Characterization of a Novel Glucosamine-6-Phosphate Deaminase from a Hyperthermophilic Archaeon

ABSTRACT A key step in amino sugar metabolism is the interconversion between fructose-6-phosphate (Fru6P) and glucosamine-6-phosphate (GlcN6P). This conversion is catalyzed in the catabolic and anabolic directions by GlcN6P deaminase and GlcN6P synthase, respectively, two enzymes that show no relationship with one another in terms of primary structure. In this study, we examined the catalytic properties and regulatory features of the glmD gene product (GlmD Tk ) present within a chitin degradation gene cluster in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Although the protein GlmD Tk was predicted as a probable sugar isomerase related to the C-terminal sugar isomerase domain of GlcN6P synthase, the recombinant GlmD Tk clearly exhibited GlcN6P deaminase activity, generating Fru6P and ammonia from GlcN6P. This enzyme also catalyzed the reverse reaction, the ammonia-dependent amination/isomerization of Fru6P to GlcN6P, whereas no GlcN6P synthase activity dependent on glutamine was observed. Kinetic analyses clarified the preference of this enzyme for the deaminase reaction rather than the reverse one, consistent with the catabolic function of GlmD Tk . In T. kodakaraensis cells, glmDTk was polycistronically transcribed together with upstream genes encoding an ABC transporter and a downstream exo-β-glucosaminidase gene (glmATk ) within the gene cluster, and their expression was induced by the chitin degradation intermediate, diacetylchitobiose. The results presented here indicate that GlmD Tk is actually a GlcN6P deaminase functioning in the entry of chitin-derived monosaccharides to glycolysis in this hyperthermophile. This enzyme is the first example of an archaeal GlcN6P deaminase and is a structurally novel type distinct from any previously known GlcN6P deaminase.

Download Full-text

Characterization of a novel 3-O-α-D-galactosyl-α-L-arabinofuranosidase for the assimilation of gum arabic AGP in Bifidobacterium longum subsp. longum

Applied and Environmental Microbiology ◽

10.1128/aem.02690-20 ◽

2021 ◽

Author(s):

Yuki Sasaki ◽

Ayako Horigome ◽

Toshitaka Odamaki ◽

Jin-Zhong Xiao ◽

Akihiro Ishiwata ◽

...

Keyword(s):

Gene Cluster ◽

Bifidobacterium Longum ◽

Gum Arabic ◽

Glycoside Hydrolase Family ◽

Type Ii ◽

Cooperative Action ◽

Genes Encoding ◽

Key Enzyme ◽

Degradative Enzymes

Gum arabic arabinogalactan (AG) protein (AGP) is a unique dietary fiber that is degraded and assimilated by only specific strains of Bifidobacterium longum subsp. longum. Here, we identified a novel 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase) from B. longum JCM7052, and classified it into the glycoside hydrolase family 39 (GH39). GAfase released α-d-Galp-(1→3)-l-Ara and β-l-Arap-(1→3)-l-Ara from gum arabic AGP and β-l-Arap-(1→3)-l-Ara from larch AGP, and the α-d-Galp-(1→3)-l-Ara release activity was found to be 594-fold higher than that of β-l-Arap-(1→3)-l-Ara. The GAfase gene was part of a gene cluster that included genes encoding a GH36 α-galactosidase candidate and ABC transporters for the assimilation of the released α-d-Galp-(1→3)-l-Ara in B. longum. Notably, when α-d-Galp-(1→3)-l-Ara was removed from gum arabic AGP, it was assimilated by both B. longum JCM7052 and the non-assimilative B. longum JCM1217, suggesting that the removal of α-d-Galp-(1→3)-l-Ara from gum arabic AGP by GAfase permitted the cooperative action with type-II AG degradative enzymes in B. longum. The present study provides new insight into the mechanism of gum arabic AGP degradation in B. longum. IMPORTANCE Bifidobacteria harbor numerous carbohydrate-active enzymes that degrade several dietary fibers in the gastrointestinal tract. B. longum JCM7052 is known to exhibit the ability to assimilate gum arabic AGP, but the key enzyme involved in the degradation of gum arabic AGP remains unidentified. Here, we cloned and characterized a GH39 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase) from B. longum JCM7052. The enzyme was responsible for the release of α-d-Galp-(1→3)-l-Ara and β-l-Arap-(1→3)-l-Ara from gum arabic AGP. The presence of a gene cluster including the GAfase gene is specifically observed in gum arabic AGP assimilative strains. However, GAfase-carrier strains may affect GAfase-noncarrier strains that express other type-II AG degradative enzymes. These findings provide insights into the bifidogenic effect of gum arabic AGP.

Download Full-text

A gene cluster involved in the biosynthesis of vanchrobactin, a chromosome-encoded siderophore produced by Vibrio anguillarum

Microbiology ◽

10.1099/mic.0.29298-0 ◽

2006 ◽

Vol 152 (12) ◽

pp. 3517-3528 ◽

Cited By ~ 35

Author(s):

Miguel Balado ◽

Carlos R. Osorio ◽

Manuel L. Lemos

Keyword(s):

Gene Cluster ◽

Vibrio Anguillarum ◽

No Production ◽

Genetic Determinants ◽

Dihydroxybenzoic Acid ◽

Peptide Synthetase ◽

Genes Encoding ◽

In Trans ◽

Culture Supernatants

Vibrio anguillarum serotype O2 strains produce a catechol siderophore named vanchrobactin, which has been identified as N-[N′-(2,3-dihydroxybenzoyl)-arginyl]-serine. This work describes a chromosomal region that harbours the genetic determinants necessary for the biosynthesis of vanchrobactin. The authors have identified the genes involved in 2,3-dihydroxybenzoic acid (DHBA) biosynthesis (vabA, vabB and vabC) and activation (vabE), and a gene (vabF) encoding a non-ribosomal peptide synthetase, which is putatively involved in the assembly of the siderophore components. Also described are the identification and characterization of genes encoding a putative vanchrobactin exporter (vabS) and a siderophore esterase (vabH). In-frame deletion mutants in vabA, vabB, vabC, vabE, vabF and vabH were impaired for growth under conditions of iron limitation, and the analysis of culture supernatants by chrome azurol-S and cross-feeding assays showed almost no production of siderophores in any of the vabABCEF mutants. In addition, deletion mutations of vabA, vabB and vabC abolished production of DHBA, as assessed by chemical and biological analyses. Complementation of each mutant with the corresponding gene provided in trans confirmed the involvement of this gene cluster in the biosynthesis of DHBA and vanchrobactin in V. anguillarum strain RV22. Based on chemical and genetic data, and on published models for other catechol siderophores, a model for vanchrobactin biosynthesis is proposed.

Download Full-text

Identification and Characterization of Genes Encoding a Putative ABC-Type Transporter Essential for Utilization of γ-Hexachlorocyclohexane in Sphingobium japonicum UT26

Journal of Bacteriology ◽

10.1128/jb.01883-06 ◽

2007 ◽

Vol 189 (10) ◽

pp. 3712-3720 ◽

Cited By ~ 37

Author(s):

Ryo Endo ◽

Yoshiyuki Ohtsubo ◽

Masataka Tsuda ◽

Yuji Nagata

Keyword(s):

Abc Transporter ◽

Sole Source ◽

Bacterial Cells ◽

New Genes ◽

Hydrophobic Compounds ◽

Dead End ◽

Genes Encoding ◽

Degradation Activity ◽

Mutant Cells

ABSTRACT Sphingobium japonicum UT26 utilizes γ-hexachlorocyclohexane (γ-HCH) as its sole source of carbon and energy. In our previous studies, we cloned and characterized genes encoding enzymes for the conversion of γ-HCH to β-ketoadipate in UT26. In this study, we analyzed a mutant obtained by transposon mutagenesis and identified and characterized new genes encoding a putative ABC-type transporter essential for the utilization of γ-HCH in strain UT26. This putative ABC transporter consists of four components, permease, ATPase, periplasmic protein, and lipoprotein, encoded by linK, linL, linM, and linN, respectively. Mutation and complementation analyses indicated that all the linKLMN genes are required, probably as a set, for γ-HCH utilization in UT26. Furthermore, the mutant cells deficient in this putative ABC transporter showed (i) higher γ-HCH degradation activity and greater accumulation of the toxic dead-end product 2,5-dichlorophenol (2,5-DCP), (ii) higher sensitivity to 2,5-DCP itself, and (iii) higher permeability of hydrophobic compounds than the wild-type cells. These results strongly suggested that LinKLMN are involved in γ-HCH utilization by controlling membrane hydrophobicity. This study clearly demonstrated that a cellular factor besides catabolic enzymes and transcriptional regulators is essential for utilization of xenobiotic compounds in bacterial cells.

Download Full-text

Characterization of the Erwinia chrysanthemi gan Locus, Involved in Galactan Catabolism

Journal of Bacteriology ◽

10.1128/jb.00845-07 ◽

2007 ◽

Vol 189 (19) ◽

pp. 7053-7061 ◽

Cited By ~ 23

Author(s):

Aurélie Delangle ◽

Anne-France Prouvost ◽

Virginie Cogez ◽

Jean-Pierre Bohin ◽

Jean-Marie Lacroix ◽

...

Keyword(s):

Abc Transporter ◽

Transport System ◽

Pathogenic Bacteria ◽

Erwinia Chrysanthemi ◽

Potato Tubers ◽

Inner Membrane ◽

Saintpaulia Ionantha ◽

Genes Encoding

ABSTRACT β-1,4-Galactan is a major component of the ramified regions of pectin. Analysis of the genome of the plant pathogenic bacteria Erwinia chrysanthemi revealed the presence of a cluster of eight genes encoding proteins potentially involved in galactan utilization. The predicted transport system would comprise a specific porin GanL and an ABC transporter made of four proteins, GanFGK2. Degradation of galactans would be catalyzed by the periplasmic 1,4-β-endogalactanase GanA, which released oligogalactans from trimer to hexamer. After their transport through the inner membrane, oligogalactans would be degraded into galactose by the cytoplasmic 1,4-β-exogalactanase GanB. Mutants affected for the porin or endogalactanase were unable to grow on galactans, but they grew on galactose and on a mixture of galactotriose, galactotetraose, galactopentaose, and galactohexaose. Mutants affected for the periplasmic galactan binding protein, the transporter ATPase, or the exogalactanase were only able to grow on galactose. Thus, the phenotypes of these mutants confirmed the functionality of the gan locus in transport and catabolism of galactans. These mutations did not affect the virulence of E. chrysanthemi on chicory leaves, potato tubers, or Saintpaulia ionantha, suggesting an accessory role of galactan utilization in the bacterial pathogeny.

Download Full-text

Streptococcal phosphotransferase system imports unsaturated hyaluronan disaccharide derived from host extracellular matrices

10.1101/480228 ◽

2018 ◽

Author(s):

Sayoko Oiki ◽

Yusuke Nakamichi ◽

Yukie Maruyama ◽

Bunzo Mikami ◽

Kousaku Murata ◽

...

Keyword(s):

Abc Transporter ◽

Molecular System ◽

Bacterial Species ◽

Three Dimensional ◽

Amino Sugar ◽

Extracellular Matrices ◽

Dimensional Structure ◽

Phosphotransferase System ◽

Streptobacillus Moniliformis ◽

Genes Encoding

ABSTRACTCertain bacterial species target the polysaccharide glycosaminoglycans (GAGs) of animal extracellular matrices for colonization and/or infection. GAGs such as hyaluronan and chondroitin sulfate consist of repeating disaccharide units of uronate and amino sugar residues, and are depolymerized to unsaturated disaccharides by bacterial extracellular or cell-surface polysaccharide lyase. The disaccharides are degraded and metabolized by cytoplasmic enzymes such as unsaturated glucuronyl hydrolase, isomerase, and reductase. The genes encoding these enzymes are assembled to form a GAG genetic cluster. Here, we demonstrate theStreptococcus agalactiaephosphotransferase system (PTS) for import of unsaturated hyaluronan disaccharide.S. agalactiaeNEM316 was found to depolymerize and assimilate hyaluronan, whereas its mutant with a disruption in PTS genes included in the GAG cluster was unable to grow on hyaluronan, while retaining the ability to depolymerize hyaluronan. Using toluene-treated wild-type cells, the PTS import activity of unsaturated hyaluronan disaccharide was significantly higher than that observed in the absence of the substrate. In contrast, the PTS mutant was unable to import unsaturated hyaluronan disaccharide, indicating that the corresponding PTS is the only importer of fragmented hyaluronan, which is suitable for PTS to phosphorylate the substrate at the C-6 position. The three-dimensional structure of streptococcal EIIA, one of the PTS components, was found to contain a Rossman-fold motif by X-ray crystallization. Docking of EIIA with another component EIIB by modeling provided structural insights into the phosphate transfer mechanism. This study is the first to identify the substrate (unsaturated hyaluronan disaccharide) recognized and imported by the streptococcal PTS.IMPORTANCE (118/120 words)The PTS identified in this work imports sulfate group-free unsaturated hyaluronan disaccharide as a result of the phosphorylation of the substrate at the C-6 position.S. agalactiaecan be indigenous to animal hyaluronan-rich tissues owing to the bacterial molecular system for fragmentation, import, degradation, and metabolism of hyaluronan. Distinct from hyaluronan, most GAGs, which are sulfated at the C-6 position, are unsuitable for PTS due to its inability to phosphorylate the substrate. More recently, we have identified a solute-binding protein-dependent ABC transporter in a pathogenicStreptobacillus moniliformisas an importer of sulfated and non-sulfated fragmented GAGs without any substrate modification. Our findings regarding PTS and ABC transporter shed light on bacterial clever colonization/infection system targeting various animal GAGs.

Download Full-text

Identifying candidate Aspergillus pathogenicity factors by annotation frequency

BMC Microbiology ◽

10.1186/s12866-020-02031-y ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Kayla K. Pennerman ◽

Guohua Yin ◽

Anthony E. Glenn ◽

Joan W. Bennett

Keyword(s):

Gc Content ◽

Sugar Metabolism ◽

Principal Component ◽

Amino Sugar ◽

Human Immune System ◽

Protein Coding ◽

Functional Annotations ◽

Pathogenicity Factors ◽

Genes Encoding ◽

Effective Use

Abstract Background Members of the genus Aspergillus display a variety of lifestyles, ranging from saprobic to pathogenic on plants and/or animals. Increased genome sequencing of economically important members of the genus permits effective use of “-omics” comparisons between closely related species and strains to identify candidate genes that may contribute to phenotypes of interest, especially relating to pathogenicity. Protein-coding genes were predicted from 216 genomes of 12 Aspergillus species, and the frequencies of various structural aspects (exon count and length, intron count and length, GC content, and codon usage) and functional annotations (InterPro, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes terms) were compared. Results Using principal component analyses, the three sets of functional annotations for each strain were clustered by species. The species clusters appeared to separate by pathogenicity on plants along the first dimensions, which accounted for over 20% of the variance. More annotations for genes encoding pectinases and secondary metabolite biosynthetic enzymes were assigned to phytopathogenic strains from species such as Aspergillus flavus. In contrast, Aspergillus fumigatus strains, which are pathogenic to animals but not plants, were assigned relatively more terms related to phosphate transferases, and carbohydrate and amino-sugar metabolism. Analyses of publicly available RNA-Seq data indicated that one A. fumigatus protein among 17 amino-sugar processing candidates, a hexokinase, was up-regulated during co-culturing with human immune system cells. Conclusion Genes encoding hexokinases and other proteins of interest may be subject to future manipulations to further refine understanding of Aspergillus pathogenicity factors.

Download Full-text

Characterization of a Gene Cluster Involved in 4-Chlorocatechol Degradation by Pseudomonas reinekei MT1

Journal of Bacteriology ◽

10.1128/jb.00331-09 ◽

2009 ◽

Vol 191 (15) ◽

pp. 4905-4915 ◽

Cited By ~ 14

Author(s):

Beatriz Cámara ◽

Patricia Nikodem ◽

Piotr Bielecki ◽

Roberto Bobadilla ◽

Howard Junca ◽

...

Keyword(s):

Gene Cluster ◽

Regulatory Gene ◽

Genomic Region ◽

Genes Encoding ◽

Catechol 1,2 Dioxygenase ◽

Dienelactone Hydrolase ◽

Intradiol Cleavage ◽

Maleylacetate Reductase ◽

Muconate Cycloisomerase

ABSTRACT Pseudomonas reinekei MT1 has previously been reported to degrade 4- and 5-chlorosalicylate by a pathway with 4-chlorocatechol, 3-chloromuconate, 4-chloromuconolactone, and maleylacetate as intermediates, and a gene cluster channeling various salicylates into an intradiol cleavage route has been reported. We now report that during growth on 5-chlorosalicylate, besides a novel (chloro)catechol 1,2-dioxygenase, C12OccaA, a novel (chloro)muconate cycloisomerase, MCIccaB, which showed features not yet reported, was induced. This cycloisomerase, which was practically inactive with muconate, evolved for the turnover of 3-substituted muconates and transforms 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, suggesting that it is a functional intermediate between chloromuconate cycloisomerases and muconate cycloisomerases. The corresponding genes, ccaA (C12OccaA) and ccaB (MCIccaB), were located in a 5.1-kb genomic region clustered with genes encoding trans-dienelactone hydrolase (ccaC) and maleylacetate reductase (ccaD) and a putative regulatory gene, ccaR, homologous to regulators of the IclR-type family. Thus, this region includes genes sufficient to enable MT1 to transform 4-chlorocatechol to 3-oxoadipate. Phylogenetic analysis showed that C12OccaA and MCIccaB are only distantly related to previously described catechol 1,2-dioxygenases and muconate cycloisomerases. Kinetic analysis indicated that MCIccaB and the previously identified C12OsalD, rather than C12OccaA, are crucial for 5-chlorosalicylate degradation. Thus, MT1 uses enzymes encoded by a completely novel gene cluster for degradation of chlorosalicylates, which, together with a gene cluster encoding enzymes for channeling salicylates into the ortho-cleavage pathway, form an effective pathway for 4- and 5-chlorosalicylate mineralization.

Download Full-text

A Member of the Second Carbohydrate Uptake Subfamily of ATP-Binding Cassette Transporters Is Responsible for Ribonucleoside Uptake in Streptococcus mutans

Journal of Bacteriology ◽

10.1128/jb.01101-06 ◽

2006 ◽

Vol 188 (23) ◽

pp. 8005-8012 ◽

Cited By ~ 20

Author(s):

Alexander J. Webb ◽

Arthur H. F. Hosie

Keyword(s):

Streptococcus Mutans ◽

Abc Transporter ◽

Atp Binding ◽

Diverse Range ◽

Adenosine Uptake ◽

Uptake Rates ◽

Genes Encoding ◽

Uptake Transporters ◽

Atp Binding Cassette

ABSTRACT Streptococcus mutans has a significant number of transporters of the ATP-binding cassette (ABC) superfamily. Members of this superfamily are involved in the translocation of a diverse range of molecules across membranes. However, the functions of many of these members remain unknown. We have investigated the role of the single S. mutans representative of the second subfamily of carbohydrate uptake transporters (CUT2) of the ABC superfamily. The genetic context of genes encoding this transporter indicates that it may have a role in ribonucleoside scavenging. Inactivation of rnsA (ATPase) or rnsB (solute binding protein) resulted in strains resistant to 5-fluorocytidine and 5-fluorouridine (toxic ribonucleoside analogues). As other ribonucleosides including cytidine, uridine, adenosine, 2-deoxyuridine, and 2-deoxycytidine protected S. mutans from 5-fluorocytidine and 5-fluorouridine toxicity, it is likely that this transporter is involved in the uptake of these molecules. Indeed, the rnsA and rnsB mutants were unable to transport [2-14C]cytidine or [2-14C]uridine and had significantly reduced [8-14C]adenosine uptake rates. Characterization of this transporter in wild-type S. mutans indicates that it is a high-affinity (Km = 1 to 2 μM) transporter of cytidine, uridine, and adenosine. The inhibition of [14C]cytidine uptake by a range of structurally related molecules indicates that the CUT2 transporter is involved in the uptake of most ribonucleosides, including 2-deoxyribonucleosides, but not ribose or nucleobases. The characterization of this permease has directly shown for the first time that an ABC transporter is involved in the uptake of ribonucleosides and extends the range of substrates known to be transported by members of the ABC transporter superfamily.

Download Full-text

Distribution of Suicin Gene Clusters in Streptococcus suis Serotype 2 Belonging to Sequence Types 25 and 28

BioMed Research International ◽

10.1155/2016/6815894 ◽

2016 ◽

Vol 2016 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

Taryn B. T. Athey ◽

Katy Vaillancourt ◽

Michel Frenette ◽

Nahuel Fittipaldi ◽

Marcelo Gottschalk ◽

...

Keyword(s):

Gene Cluster ◽

Streptococcus Suis ◽

Gene Clusters ◽

Sequence Type ◽

Purification And Characterization ◽

Heterogeneous Distribution ◽

Genes Encoding ◽

Sequence Types ◽

Suis Serotype

Recently, we reported the purification and characterization of three distinct lantibiotics (named suicin 90-1330, suicin 3908, and suicin 65) produced by Streptococcus suis. In this study, we investigated the distribution of the three suicin lantibiotic gene clusters among serotype 2 S. suis strains belonging to sequence type (ST) 25 and ST28, the two dominant STs identified in North America. The genomes of 102 strains were interrogated for the presence of suicin gene clusters encoding suicins 90-1330, 3908, and 65. The gene cluster encoding suicin 65 was the most prevalent and mainly found among ST25 strains. In contrast, none of the genes related to suicin 90-1330 production were identified in 51 ST25 strains nor in 35/51 ST28 strains. However, the complete suicin 90-1330 gene cluster was found in ten ST28 strains, although some genes in the cluster were truncated in three of these isolates. The vast majority (101/102) of S. suis strains did not possess any of the genes encoding suicin 3908. In conclusion, this study indicates heterogeneous distribution of suicin genes in S. suis.

Download Full-text