Expression, purification and preliminary crystallographic analysis of bacterial transmembrane protein EfeU for iron import.

The bacterial Efe system functions as an importer of free Fe2+ into cells independently of iron-chelating compounds such as siderophores and consisted of iron-binding protein EfeO, peroxidase EfeB, and transmembrane permease EfeU. While we and other researchers reported crystal structures of EfeO and EfeB, that of EfeU remains undetermined. In this study, we constructed expression system of EfeU derived from Escherichia coli, selected E. coli Rosetta-gami 2 (DE3) as an expression host, and succeeded in purification of the proteins which were indicated to form an oligomer by blue native PAGE. We obtained preliminary data of the X-ray crystallography, suggesting that expression and purification methods we established in this study enable structural analysis of the bacterial Efe system.

Crystal Structures of Lacticaseibacillus 4-Deoxy-L-threo-5-hexosulose-uronate Ketol-isomerase KduI in Complex with Substrate Analogs.

Some probiotics including lactobacilli, colonize host animal cells by targeting glycosaminoglycans (GAGs), such as heparin, located in the extracellular matrix. Recent studies have shown that several lactic acid bacteria degrade GAGs. Here we show the structure/function relationship of Lacticaseibacillus rhamnosus 4-deoxy-L-threo-5-hexosulose-uronate ketol-isomerase (KduI) crucial for metabolism of unsaturated glucuronic acid produced through degradation of GAGs. Crystal structures of ligand-free and bound KduIs were determined by X-ray crystallography and the enzyme was found to consist of six identical subunits and adopt a ß-helix as a basic scaffold. Ligands structurally similar to the substrate were bound to the cleft of each enzyme subunit. Several residues located in the cleft interacted with ligands through hydrogen bonds and/or C-C contacts. In addition to substrate analogs, a metal ion coordinated to four residues, His198, His200, Glu205, and His248, in the cleft, and the enzyme activity was significantly inhibited by a chelator, ethylenediaminetetraacetic acid. Site-directed mutants in Arg163, Ile165, Thr184, Thr194, His200, Arg203, Tyr207, Met262, and Tyr269 in the cleft exhibited little enzyme activity, indicating that these residues and the metal ion constituted an active site in the cleft. This is the first report on the active site structure of KduI based on the ligand-bound complex.

Intracellular production of reactive oxygen species and a DAF-FM-related compound in Aspergillus fumigatus in response to antifungal agent exposure.

Fungi are ubiquitously present in our living environment and are responsible for crop and infectious diseases. Developing new antifungal agents is constantly needed for their effective control. Here, we investigated fungal cellular responses to an array of antifungal compounds, including plant- and bacteria-derived antifungal compounds. The pathogenic fungus Aspergillus fumigatus generated reactive oxygen species in its hyphae after exposure to the antifungal compounds thymol, farnesol, citral, nerol, salicylic acid, phenazine-1-carbonic acid, and pyocyanin, as well as under oxidative and high-temperature stress conditions. The production of nitric oxide (NO) was determined using diaminofluorescein-FM diacetate (DAF-FM DA) and occurred in response to antifungal compounds and stress conditions. The application of reactive oxygen species or NO scavengers partly suppressed the inhibitory effects of farnesol on germination. However, NO production was not detected in the hyphae using the Greiss method. An LC/MS analysis also failed to detect DAF-FM-T, a theoretical product derived from DAF-FM DA and NO, in the hyphae after antifungal treatments. Thus, the cellular state after exposure to antifungal agents may be more complex than previously believed, and the role of NO in fungal cells needs to be investigated further.

Enhanced propagation of Granulicatella adiacens from human oral microbiota by hyaluronan.

Host determinants for formation/composition of human oral microbiota remain to be clarified, although microorganisms entering the mouth cannot necessarily colonize the oral environment. Here we show that human oral-abundant bacteria degraded host glycosaminoglycans (GAGs) in saliva and gingiva, and certain bacteria significantly grew on hyaluronan (HA), a kind of GAGs. Microbial communities from teeth or gingiva of healthy donors assimilated HA. Metagenomic analysis of human oral microbiota under different carbon sources revealed HA-driven Granulicatella growth. HA-degrading bacterial strains independently isolated from teeth and gingiva were identified as Granulicatella adiacens producing extracellular 130 kDa polysaccharide lyase as a HA-degrading enzyme encoded in a peculiar GAG genetic cluster containing genes for isomerase KduI and dehydrogenase DhuD. These findings demonstrated that GAGs are one of the host determinants for formation/composition of oral microbiota not only for colonization but also for the adaptation to the host niche. Especially, HA enhanced the G. adiacens propagation.

Identification and Functional Analysis of GTP Cyclohydrolase II in Candida glabrata in Response to Nitrosative Stress.

Reactive nitrogen species (RNS) are signal molecules involved in various biological events; however, excess levels of RNS cause nitrosative stress, leading to cell death and/or cellular dysfunction. During the process of infection, pathogens are exposed to nitrosative stress induced by host-derived RNS. Therefore, the nitrosative stress resistance mechanisms of pathogenic microorganisms are important for their infection and pathogenicity, and could be promising targets for antibiotics. Previously, we demonstrated that the RIB1 gene encoding GTP cyclohydrolase II (GCH2), which catalyzes the first step of the riboflavin biosynthesis pathway, is important for nitrosative stress resistance in the yeast Saccharomyces cerevisiae. Here, we identified and characterized the RIB1 gene in the opportunistic pathogenic yeast Candida glabrata. Our genetic and biochemical analyses indicated that the open reading frame of CAGL0F04279g functions as RIB1 in C. glabrata (CgRIB1). Subsequently, we analyzed the effect of CgRIB1 on nitrosative stress resistance by a growth test in the presence of RNS. Overexpression or deletion of CgRIB1 increased or decreased the nitrosative stress resistance of C. glabrata, respectively, indicating that GCH2 confers nitrosative stress resistance on yeast cells. Moreover, we showed that the proliferation of C. glabrata in cultures of macrophage-like cells required the GCH2-dependent nitrosative stress detoxifying mechanism. Additionally, an infection assay using silkworms as model host organisms indicated that CgRIB1 is indispensable for the virulence of C. glabrata. Our findings suggest that the GCH2-dependent nitrosative stress detoxifying mechanism is a promising target for the development of novel antibiotics.

Wide distribution of resistance to the fungicides fludioxonil and iprodione in Penicillium species.

Fludioxonil and iprodione are effective fungicides widely used for crop protection and are essential for controlling plant pathogenic fungi. The emergence of fungicide-resistant strains of targeted pathogens is regularly monitored, and several cases have been reported. Non-targeted fungi may also be exposed to the fungicide residues in agricultural fields. However, there are no comprehensive reports on fungicide-resistant strains of non-targeted fungi. Here, we surveyed 99 strains, representing 12 Penicillium species, that were isolated from a variety of environments, including foods, dead bodies, and clinical samples. Among the Penicillium strains, including non-pathogenic P. chrysogenum and P. camembertii, as well as postharvest pathogens P. expansum and P. digitatum, 14 and 20 showed resistance to fludioxonil and iprodione, respectively, and 6 showed multi-drug resistance to the fungicides. Sequence analyses revealed that some strains of P. chrysogenum and Penicillium oxalicum had mutations in NikA, a group III histidine kinase of the high-osmolarity glycerol pathway, which is the mode of action for fludioxonil and iprodione. The single nucleotide polymorphisms of G693D and T1318P in P. chrysogenum and T960S in P. oxalicum were only present in the fludioxonil- or iprodione-resistant strains. These strains also exhibited resistance to pyrrolnitrin, which is the lead compound in fludioxonil and is naturally produced by some Pseudomonas species. This study demonstrated that non-targeted Penicillium strains distributed throughout the environment possess fungicide resistance.

Intimate genetic relationships and fungicide resistance in multiple strains of Aspergillus fumigatus isolated from a plant bulb.

Fungal infections are increasingly dangerous because of environmentally dispersed resistance to antifungal drugs. Azoles are commonly used antifungal drugs, but they are also used as fungicides in agriculture, which may enable enrichment of azole-resistant strains of the human pathogen Aspergillus fumigatus in the environment. Understanding of environmental dissemination and enrichment of genetic variation associated with azole resistance in A. fumigatus is required to suppress resistant strains. Here, we focused on eight strains of azole-resistant A. fumigatus isolated from a single tulip bulb for sale in Japan. This set includes strains with TR34 /L98H/T289A/I364V/G448S and TR46 /Y121F/T289A/S363P/I364V/G448S mutations in the cyp51A gene, which showed higher tolerance to several azoles than strains harbouring TR46 /Y121F/T289A mutation. The strains were typed by microsatellite typing, single nucleotide polymorphism profiles, and mitochondrial and nuclear genome analyses. The strains grouped differently using each typing method, suggesting historical genetic recombination among the strains. Our data also revealed that some strains isolated from the tulip bulb showed tolerance to other classes of fungicides, such as QoI and carbendazim, followed by related amino acid alterations in the target proteins. Considering spatial-temporal factors, plant bulbs are an excellent environmental niche for fungal strains to encounter partners, and to obtain and spread resistance-associated mutations.

Erratum: Discovery of divided RdRp sequences and a hitherto unknown genomic complexity in fungal viruses.

[This corrects the article DOI: 10.1093/ve/veaa101.].

Discovery of divided RdRp sequences and a hitherto unknown genomic complexity in fungal viruses.

By identifying variations in viral RNA genomes, cutting-edge metagenome technology has potential to reshape current concepts about the evolution of RNA viruses. This technology, however, cannot process low-homology genomic regions properly, leaving the true diversity of RNA viruses unappreciated. To overcome this technological limitation, we applied an advanced method, Fragmented and Primer-Ligated Double-stranded (ds) RNA Sequencing (FLDS), to screen RNA viruses from 155 fungal isolates, which allowed us to obtain complete viral genomes in a homology-independent manner. We created a high-quality catalog of 19 RNA viruses (12 viral species) that infect Aspergillus isolates. Among them, nine viruses were not detectable by the conventional methodology involving agarose gel electrophoresis of dsRNA, a hallmark of RNA virus infections. Segmented genome structures were determined in 42 per cent of the viruses. Some RNA viruses had novel genome architectures; one contained a dual methyltransferase domain and another had a separated RNA-dependent RNA polymerase (RdRp) gene. A virus from a different fungal taxon (Pyricularia) had an RdRp sequence that was separated on different segments, suggesting that a divided RdRp is widely present among fungal viruses, despite the belief that all RNA viruses encode RdRp as a single gene. These findings illustrate the previously hidden diversity and evolution of RNA viruses, and prompt reconsideration of the structural plasticity of RdRp.

Splitting of RNA-dependent RNA polymerase is common in Narnaviridae: Identification of a type II divided RdRp from deep-sea fungal isolates.

Until recently, it was accepted that RNA-dependent RNA polymerase (RdRp) is the only essential gene for non-retro RNA viruses and is encoded by a single open reading frame (ORF) in their genomes. However, divided RdRps that are coded by two ORFs were discovered in fungal RNA viruses in a few independent reports. This discovery showed higher plasticity of viral RdRp than was expected. Among these divided RdRps, the division site was common; specifically, the first part of the RdRp contains motifs F, A, and B, whereas the latter part possesses motifs C and D. These RdRps are designated as type I divided RdRp and have been limited to viruses in a specific clade of Narnaviridae. In this study, to further understand the plasticity of RdRp, we explored viruses from deep sea-derived fungal strains as an untapped resource with a focus on Aspergillus section Versicolores. Seven strains were found to be infected by a total of 13 viruses, and the viral RNA genomes were determined by fragmented and primer-ligated double-stranded RNA sequencing technology. Among them, six strains belong to Narnaviridae. One of the strains, Aspergillus tennesseensis narnavirus 1, which infects an Aspergillus tennesseensis, has a divided RdRp with a new division site (referred to as type II divided RdRp). A couple of sequences for possible type II divided RdRps were also detected in public metagenomic data sets. Our findings reveal that different types of divisions in RdRp are present in the virosphere, and two types of RdRp splitting occurred independently within Narnaviridae.

Bacterial inducible expression of plant cell wall-binding protein YesO through conflict between Glycine max and saprophytic Bacillus subtilis.

Saprophytic bacteria and plants compete for limited nutrient sources. Bacillus subtilis grows well on steamed soybeans Glycine max to produce the fermented food, natto. Here we focus on bacterial responses in conflict between B. subtilis and G. max. B. subtilis cells maintained high growth rates specifically on non-germinating, dead soybean seeds. On the other hand, viable soybean seeds with germinating capability attenuated the initial growth of B. subtilis. Thus, B. subtilis cells may trigger saprophytic growth in response to the physiological status of G. max. Scanning electron microscope observation indicated that B. subtilis cells on steamed soybeans undergo morphological changes to form apertures, demonstrating cell remodeling during saprophytic growth. Further, transcriptomic analysis of B. subtilis revealed upregulation of the gene cluster, yesOPQR, in colonies growing on steamed soybeans. Recombinant YesO protein, a putative, solute-binding protein for the ATP-binding cassette transporter system, exhibited an affinity for pectin-derived oligosaccharide from plant cell wall. The crystal structure of YesO, in complex with the pectin oligosaccharide, was determined at 1.58 Å resolution. This study expands our knowledge of defensive and offensive strategies in interspecies competition, which may be promising targets for crop protection and fermented food production.

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

Certain 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 the Streptococcus agalactiae phosphotransferase system (PTS) for import of unsaturated hyaluronan disaccharide. S. agalactiae NEM316 was found to depolymerize and assimilate hyaluronan, whereas its mutant with a disruption in the 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 activity for import 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. This is distinct from Streptobacillus moniliformis ATP-binding cassette transporter for import of sulfated and non-sulfated fragmented GAGs without substrate modification. 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. The PTS and ABC transporter for import of GAGs shed light on bacterial clever colonization/infection system targeting various animal polysaccharides.

Substrate recognition by bacterial solute-binding protein is responsible for import of extracellular hyaluronan and chondroitin sulfate from the animal host.

Glycosaminoglycans (GAGs) such as hyaluronan and chondroitin in animal extracellular matrices contain disaccharide-repeating units. In a Gram-negative pathogenic Streptobacillus moniliformis, which belongs to Fusobacteria phylum and resides in rodent oral cavities, the solute-binding protein (Smon0123)-dependent ATP-binding cassette transporter imports unsaturated hyaluronan/chondroitin disaccharides into the cytoplasm after GAG lyase-dependent depolymerization. Here we show substrate recognition of unsaturated hyaluronan disaccharide by Smon0123. Moreover, Smon0123 exhibited no affinity for unsaturated chondroitin disaccharides containing three sulfate groups, distinct from non-sulfated, mono-sulfated, and di-sulfated chondroitin disaccharides previously identified as substrates. Crystal structure of Smon0123 with unsaturated hyaluronan disaccharide demonstrates that several residues, including Trp284 and Glu410, are crucial for binding to unsaturated hyaluronan/chondroitin disaccharides, whereas arrangements of water molecules at binding sites are found to be substrate dependent through comparison with substrate-bound structures determined previously. These residues are well conserved in Smon0123-like proteins of fusobacteria, and probably facilitate the fusobacterial residence in hyaluronan-rich oral cavities.

Probiotics in human gut microbiota can degrade host glycosaminoglycans.

Glycosaminoglycans (GAGs) (e.g. heparin, chondroitin sulfate, and hyaluronan) show various significant physiological functions as a major component of extracellular matrix in animals. Some bacteria target GAGs for adhesion and/or infection to host cells, although no probiotics have been known to degrade GAGs. Here, we show GAG degradation by probiotics from human gut microbiota and their adhesion to human intestinal cells through a GAG. GAG-degrading bacteria were isolated from human faeces and identified as Enterococcus faecium, and some typical probiotics such as Lactobacillus casei, Lactobacillus rhamnosus and Enterococcus faecalis were also found to degrade heparin. GAG-degrading lactobacilli and enterococci including the isolated E. faecium possessed a genetic cluster encoding GAG-degrading/metabolising enzymes in the bacterial genome. KduI and KduD enzymes encoded in the GAG cluster of L. rhamnosus functioned as 4-deoxy-l-threo-5-hexosulose-uronate ketol-isomerase and 2-keto-3-deoxy-d-gluconate dehydrogenase, respectively, both of which were crucial for GAG metabolism. GAG-degrading L. rhamnosus and E. faecium attached to human intestinal Caco-2 cells via heparin. Some species of Bacteroides, considered to be the next generation probiotics, degraded chondroitin sulfate C and hyaluronan, and genes coding for the Bacteroides GAG-degrading enzyme were frequently detected from human gut microbiota. This is the first report on GAG-degrading probiotics in human gut microbiota.

Alternative substrate-bound conformation of bacterial solute-binding protein involved in the import of mammalian host glycosaminoglycans.

Glycosaminoglycans (GAGs), constituted by repeating uronate and amino sugar units, are major components of mammalian extracellular matrices. Some indigenous and pathogenic bacteria target GAGs for colonization to and/or infection of host mammalian cells. In Gram-negative pathogenic Streptobacillus moniliformis, the solute-binding protein (Smon0123)-dependent ATP-binding cassette (ABC) transporter incorporates unsaturated GAG disaccharides into the cytoplasm after depolymerization by polysaccharide lyase. Smon0123, composed of N and C domains, adopts either a substrate-free open or a substrate-bound closed form by approaching two domains at 47° in comparison with the open form. Here we show an alternative 39°-closed conformation of Smon0123 bound to unsaturated chondroitin disaccharide sulfated at the C-4 and C-6 positions of N-acetyl-d-galactosamine residue (CΔ4S6S). In CΔ4S6S-bound Smon0123, Arg204 and Lys210 around the two sulfate groups were located at different positions from those at other substrate-bound 47°-closed conformations. Therefore, the two sulfate groups in CΔ4S6S shifted substrate-binding residue arrangements, causing dynamic conformational change. Smon0123 showed less affinity with CΔ4S6S than with non-sulfated and monosulfated substrates. ATPase activity of the Smon0123-dependent ABC transporter in the presence of CΔ4S6S was lower than that in the presence of other unsaturated chondroitin disaccharides, suggesting that CΔ4S6S-bound Smon0123 was unpreferable for docking with the ABC transporter.

A bacterial ABC transporter enables import of mammalian host glycosaminoglycans.

Glycosaminoglycans (GAGs), such as hyaluronan, chondroitin sulfate, and heparin, constitute mammalian extracellular matrices. The uronate and amino sugar residues in hyaluronan and chondroitin sulfate are linked by 1,3-glycoside bond, while heparin contains 1,4-glycoside bond. Some bacteria target GAGs as means of establishing colonization and/or infection, and bacterial degradation mechanisms of GAGs have been well characterized. However, little is known about the bacterial import of GAGs. Here, we show a GAG import system, comprised of a solute-binding protein (Smon0123)-dependent ATP-binding cassette (ABC) transporter, in the pathogenic Streptobacillus moniliformis. A genetic cluster responsible for depolymerization, degradation, and metabolism of GAGs as well as the ABC transporter system was found in the S. moniliformis genome. This bacterium degraded hyaluronan and chondroitin sulfate with an expression of the genetic cluster, while heparin repressed the bacterial growth. The purified recombinant Smon0123 exhibited an affinity with disaccharides generated from hyaluronan and chondroitin sulfate. X-ray crystallography indicated binding mode of Smon0123 to GAG disaccharides. The purified recombinant ABC transporter as a tetramer (Smon0121-Smon0122/Smon0120-Smon0120) reconstructed in liposomes enhanced its ATPase activity in the presence of Smon0123 and GAG disaccharides. This is the first report that has molecularly depicted a bacterial import system of both sulfated and non-sulfated GAGs.

Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.

Structural determinants in bacterial 2-keto-3-deoxy-D-gluconate dehydrogenase KduD for dual-coenzyme specificity.

Short-chain dehydrogenase/reductase (SDR) is distributed in many organisms, from bacteria to humans, and has significant roles in metabolism of carbohydrates, lipids, amino acids, and other biomolecules. An important intermediate in acidic polysaccharide metabolism is 2-keto-3-deoxy-d-gluconate (KDG). Recently, two short and long loops in Sphingomonas KDG-producing SDR enzymes (NADPH-dependent A1-R and NADH-dependent A1-R') involved in alginate metabolism were shown to be crucial for NADPH or NADH coenzyme specificity. Two SDR family enzymes-KduD from Pectobacterium carotovorum (PcaKduD) and DhuD from Streptococcus pyogenes (SpyDhuD)-prefer NADH as coenzyme, although only PcaKduD can utilize both NADPH and NADH. Both enzymes reduce 2,5-diketo-3-deoxy-d-gluconate to produce KDG. Tertiary and quaternary structures of SpyDhuD and PcaKduD and its complex with NADH were determined at high resolution (approximately 1.6 Å) by X-ray crystallography. Both PcaKduD and SpyDhuD consist of a three-layered structure, α/ß/α, with a coenzyme-binding site in the Rossmann fold; similar to enzymes A1-R and A1-R', both arrange the two short and long loops close to the coenzyme-binding site. The primary structures of the two loops in PcaKduD and SpyDhuD were similar to those in A1-R' but not A1-R. Charge neutrality and moderate space at the binding site of the nucleoside ribose 2' coenzyme region were determined to be structurally crucial for dual-coenzyme specificity in PcaKduD by structural comparison of the NADH- and NADPH-specific SDR enzymes. The corresponding site in SpyDhuD was negatively charged and spatially shallow. This is the first reported study on structural determinants in SDR family KduD related to dual-coenzyme specificity. Proteins 2016; 84:934-947. © 2016 Wiley Periodicals, Inc.

Metabolic fate of unsaturated glucuronic/iduronic acids from glycosaminoglycans: molecular identification and structure determination of streptococcal isomerase and dehydrogenase.

Glycosaminoglycans in mammalian extracellular matrices are degraded to their constituents, unsaturated uronic (glucuronic/iduronic) acids and amino sugars, through successive reactions of bacterial polysaccharide lyase and unsaturated glucuronyl hydrolase. Genes coding for glycosaminoglycan-acting lyase, unsaturated glucuronyl hydrolase, and the phosphotransferase system are assembled into a cluster in the genome of pathogenic bacteria, such as streptococci and clostridia. Here, we studied the streptococcal metabolic pathway of unsaturated uronic acids and the structure/function relationship of its relevant isomerase and dehydrogenase. Two proteins (gbs1892 and gbs1891) of Streptococcus agalactiae strain NEM316 were overexpressed in Escherichia coli, purified, and characterized. 4-Deoxy-l-threo-5-hexosulose-uronate (Dhu) nonenzymatically generated from unsaturated uronic acids was converted to 2-keto-3-deoxy-d-gluconate via 3-deoxy-d-glycero-2,5-hexodiulosonate through successive reactions of gbs1892 isomerase (DhuI) and gbs1891 NADH-dependent reductase/dehydrogenase (DhuD). DhuI and DhuD enzymatically corresponded to 4-deoxy-l-threo-5-hexosulose-uronate ketol-isomerase (KduI) and 2-keto-3-deoxy-d-gluconate dehydrogenase (KduD), respectively, involved in pectin metabolism, although no or low sequence identity was observed between DhuI and KduI or between DhuD and KduD, respectively. Genes for DhuI and DhuD were found to be included in the streptococcal genetic cluster, whereas KduI and KduD are encoded in clostridia. Tertiary and quaternary structures of DhuI and DhuD were determined by x-ray crystallography. Distinct from KduI ß-barrels, DhuI adopts an α/ß/α-barrel structure as a basic scaffold similar to that of ribose 5-phosphate isomerase. The structure of DhuD is unable to accommodate the substrate/cofactor, suggesting that conformational changes are essential to trigger enzyme catalysis. This is the first report on the bacterial metabolism of glycosaminoglycan-derived unsaturated uronic acids by isomerase and dehydrogenase.

Localization of Tektin 1 at both acrosome and flagella of mouse and bull spermatozoa.

Tektins (TEKTs) are constitutive filamentous proteins of microtubules in cilia, flagella, basal bodies, and centrioles. In mammals, five TEKTs (TEKT1, 2, 3, 4, and 5) have been identified in testis and spermatozoa. With the exception of TEKT1, these TEKTs have been reported to be present in spermatozoa with predominant localization at the peri-axoneme structures of flagella, i.e., mitochondria and outer dense fibers. In the present study, we produced an antibody against TEKT1 to examine the localization of TEKT1 in mouse, bull, and rat spermatozoa. By immunoblot analyses and immunofluorescence microscopy, we found TEKT1 to be present in sperm flagella and at the apical region of acrosome cap in spermatozoa of all these species. Acrosome-associated TEKT1 disappeared after in vitro acrosome reaction in mouse spermatozoa. These observations suggest another potential role for TEKT1 as a cytoskeletal element in the sperm head, or as a molecule involved in acrosome-related phenomena, such as acrosome reaction.