP by either inactivating the gene dr0862, encoding phytoene synthase, or substituting the native GPP synthase with that of E. coli. These manipulations resulted in a D. radiodurans strain capable of producing 3.2 ± 0.2 mg/L pinene in a minimal medium supplemented with glycerol, with a yield of 0.13 ± 0.04 mg/g glycerol in shake flask cultures. Additionally, our results indicated a higher tolerance of D. radiodurans towards pinene as compared to E. coli. Conclusions: In this study, we successfully engineered the extremophile bacterium D. radiodurans to produce pinene. This is the first study demonstrating the use of D. radiodurans as a cell factory for the production of terpenoid molecules. Besides, its high resistance to pinene makes D. radiodurans a suitable host for further engineering efforts to increase pinene titer as well as a candidate for the production of the other terpenoid molecules. [ABSTRACT FROM AUTHOR]

The development of carbon capture and storage technologies has resulted in a rising interest in the use of carbonic anhydrases (CAs) for CO2 fixation at elevated temperatures. In this study, we chose to rationally engineer the α-CA (NtCA) from the thermophilic bacterium Nitratiruptor tergarcus, which has been previously suggested to be thermostable by in silico studies. Using a combination of analyses with the DEEPDDG software and available structural knowledge, we selected residues in three regions, namely, the catalytic pocket, the dimeric interface and the surface, in order to increase thermostability and CO2 hydration activity. A total of 13 specific mutations, affecting seven amino acids, were assessed. Single, double and quadruple mutants were produced in Escherichia coli and analyzed. The best-performing mutations that led to improvements in both activity and stability were D168K, a surface mutation, and R210L, a mutation in the dimeric interface. Apart from these, most mutants showed improved thermostability, with mutants R210K and N88K_R210L showing substantial improvements in activity, up to 11-fold. Molecular dynamics simulations, focusing particularly on residue fluctuations, conformational changes and hydrogen bond analysis, elucidated the structural changes imposed by the mutations. Successful engineering of NtCA provided valuable lessons for further engineering of α-CAs. [ABSTRACT FROM AUTHOR]

Protein phosphorylation regulates a large variety of biological processes in all living cells. In pathogenic bacteria, the study of serine, threonine, and tyrosine (Ser/Thr/Tyr) phosphorylation has shed light on the course of infectious diseases, from adherence to host cells to pathogen virulence, replication, and persistence. Mass spectrometry (MS)‐based phosphoproteomics has provided global maps of Ser/Thr/Tyr phosphosites in bacterial pathogens. Despite recent developments, a quantitative and dynamic view of phosphorylation events that occur during bacterial pathogenesis is currently lacking. Temporal, spatial, and subpopulation resolution of phosphorylation data is required to identify key regulatory nodes underlying bacterial pathogenesis. Herein, we discuss how technological improvements in sample handling, MS instrumentation, data processing, and machine learning should improve bacterial phosphoproteomic datasets and the information extracted from them. Such information is expected to significantly extend the current knowledge of Ser/Thr/Tyr phosphorylation in pathogenic bacteria and should ultimately contribute to the design of novel strategies to combat bacterial infections. [ABSTRACT FROM AUTHOR]

We have previously shown that galacto-rhamnogalacturonan fibers can be enzymatically extracted from potato pulp and that these fibers have potential for exerting a prebiotic effect in piglets. The spore-forming Bacillus species are widely used as probiotics in feed supplements for pigs. In this study, we evaluated the option for further functionalizing Bacillus feed supplements by selecting strains possessing the enzymes required for extraction of the potentially prebiotic fibers. We established that it would require production and secretion of pectin lyase and/or polygalacturonase but no or limited secretion of galactanase and β-galactosidase. By screening a library of 158 Bacillus species isolated from feces and soil, we demonstrated that especially strains of Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus mojavensis have the necessary enzyme profile and thus the capability to degrade polygalacturonan. Using an in vitro porcine gastrointestinal model system, we revealed that specifically strains of B. mojavensis were able to efficiently release galacto-rhamnogalacturonan from potato pulp under simulated gastrointestinal conditions. The work thus demonstrated the feasibility of producing prebiotic fibers via a feed containing Bacillus spores and potato pulp and identified candidates for future in vivo evaluation in piglets. [ABSTRACT FROM AUTHOR]

Rhamnogalacturonan I (RGI) modifying enzymes catalyse the degradation of the RGI backbone and encompass enzymes specific for either the α1,2-bond linking galacturonic acid to rhamnose or the α1,4-bond linking rhamnose to galacturonic acid in the RGI backbone. The first microbial enzyme found to be able to catalyse the degradation of the RGI backbone, an endo-hydrolase (EC 3.2.1.171) derived from Aspergillus aculeatus , was discovered 25 years ago. Today the group of RGI modifying enzymes encompasses endo- and exo-hydrolases as well as lyases. The RGI hydrolases, EC 3.2.1.171–EC 3.2.1.174, have been described to be produced by Aspergillus spp. and Bacillus subtilis and are categorized in glycosyl hydrolase families 28 and 105. The RGI lyases, EC 4.2.2.23–EC 4.2.2.24, have been isolated from different fungi and bacterial species and are categorized in polysaccharide lyase families 4 and 11. This review brings together the available knowledge of the RGI modifying enzymes and provides a detailed overview of biocatalytic reaction characteristics, classification, structure-function traits, and analyses the protein properties of these enzymes by multiple sequence alignments in neighbour-joining phylogenetic trees. Some recently detected unique structural features and dependence of calcium for activity of some of these enzymes (notably the lyases) are discussed and newly published results regarding improvement of their thermostability by protein engineering are highlighted. Knowledge of these enzymes is important for understanding microbial plant cell wall degradation and for advancing enzymatic processing and biorefining of pectinaceous plant biomass. [ABSTRACT FROM AUTHOR]

This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6–7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation) or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2’-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides. [ABSTRACT FROM AUTHOR]

Several bacterial sialyltransferases have been reported to be multifunctional also catalysing sialidase and trans-sialidase reactions. In this study, we examined the trans-sialylation efficacy and regioselectivity of mutants of the multifunctional Pasteurella multocida sialyltransferase (PmST) for catalysing the synthesis of 3′- and 6′-sialyllactose using casein glycomacropeptide as sialyl-donor and lactose as acceptor. The mutation P34H led to a 980-fold increase in α-2,6-sialyltransferase activity (with cytidine-5′-monophospho- N -acetylneuraminic acid as donor), while its α-2,3-sialyltransferase activity was abolished. Histidine in this position is conserved in α-2,6-sialyltransferases and has been suggested, and recently confirmed, to be the determinant for strict regiospecificity in the sialyltransferase reaction. Our data verified this theorem. In trans-sialidase reactions, the P34H mutant displayed a distinct preference for 6′-sialyllactose synthesis but low levels of 3′-sialyllactose were also produced. The sialyllactose yield was however lower than when using PmST WT under optimal conditions for 6′-sialyllactose formation. The discrepancy in regiospecificity between the two reactions could indicate subtle differences in the substrate binding site in the two reactions. In contrast, the two mutations E271F and R313Y led to preferential synthesis of 3′-sialyllactose over 6′-sialyllactose and the double mutant (PmST E271F/R313Y ) exhibited the highest α-2,3-regioselectivity via reduced sialidase and α-2,6-trans-sialidase activity. The double mutant PmST E271F/R313Y thus showed the highest α-2,3-regioselectivity and constitutes an interesting enzyme for regioselective synthesis of α-2,3-sialylated glycans. This study has expanded the understanding of the structure-function relationship of multifunctional, bacterial sialyltransferases and provided new enzymes for regioselective glycan sialylation. [ABSTRACT FROM AUTHOR]

Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of α-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by β-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM 13/ATCC14580) was examined by using a combinatorial protein engineering approach exploring additive effects of single amino acid substitutions. These were selected by using a consensus approach together with assessing protein stability changes (PoPMuSiC) and B-factor iterative test (B-FIT). The second-generation mutants involved combinations of two to seven individually favorable single mutations. Thermal stability was examined as half-life at 60 °C and by recording of thermal transitions by circular dichroism. Surprisingly, the biggest increment in thermal stability was achieved by producing the wild-type RGI lyase in Bacillus subtilis as opposed to in Pichia pastoris; this effect is suggested to be a negative result of glycosylation of the P. pastoris expressed enzyme. A ~ twofold improvement in thermal stability at 60 °C, accompanied by less significant increases in T of the enzyme mutants, were obtained due to additive stabilizing effects of single amino acid mutations (E434L, G55V, and G326E) compared to the wild type. The crystal structure of the B. licheniformis wild-type RGI lyase was also determined; the structural analysis corroborated that especially mutation of charged amino acids to hydrophobic ones in surface-exposed loops produced favorable thermal stability effects. [ABSTRACT FROM AUTHOR]

Highlights: [•] PmST, a Pasteurella multocida sialyltransferase expressed in Escherichia coli exhibited both α-2,3- and α-2,6-trans-sialidase activities. [•] Optimum conditions were identified for maximum production of 3′- and 6′-sialyllactoses by PmST-catalyzed trans-sialylation using casein glycomacropeptide as the donor for sialic acid. [•] The regioselective synthesis of sialyllactose could be achieved by regulating reaction time. [•] The kinetic parameter for α-2,6-trans-sialidase activity of PmST was estimated. [•] PmST catalyzed α-2,3 as well as α-2,6 sialylation of prebiotic galactooligosaccharides. [ABSTRACT FROM AUTHOR]

This paper reports rational engineering of Trypanosoma rangeli sialidase to develop an effective enzyme for a potentially important type of reactivity: production of sialylated prebiotic glycans. The Trypanosoma cruzi trans-sialidase and the homologous T. rangeli sialidase has previously been used to investigate the structural requirements for trans-sialidase activity. We observed that the T. cruzi trans-sialidase has a seven-amino-acid motif (197–203) at the border of the substrate binding cleft. The motif differs substantially in chemical properties and substitution probability from the homologous sialidase, and we hypothesised that this motif is important for trans-sialidase activity. The 197–203 motif is strongly positively charged with a marked change in hydrogen bond donor capacity as compared to the sialidase. To investigate the role of this motif, we expressed and characterised a T. rangeli sialidase mutant, Tr13. Conditions for efficient trans-sialylation were determined, and Tr13's acceptor specificity demonstrated promiscuity with respect to the acceptor molecule enabling sialylation of glycans containing terminal galactose and glucose and even monomers of glucose and fucose. Sialic acid is important in association with human milk oligosaccharides, and Tr13 was shown to sialylate a number of established and potential prebiotics. Initial evaluation of prebiotic potential using pure cultures demonstrated, albeit not selectively, growth of Bifidobacteria. Since the 197–203 motif stands out in the native trans-sialidase, is markedly different from the wild-type sialidase compared to previous mutants, and is shown here to confer efficient and broad trans-sialidase activity, we suggest that this motif can serve as a framework for future optimization of trans-sialylation towards prebiotic production. [ABSTRACT FROM AUTHOR]

Bacillus subtilis two-component system DegS/U is well known for the complexity of its regulation. The cytosolic sensory kinase DegS does not receive a single predominant input signal like most two-component kinases, instead it integrates a wide array of metabolic inputs that modulate its activity. The phosphorylation state of the response regulator DegU also does not confer a straightforward "on/off" response; it is fine-tuned and at different levels triggers different sub-regulons. Here we describe serine phosphorylation of the DegS sensing domain, which stimulates its kinase activity. We demonstrate that DegS phosphorylation can be carried out by at least two B. subtilis Hanks-type kinases in vitro, and this stimulates the phosphate transfer towards DegU. The consequences of this process were studied in vivo, using phosphomimetic (Ser76Asp) and non-phosphorylatable (Ser76Ala) mutants of DegS. In a number of physiological assays focused on different processes regulated by DegU, DegS S76D phosphomimetic mutant behaved like a strain with intermediate levels of DegU phosphorylation, whereas DegS S76A behaved like a strain with lower levels of DegU phophorylation. These findings suggest a link between DegS phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this posttranslational modification as an additional trigger for this two-component system. [ABSTRACT FROM AUTHOR]

Bacillus subtilis BY-kinase PtkA was previously shown to phosphorylate, and thereby regulate the activity of two classes of protein substrates: UDP-glucose dehydrogenases and single-stranded DNA-binding proteins. Our recent phosphoproteome study identified nine new tyrosine-phosphorylated proteins in B. subtilis. We found that the majority of these proteins could be phosphorylated by PtkA in vitro. Among these new substrates, single-stranded DNA exonuclease YorK, and aspartate semialdehyde dehydrogenase Asd were activated by PtkA-dependent phosphorylation. Because enzyme activity was not affected in other cases, we used fluorescent protein tags to study the impact of PtkA on localization of these proteins in vivo. For several substrates colocalization with PtkA was observed, and more importantly, the localization pattern of the proteins enolase, YjoA, YnfE, YvyG, Ugd and SsbA was dramatically altered in Δ ptkA background. Our results confirm that PtkA can control enzyme activity of its substrates in some cases, but also reveal a new mode of action for PtkA, namely ensuring correct cellular localization of its targets. [ABSTRACT FROM AUTHOR]

Abstract: Recent advances in mass spectrometry allowed the charting of bacterial serine/threonine/tyrosine phosphoproteomes with unprecedented accuracy, including the acquisition of a large number of phosphorylation sites. Phosphorylated bacterial proteins are involved in some key housekeeping processes, and their phosphorylation is expected to play an important regulatory role. When coupled to stable isotope labeling by amino acids in cell culture (SILAC), high-resolution mass spectrometry allows the detection of changes in the occupancy of phosphorylation sites in response to various stimuli. This and similar approaches promise to lead bacterial phosphoproteomics into the era of systems biology, where the entire phosphorylation-based regulatory networks will be charted, modelled, and ultimately engineered to obtain desired properties. [Copyright &y& Elsevier]

With multidrug-resistant bacterial pathogens on the rise, there is a strong research focus on alternative antibacterial treatments that could replace or complement classical antibiotics. Metallic nanoparticles, and in particular silver nanoparticles (AgNPs), have been shown to kill bacterial biofilms effectively, but their chemical synthesis often involves environmentally unfriendly by-products. Recent studies have shown that microbial and plant extracts can be used for the environmentally friendly synthesis of AgNPs. Herein we report a procedure for producing AgNPs using a putative Cedecea sp. strain isolated from soil. The isolated bacterial strain showed a remarkable potential for producing spherical, crystalline and stable AgNPs characterized by UV–visible spectroscopy, transmission electron microscopy, dynamic light scattering, and Fourier transform infrared spectroscopy. The concentration of produced nanoparticles was 1.31 µg/µl with a negative surface charge of − 15.3 mV and nanoparticles size ranging from 10–40 nm. The AgNPs was tested against four pathogenic microorganisms S. epidermidis, S. aureus, E. coli and P. aeruginosa. The nanoparticles exhibited strong minimum inhibitory concentration (MIC) values of 12.5 and 6.25 µg/µl and minimum bactericidal concentration (MBC) values of 12.5 and 12.5 µg/mL against E. coli and P. aeruginosa, respectively. One distinguishing feature of AgNPs produced by Cedecea sp. extracts is their extreme stability. Inductively coupled plasma mass spectrometry and thermogravimetric analysis demonstrated that the produced AgNPs are stable for periods exceeding one year. This means that their strong antibacterial effects, demonstrated against E. coli and P. aeruginosa biofilms, can be expected to persist during extended periods. [ABSTRACT FROM AUTHOR]

A sialidase (EC 3.2.1.18) from the non-pathogenic Trypanosoma rangeli, TrSA, has been shown to exert trans-sialidase activity after mutation of five specific amino acids in the active site (M96V, A98P, S120Y, G249Y, Q284P) to form the so-called TrSA5mut enzyme. By computational and hypothesis driven approaches additional mutations enhancing the trans-sialidase activity have been suggested. In the present work, we made a systematic combination of these mutations leading to seven new variants of the T. rangeli sialidase, having 6–16 targeted amino acid mutations. The resulting enzyme variants were analyzed via kinetics for their ability to carry out trans-sialidase reaction using CGMP and D-lactose as substrates. The sialidase variants with 15 and 16 mutations, respectively, exhibited significantly improved trans-sialidase activity for D-lactose sialylation. Our results corroborate, that computational studies of trans-glycosylation can be a valuable input in the design of novel trans-glycosidases, but also highlight the importance of experimental validation in order to assess the performance. In conclusion, two of the seven mutants displayed a dramatic switch in specificity from hydrolysis towards trans-sialylation and constitute the most potent trans-sialidase mutants of TrSA described in literature to date. [ABSTRACT FROM AUTHOR]

Enterococci comprise a group of lactic acid bacteria (LAB) with considerable potential to serve as food fermentation microorganisms. Unfortunately, enterococci have received a lot of negative attention, due to the occurrence of pathogenic and multidrug resistant strains. In this study, we used genomics to select safe candidates among the forty-four studied enterococcal isolates. The genomes of the forty-four strains were fully sequenced and assessed for presence of virulence and antibiotic resistance genes. Nineteen isolates belonging to the species Enterococcus lactis, Enterococcus faecium, Enterococcus durans , and Enterococcus thailandicus , were deemed safe from the genome analysis. The presence of secondary metabolite gene clusters for bacteriocins was assessed, and twelve candidates were found to secrete antimicrobial compounds effective against Listeria monocytogenes isolated from cheese and Staphylococcus aureus. Physiological characterization revealed nineteen industrial potentials; all strains grew well at 42 °C and acidified 1.5 hours faster than their mesophilic counterpart Lactococcus lactis , with which they share metabolism and flavor forming ability. We conclude that a large fraction of the examined enterococci were safe and could serve as excellent food fermentation microorganisms with inherent bioprotective abilities. [ABSTRACT FROM AUTHOR]

Highlights: [•] A mutated sialidase from Trypanosoma rangeli was expressed in Pichia pastoris. [•] The sialidase mutant was tuned for trans-sialidase activity by optimizing reaction conditions. [•] 3′-Sialyllactose and other sialylated HMO molecules were produced via trans-sialidase biocatalysis. [•] Casein glycomacropeptide was used as substrate for the trans-sialidase mutant biocatalysis. [ABSTRACT FROM AUTHOR]

Rhamnogalacturonan I lyase (RGI lyase) (EC 4.2.2.-) catalyzes the cleavage of rhamnogalacturonan I in pectins by β-elimination. In this study the thermal stability of a RGI lyase (PL 11) originating from Bacillus licheniformis DSM 13/ATCC14580 was increased by a targeted protein engineering approach involving single amino acid substitution. Nine individual amino acids were selected as targets for site-saturated mutagenesis by the use of a predictive consensus approach in combination with prediction of protein mutant stability changes and B-factor iteration testing. After extensive experimental verification of the thermal stability of the designed mutants versus the original wild-type RGI lyase, several promising single point mutations were obtained, particularly in position Glu434 on the surface of the enzyme protein. The best mutant, Glu434Leu, produced a half-life of 31 min at 60 °C, corresponding to a 1.6-fold improvement of the thermal stability compared to the original RGI lyase. Gly55Val was the second best mutation with a thermostability half-life increase of 27 min at 60 °C, and the best mutations following were Glu434Trp, Glu434Phe, and Glu434Tyr, respectively. The data verify the applicability of a combinatorial predictive approach for designing a small site saturation library for improving enzyme thermostability. In addition, new thermostable RGI lyases suitable for enzymatic upgrading of pectinaceous plant biomass materials at elevated temperatures were produced. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides (HMOs) constitute a unique family of bioactive lactose-based molecules present in human breast milk. HMOs are of major importance for infant health and development but also virtually absent from bovine milk used for infant formula. Among the HMOs, the fucosylated species are the most abundant. Transfucosylation catalysed by retaining α- l -fucosidases is a new route for manufacturing biomimetic HMOs. Seven α- l -fucosidases from glycosyl hydrolase family 29 were expressed, characterized in terms of substrate specificity and thermal stability, and shown to be able to catalyse transfucosylation. The α- l -1,3/4-fucosidase CpAfc2 from Clostridium perfringens efficiently catalysed the formation of the more complex human milk oligosaccharide structure lacto- N- fucopentaose II (LNFP II) using 3-fucosyllactose as fucosyl donor and lacto- N -tetraose as acceptor with a 39% yield. α- l -Fucosidases FgFCO1 from Fusarium graminearum and Mfuc5 from a soil metagenome were able to catalyse transfucosylation of lactose using citrus xyloglucan as fucosyl donor. FgFCO1 catalysed formation of 2′-fucosyllactose, whereas Mfuc5 catalysis mainly produced an unidentified, non-HMO fucosyllactose, reaching molar yields based on the donor substrate of 14% and 18%, respectively. [ABSTRACT FROM AUTHOR]

The main family of serine/threonine/tyrosine protein kinases present in eukarya was defined and described by Hanks et al . in 1988 ( Science , 241 , 42–52). It was initially believed that these kinases do not exist in bacteria, but extensive genome sequencing revealed their existence in many bacteria. For historical reasons, the term “eukaryotic-type kinases” propagated in the literature to describe bacterial members of this protein family. Here, we argue that this term should be abandoned as a misnomer, and we provide several lines of evidence to support this claim. Our comprehensive phylostratigraphic analysis suggests that Hanks-type kinases present in eukarya, bacteria and archaea all share a common evolutionary origin in the lineage leading to the last universal common ancestor (LUCA). We found no evidence to suggest substantial horizontal transfer of genes encoding Hanks-type kinases from eukarya to bacteria. Moreover, our systematic structural comparison suggests that bacterial Hanks-type kinases resemble their eukaryal counterparts very closely, while their structures appear to be dissimilar from other kinase families of bacterial origin. This indicates that a convergent evolution scenario, by which bacterial kinases could have evolved a kinase domain similar to that of eukaryal Hanks-type kinases, is not very likely. Overall, our results strongly support a monophyletic origin of all Hanks-type kinases, and we therefore propose that this term should be adopted as a universal name for this protein family. [ABSTRACT FROM AUTHOR]

Sialidases (3.2.1.18) may exhibit trans-sialidase activity to catalyze sialylation of lactose if the active site topology is congruent with that of the Trypanosoma cruzi trans-sialidase (EC 2.4.1.-). The present work was undertaken to test the hypothesis that a particular aromatic sandwich structure of two amino acids proximal to the active site of the T. cruzi trans-sialidase infers trans-sialidase activity. On this basis, four enzymes with putative trans-sialidase activity were identified through an iterative alignment from 2909 native sialidases available in GenBank, which were cloned and expressed in Escherichia coli. Of these, one enzyme, SialH, derived from Haemophilus parasuis had an aromatic sandwich structure on the protein surface facing the end of the catalytic site (Phe168; Trp366), and was indeed found to exhibit trans-sialidase activity. SialH catalyzed production of the human milk oligosaccharide 3’-sialyllactose as well as the novel trans-sialylation product 3-sialyllactose using casein glycomacropeptide as sialyl donor and lactose as acceptor. The findings corroborated that Tyr119 and Trp312 in the T. cruzi trans-sialidase are part of an aromatic sandwich structure that confers trans-sialylation activity for lactose sialylation. The in silico identification of trans-glycosidase activity by rational active site topology alignment thus proved to be a quick tool for selecting putative trans-sialidases amongst a large group of glycosyl hydrolases. The approach moreover provided data that help understand structure-function relations of trans-sialidases. [ABSTRACT FROM AUTHOR]

A mutant Trypanosoma rangeli sialidase, Tr7, expressed in Pichia pastoris, exhibits significant transsialidase activity, and has been used for analytical-scale production of sialylated human milk oligosaccharides. Mass spectrometry-based site-specific N-glycoprofiling of Tr7 showed that heterogeneous high-mannose type N-glycans were present at all the five potential N-linked glycosites. N-linked glycans in Tr7 were predominantly neutral oligosaccharides with compositions Man8-16GlcNAc2, but also mono- and di-phosphorylated oligosaccharides in the forms of Man9-15P1GlcNAc2 and Man9-14P2GlcNAc2, respectively. Some phosphorylated N-linked glycans further contained an additional HexNAc, which has not previously been reported in P. pastorisexpressed proteins. We compiled a method pipeline that combined hydrophilic interaction liquid chromatography enrichment of glycopeptides, high accuracy mass spectrometry and automated interpretation of the mass spectra with in-house developed "MassAI" software, which proved efficient in glycan site microheterogeneity analysis. Functional analysis showed that the deglycosylated Tr7 retained more than 90% of both the sialidase and trans-sialidase activities relative to the glycosylated Tr7. [ABSTRACT FROM AUTHOR]

AbrB is a global gene regulator involved in transition phase phenomena in B acillus subtilis. It participates in a complex regulatory network governing the expression of stationary-phase functions. AbrB was previously found to be phosphorylated on serine 86 located close to its C-terminal oligomerization domain. Here we report that AbrB can be phosphorylated by three B . subtilis serine/threonine kinases expressed during the transition and stationary phase: PrkC, PrkD and YabT. Our in vitro findings suggest that AbrB phosphorylation impedes its DNA binding and abolishes binding cooperativity. In vivo we established that a phospho-mimetic mutation abrB S86 D leads to a significant loss of AbrB control over several key target functions: exoprotease production, competence development and sporulation. A wider transcriptome analysis of abrB S86 D and S86 A mutant strains revealed deregulation of a large number of target genes. We therefore propose that AbrB phosphorylation serves as an additional input for fine-tuning the activity of this ambiactive gene regulator. [ABSTRACT FROM AUTHOR]