scholarly journals Physiological Adaptation of Desulfitobacterium hafniense Strain TCE1 to Tetrachloroethene Respiration

2011 ◽  
Vol 77 (11) ◽  
pp. 3853-3859 ◽  
Author(s):  
Laure Prat ◽  
Julien Maillard ◽  
Régis Grimaud ◽  
Christof Holliger
Keyword(s):  
Electron Acceptor ◽  
Growth Conditions ◽  
Mass Spectrometry Analysis ◽  
Physiological Adaptation ◽  
Terminal Electron Acceptor ◽  
Organohalide Respiration ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Metabolic Versatility ◽  
Desulfitobacterium Hafniense

ABSTRACTDesulfitobacteriumspp. are ubiquitous organisms with a broad metabolic versatility, and some isolates have the ability to use tetrachloroethene (PCE) as terminal electron acceptor. In order to identify proteins involved in this organohalide respiration process, a comparative proteomic analysis was performed. Soluble and membrane-associated proteins obtained from cells ofDesulfitobacterium hafniensestrain TCE1 that were growing on different combinations of the electron donors lactate and hydrogen and the electron acceptors PCE and fumarate were analyzed. Among proteins increasingly expressed in the presence of PCE compared to fumarate as electron acceptor, a total of 57 proteins were identified by mass spectrometry analysis, revealing proteins involved in stress response and associated regulation pathways, such as PspA, GroEL, and CodY, and also proteins potentially participating in carbon and energy metabolism, such as proteins of the Wood-Ljungdahl pathway and electron transfer flavoproteins. These proteomic results suggest thatD. hafniensestrain TCE1 adapts its physiology to face the relative unfavorable growth conditions during an apparent opportunistic organohalide respiration.

Magnetovibrio blakemorei gen. nov., sp. nov., a magnetotactic bacterium ( Alphaproteobacteria : Rhodospirillaceae ) isolated from a salt marsh

2013 ◽  
Vol 63 (Pt_5) ◽  
pp. 1824-1833 ◽  
Author(s):  
Dennis A. Bazylinski ◽  
Timothy J. Williams ◽  
Christopher T. Lefèvre ◽  
Denis Trubitsyn ◽  
Jiasong Fang ◽  
...  
Keyword(s):  
Salt Marsh ◽  
Electron Acceptor ◽  
Anaerobic Growth ◽  
Rrna Gene ◽  
Magnetotactic Bacterium ◽  
Single Chain ◽  
Circular Chromosome ◽  
Terminal Electron Acceptor ◽  
Content Type ◽  
Link Type

A magnetotactic bacterium, designated strain MV-1T, was isolated from sulfide-rich sediments in a salt marsh near Boston, MA, USA. Cells of strain MV-1T were Gram-negative, and vibrioid to helicoid in morphology. Cells were motile by means of a single polar flagellum. The cells appeared to display a transitional state between axial and polar magnetotaxis: cells swam in both directions, but generally had longer excursions in one direction than the other. Cells possessed a single chain of magnetosomes containing truncated hexaoctahedral crystals of magnetite, positioned along the long axis of the cell. Strain MV-1T was a microaerophile that was also capable of anaerobic growth on some nitrogen oxides. Salinities greater than 10 % seawater were required for growth. Strain MV-1T exhibited chemolithoautotrophic growth on thiosulfate and sulfide with oxygen as the terminal electron acceptor (microaerobic growth) and on thiosulfate using nitrous oxide (N2O) as the terminal electron acceptor (anaerobic growth). Chemo-organoautotrophic and methylotrophic growth was supported by formate under microaerobic conditions. Autotrophic growth occurred via the Calvin–Benson–Bassham cycle. Chemo-organoheterotrophic growth was supported by various organic acids and amino acids, under microaerobic and anaerobic conditions. Optimal growth occurred at pH 7.0 and 26–28 °C. The genome of strain MV-1T consisted of a single, circular chromosome, about 3.7 Mb in size, with a G+C content of 52.9–53.5 mol%.Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MV-1T belongs to the family Rhodospirillaceae within the Alphaproteobacteria , but is not closely related to the genus Magnetospirillum . The name Magnetovibrio blakemorei gen. nov., sp. nov. is proposed for strain MV-1T. The type strain of Magnetovibrio blakemorei is MV-1T ( = ATCC BAA-1436T  = DSM 18854T).

scholarly journals Thiol Peroxidase Is an Important Component of Streptococcus pneumoniae in Oxygenated Environments

2012 ◽  
Vol 80 (12) ◽  
pp. 4333-4343 ◽  
Author(s):  
Barak Hajaj ◽  
Hasan Yesilkaya ◽  
Rachel Benisty ◽  
Maayan David ◽  
Peter W. Andrew ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Mass Spectrometry Analysis ◽  
Activity Levels ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Cellular Processes ◽  
Thiol Peroxidase ◽  
Fine Tune

ABSTRACTStreptococcus pneumoniaeis an aerotolerant Gram-positive bacterium that causes an array of diseases, including pneumonia, otitis media, and meningitis. During aerobic growth,S. pneumoniaeproduces high levels of H2O2. SinceS. pneumoniaelacks catalase, the question of how it controls H2O2levels is of critical importance. Thepsalocus encodes an ABC Mn2+-permease complex (psaBCA) and a putative thiol peroxidase,tpxD. This study shows thattpxDencodes a functional thiol peroxidase involved in the adjustment of H2O2homeostasis in the cell. Kinetic experiments showed that recombinant TpxD removed H2O2efficiently. However,in vivoexperiments revealed that TpxD detoxifies only a fraction of the H2O2generated by the pneumococcus. Mass spectrometry analysis demonstrated that TpxD Cys58undergoes selective oxidationin vivo, under conditions where H2O2is formed, confirming the thiol peroxidase activity. Levels of TpxD expression and synthesisin vitrowere significantly increased in cells grown under aerobic versus anaerobic conditions. The challenge with D39 and TIGR4 with H2O2resulted intpxDupregulation, whilepsaBCAexpression was oppositely affected. However, the challenge of ΔtpxDmutants with H2O2did not affectpsaBCA, implying that TpxD is involved in the regulation of thepsaoperon, in addition to its scavenging activity. Virulence studies demonstrated a notable difference in the survival time of mice infected intranasally with D39 compared to that of mice infected intranasally with D39ΔtpxD. However, when bacteria were administered directly into the blood, this difference disappeared. The findings of this study suggest that TpxD constitutes a component of the organism's fundamental strategy to fine-tune cellular processes in response to H2O2.

scholarly journals Dissection of the Role of Pili and Type 2 and 3 Secretion Systems in Adherence and Biofilm Formation of an Atypical Enteropathogenic Escherichia coli Strain

2013 ◽  
Vol 81 (10) ◽  
pp. 3793-3802 ◽  
Author(s):  
Rodrigo T. Hernandes ◽  
Miguel A. De la Cruz ◽  
Denise Yamamoto ◽  
Jorge A. Girón ◽  
Tânia A. T. Gomes
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Coli Strain ◽  
Mass Spectrometry Analysis ◽  
Secretion Systems ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Rigid Rod

ABSTRACTAtypical enteropathogenicEscherichia coli(aEPEC) strains are diarrheal pathogens that lack bundle-forming pilus production but possess the virulence-associated locus of enterocyte effacement. aEPEC strain 1551-2 produces localized adherence (LA) on HeLa cells; however, its isogenic intimin (eae) mutant produces a diffuse-adherence (DA) pattern. In this study, we aimed to identify the DA-associated adhesin of the 1551-2eaemutant. Electron microscopy of 1551-2 identified rigid rod-like pili composed of an 18-kDa protein, which was identified as the major pilin subunit of type 1 pilus (T1P) by mass spectrometry analysis. Deletion offimAin 1551-2 affected biofilm formation but had no effect on adherence properties. Analysis of secreted proteins in supernatants of this strain identified a 150-kDa protein corresponding to SslE, a type 2 secreted protein that was recently reported to be involved in biofilm formation of rabbit and human EPEC strains. However, neither adherence nor biofilm formation was affected in a 1551-2sslEmutant. We then investigated the role of the EspA filament associated with the type 3 secretion system (T3SS) in DA by generating a doubleeae espAmutant. This strain was no longer adherent, strongly suggesting that the T3SS translocon is the DA adhesin. In agreement with these results, specific anti-EspA antibodies blocked adherence of the 1551-2eaemutant. Our data support a role for intimin in LA, for the T3SS translocon in DA, and for T1P in biofilm formation, all of which may act in concert to facilitate host intestinal colonization by aEPEC strains.

scholarly journals Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other IdentifiedEnterococcus faecalisPhage Endolysins

2019 ◽  
Vol 85 (13) ◽  
Author(s):  
Hongming Zhang ◽  
Bettina A. Buttaro ◽  
Derrick E. Fouts ◽  
Salar Sanjari ◽  
Bradley S. Evans ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecalis ◽  
Amino Acid Identity ◽  
Lytic Infection ◽  
Mass Spectrometry Analysis ◽  
Lytic Enzyme ◽  
Lytic Enzymes ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Wide Range

ABSTRACTϕEf11 is a temperateSiphoviridaebacteriophage that infects strains ofEnterococcus faecalis. The ϕEf11 genome, encompassing 65 open reading frames (ORFs), is contained within 42,822 bp of DNA. Within this genome, a module of six lysis-related genes was identified. Based upon sequence homology, one of these six genes, ORF28, was predicted to code for anN-acetylmuramoyl-l-alanine amidase endolysin of 46.133 kDa, composed of 421 amino acids. The PCR-amplified ORF28 was cloned and expressed, and the resulting gene product was affinity purified to homogeneity. The purified protein was obtained from a fusion protein that exhibited a molecular mass of 72.5 kDa, consistent with a 46.1-kDa protein combined with a fused 26.5-kDa glutathioneS-transferase tag. It produced rapid, profound lysis inE. faecalispopulations and was active against 73 of 103 (71%)E. faecalisstrains tested. In addition, it caused substantial destruction ofE. faecalisbiofilms. The lysin was quite stable, retaining its activity for three years in refrigerated storage, was stable over a wide range of pHs, and was unaffected by the presence of a reducing agent; however, it was inhibited by increasing concentrations of Ca2+. Liquid chromatography-mass spectrometry analysis ofE. faecaliscell wall digestion products produced by the ORF28 endolysin indicated that the lysin acted as anN-acetylmuramidase, an endo-β-N-acetylglucosaminidase, and an endopeptidase, rather than anN-acetylmuramoyl-l-alanine amidase. The ϕEf11 ORF28 lysin shared 10% to 37% amino acid identity with the lytic enzymes of all other characterizedE. faecalisbacteriophages.IMPORTANCEThe emergence of multidrug-resistant pathogenic microorganisms has brought increasing attention to the urgent need for the development of alternative antimicrobial strategies. One such alternative to conventional antibiotics employs lytic enzymes (endolysins) that are produced by bacteriophages in the course of lytic infection. During lytic infection by a bacteriophage, these enzymes hydrolyze the cell wall peptidoglycan, resulting in the lysis of the host cell. However, external endolysin application can result in lysis from without. In this study, we have cloned, expressed, purified, and characterized an endolysin produced by a bacteriophage infecting strains ofEnterococcus faecalis. The lysin is broadly active against most of the testedE. faecalisstrains and exhibits multifunctional enzymatic specificities that differ from all other characterized endolysins produced byE. faecalisbacteriophages.

scholarly journals Transcriptomic and Proteomic Responses of the Organohalide-Respiring Bacterium Desulfoluna spongiiphila to Growth with 2,6-Dibromophenol as the Electron Acceptor

2019 ◽  
Vol 86 (5) ◽  
Author(s):  
Jie Liu ◽  
Lorenz Adrian ◽  
Max M. Häggblom
Keyword(s):  
Gene Expression ◽  
Secondary Metabolites ◽  
Electron Acceptor ◽  
Marine Sponge ◽  
Reductive Dehalogenation ◽  
Important Process ◽  
Organohalide Respiration ◽  
Content Type ◽  
Reductive Dehalogenase ◽  
Significant Difference

ABSTRACT Organohalide respiration is an important process in the global halogen cycle and for bioremediation. In this study, we compared the global transcriptomic and proteomic analyses of Desulfoluna spongiiphila strain AA1, an organohalide-respiring member of the Desulfobacterota isolated from a marine sponge, with 2,6-dibromophenol or with sulfate as an electron acceptor. The most significant difference of the transcriptomic analysis was the expression of one reductive dehalogenase gene cluster (rdh16), which was significantly upregulated with the addition of 2,6-dibromophenol. The corresponding protein, reductive dehalogenase RdhA16032, was detected in the proteome under treatment with 2,6-dibromophenol but not with sulfate only. There was no significant difference in corrinoid biosynthesis gene expression levels between the two treatments, indicating that the production of corrinoid in D. spongiiphila is constitutive or not specific for organohalide versus sulfate respiration. Electron-transporting proteins or mediators unique for reductive dehalogenation were not revealed in our analysis, and we hypothesize that reductive dehalogenation may share an electron-transporting system with sulfate reduction. The metabolism of D. spongiiphila, predicted from transcriptomic and proteomic results, demonstrates high metabolic versatility and provides insights into the survival strategies of a marine sponge symbiont in an environment rich in organohalide compounds and other secondary metabolites. IMPORTANCE Respiratory reductive dehalogenation is an important process in the overall cycling of both anthropogenic and natural organohalide compounds. Marine sponges produce a vast array of bioactive compounds as secondary metabolites, including diverse halogenated compounds that may enrich for dehalogenating bacteria. Desulfoluna spongiiphila strain AA1 was originally enriched and isolated from the marine sponge Aplysina aerophoba and can grow with both brominated compounds and sulfate as electron acceptors for respiration. An understanding of the overall gene expression and the protein production profile in response to organohalides is needed to identify the full complement of genes or enzymes involved in organohalide respiration. Elucidating the metabolic capacity of this sponge-associated bacterium lays the foundation for understanding how dehalogenating bacteria may control the fate of organohalide compounds in sponges and their role in a symbiotic organobromine cycle.

scholarly journals Evidence that Biosynthesis of the Second and Third Sugars of the Archaellin Tetrasaccharide in the Archaeon Methanococcus maripaludis Occurs by the Same Pathway Used by Pseudomonas aeruginosa To Make a Di-N-Acetylated Sugar

2015 ◽  
Vol 197 (9) ◽  
pp. 1668-1680 ◽  
Author(s):  
Sarah Siu ◽  
Anna Robotham ◽  
Susan M. Logan ◽  
John F. Kelly ◽  
Kaoru Uchida ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mass Spectroscopy ◽  
Specific Antigen ◽  
Mass Spectrometry Analysis ◽  
Methanococcus Maripaludis ◽  
Western Blots ◽  
Bacterial Gene ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Type Iv

ABSTRACTMethanococcus maripaludishas two surface appendages, archaella and type IV pili, which are composed of glycoprotein subunits. Archaellins are modified with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. The pilin glycan has an additional hexose attached to GalNAc. In this study, genes located in two adjacent, divergently transcribed operons (mmp0350-mmp0354andmmp0359-mmp0355) were targeted for study based on annotations suggesting their involvement in biosynthesis of N-glycan sugars. Mutants carrying deletions inmmp0350,mmp0351,mmp0352, ormmp0353were nonarchaellated and synthesized archaellins modified with a 1-sugar glycan, as estimated from Western blots. Mass spectroscopy analysis of pili purified from the Δmmp0352strain confirmed a glycan with only GalNAc, suggestingmmp0350tommp0353were all involved in biosynthesis of the second sugar (GlcNAc3NAcA). The Δmmp0357mutant was archaellated and had archaellins with a 2-sugar glycan, as confirmed by mass spectroscopy of purified archaella, indicating a role for MMP0357 in biosynthesis of the third sugar (ManNAc3NAmA6Thr).M. maripaludismmp0350,mmp0351,mmp0352,mmp0353, andmmp0357are proposed to be functionally equivalent toPseudomonas aeruginosawbpABEDI, involved in converting UDP-N-acetylglucosamine to UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, an O5-specific antigen sugar. Cross-domain complementation of the final step of theP. aeruginosapathway withmmp0357supports this hypothesis.IMPORTANCEThis work identifies a series of genes in adjacent operons that are shown to encode the enzymes that complete the entire pathway for generation of the second and third sugars of the N-linked tetrasaccharide that modifies archaellins ofMethanococcus maripaludis. This posttranslational modification of archaellins is important, as it is necessary for archaellum assembly. Pilins are modified with a different N-glycan consisting of the archaellin tetrasaccharide but with an additional hexose attached to the linking sugar. Mass spectrometry analysis of the pili of one mutant strain provided insight into how this different glycan might ultimately be assembled. This study includes a rare example of an archaeal gene functionally replacing a bacterial gene in a complex sugar biosynthesis pathway.

scholarly journals Nonribosomal Peptide Synthetase GenespesLandpes1Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

2012 ◽  
Vol 78 (9) ◽  
pp. 3166-3176 ◽  
Author(s):  
Karen A. O'Hanlon ◽  
Lorna Gallagher ◽  
Markus Schrettl ◽  
Christoph Jöchl ◽  
Kevin Kavanagh ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Opportunistic Pathogen ◽  
Mass Spectrometry Analysis ◽  
Infection Model ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Peptide Synthetases ◽  
Increased Sensitivity

ABSTRACTThe identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen,Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway inA. fumigatus. Deletion of eitherpesL(ΔpesL) orpes1(Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H2O2, and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion ofpesLresulted in severely reduced virulence in an invertebrate infection model (P< 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster forA. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in bothA. fumigatuswild-type and ΔpesLstrains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesisin vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature.

scholarly journals Vacuolar Protein Sorting Protein 13A, TtVPS13A, Localizes to the Tetrahymena thermophila Phagosome Membrane and Is Required for Efficient Phagocytosis

2011 ◽  
Vol 10 (9) ◽  
pp. 1207-1218 ◽  
Author(s):  
Haresha S. Samaranayake ◽  
Ann E. Cowan ◽  
Lawrence A. Klobutcher
Keyword(s):  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Genetic Disorders ◽  
Protein Sorting ◽  
Mass Spectrometry Analysis ◽  
Vacuolar Protein Sorting ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Green Fluorescent ◽  
Vacuolar Protein

ABSTRACT Vacuolar protein sorting 13 (VPS13) proteins have been studied in a number of organisms, and mutations in VPS13 genes have been implicated in two human genetic disorders, but the function of these proteins is poorly understood. The TtVPS13A protein was previously identified in a mass spectrometry analysis of the Tetrahymena thermophila phagosome proteome (M. E. Jacobs et al., Eukaryot. Cell 5:1990–2000, 2006), suggesting that it is involved in phagocytosis. In this study, we analyzed the structure of the macronuclear TtVPS13A gene, which was found to be composed of 17 exons spanning 12.5 kb and was predicted to encode a protein of 3,475 amino acids (aa). A strain expressing a TtVPS13A-green fluorescent protein (GFP) fusion protein was constructed, and the protein was found to associate with the phagosome membrane during the entire cycle of phagocytosis. In addition, Tetrahymena cells with a TtVPS13A knockout mutation displayed impaired phagocytosis. Specifically, they grew slowly under conditions where phagocytosis is essential, they formed few phagosomes, and the digestion of phagosomal contents was delayed compared to wild-type cells. Overall, these results provide evidence that the TtVPS13A protein is required for efficient phagocytosis.

scholarly journals Dehalococcoides mccartyi Strain DCMB5 Respires a Broad Spectrum of Chlorinated Aromatic Compounds

2014 ◽  
Vol 81 (2) ◽  
pp. 587-596 ◽  
Author(s):  
Marlén Pöritz ◽  
Christian L. Schiffmann ◽  
Gerd Hause ◽  
Ulrike Heinemann ◽  
Jana Seifert ◽  
...  
Keyword(s):  
Electron Acceptor ◽  
Aromatic Compounds ◽  
Lag Phase ◽  
Organohalide Respiration ◽  
Content Type ◽  
Reductive Dehalogenase ◽  
Dehalococcoides Mccartyi ◽  
Type Iv ◽  
Transmission Electron ◽  
Almost All

ABSTRACTPolyhalogenated aromatic compounds are harmful environmental contaminants and tend to persist in anoxic soils and sediments.Dehalococcoides mccartyistrain DCMB5, a strain originating from dioxin-polluted river sediment, was examined for its capacity to dehalogenate diverse chloroaromatic compounds. Strain DCMB5 used hexachlorobenzenes, pentachlorobenzenes, all three tetrachlorobenzenes, and 1,2,3-trichlorobenzene as well as 1,2,3,4-tetra- and 1,2,4-trichlorodibenzo-p-dioxin as electron acceptors for organohalide respiration. In addition, 1,2,3-trichlorodibenzo-p-dioxin and 1,3-, 1,2-, and 1,4-dichlorodibenzo-p-dioxin were dechlorinated, the latter to the nonchlorinated congener with a remarkably short lag phase of 1 to 4 days following transfer. Strain DCMB5 also dechlorinated pentachlorophenol and almost all tetra- and trichlorophenols. Tetrachloroethene was dechlorinated to trichloroethene and served as an electron acceptor for growth. To relate selected dechlorination activities to the expression of specific reductive dehalogenase genes, the proteomes of 1,2,3-trichlorobenzene-, pentachlorobenzene-, and tetrachloroethene-dechlorinating cultures were analyzed. Dcmb_86, an ortholog of the chlorobenzene reductive dehalogenase CbrA, was the most abundant reductive dehalogenase during growth with each electron acceptor, suggesting its pivotal role in organohalide respiration of strain DCMB5. Dcmb_1041 was specifically induced, however, by both chlorobenzenes, whereas 3 putative reductive dehalogenases, Dcmb_1434, Dcmb_1339, and Dcmb_1383, were detected only in tetrachloroethene-grown cells. The proteomes also harbored a type IV pilus protein and the components for its assembly, disassembly, and secretion. In addition, transmission electron microscopy of DCMB5 revealed an irregular mode of cell division as well as the presence of pili, indicating that pilus formation is a feature ofD. mccartyiduring organohalide respiration.

scholarly journals GlcNAc De-N-Acetylase from the Hyperthermophilic ArchaeonSulfolobus solfataricus

2018 ◽  
Vol 85 (2) ◽  
Author(s):  
Roberta Iacono ◽  
Andrea Strazzulli ◽  
Luisa Maurelli ◽  
Nicola Curci ◽  
Angela Casillo ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Recombinant Enzyme ◽  
Mass Spectrometry Analysis ◽  
Genomic Context ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Additional Information ◽  
The One ◽  
Hydrolysis Of

ABSTRACTSulfolobus solfataricusis an aerobic crenarchaeal hyperthermophile with optimum growth at temperatures greater than 80°C and pH 2 to 4. Within the crenarchaeal group ofSulfolobales,N-acetylglucosamine (GlcNAc) has been shown to be a component of exopolysaccharides, forming their biofilms, and of theN-glycan decorating some proteins. The metabolism of GlcNAc is still poorly understood inArchaea, and one approach to gaining additional information is through the identification and functional characterization of carbohydrate active enzymes (CAZymes) involved in the modification of GlcNAc. The screening ofS. solfataricusextracts allowed the detection of a novel α-N-acetylglucosaminidase (α-GlcNAcase) activity, which has never been identified inArchaea. Mass spectrometry analysis of the purified activity showed a protein encoded by thesso2901gene. Interestingly, the purified recombinant enzyme, which was characterized in detail, revealed a novel de-N-acetylase activity specific for GlcNAc and derivatives. Thus, assays to identify an α-GlcNAcase found a GlcNAc de-N-acetylase instead. The α-GlcNAcase activity observed inS. solfataricusextracts did occur when SSO2901 was used in combination with an α-glucosidase. Furthermore, the inspection of the genomic context and the preliminary characterization of a putative glycosyltransferase immediately upstream ofsso2901(sso2900) suggest the involvement of these enzymes in the GlcNAc metabolism inS. solfataricus.IMPORTANCEIn this study, a preliminary screening of cellular extracts ofS. solfataricusallowed the identification of an α-N-acetylglucosaminidase activity. However, the characterization of the corresponding recombinant enzyme revealed a novel GlcNAc de-N-acetylase, which, in cooperation with the α-glucosidase, catalyzed the hydrolysis of O-α-GlcNAc glycosides. In addition, we show that the product of a gene flanking the one encoding the de-N-acetylase is a putative glycosyltransferase, suggesting the involvement of the two enzymes in the metabolism of GlcNAc. The discovery and functional analysis of novel enzymatic activities involved in the modification of this essential sugar represent a powerful strategy to shed light on the physiology and metabolism ofArchaea.

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