Engineered AAV13 variants with enhanced transduction and confined spread.

Precise targeting of specific regions within the central nervous system (CNS) is crucial for both scientific research and gene therapy in the context of brain diseases. Adeno-associated virus 13 (AAV13) is known for its restricted diffusion range within the CNS, making it an ideal choice for precise labeling and administration within small brain regions. However, AAV13 mediates relatively low expression of target genes. Here, we introduced specifically engineered modifications to the AAV13 capsid protein to enhance its transduction efficiency. We first constructed AAV13-YF by mutating tyrosine to phenylalanine on the surface of the AAV13 capsid. We then inserted the 7m8 peptide, known to enhance cell transduction, into positions 587/588 and 585/586 of the AAV13 capsid, resulting in two distinct variants named AAV13-587-7m8 and AAV13-585-7m8, respectively. We found that AAV13-YF exhibited superior in vitro infectivity in HEK293T cells compared to AAV13, while AAV13-587-7m8 and AAV13-585-7m8 showed enhanced CNS infection capabilities in C57BL/6 mice, with AAV13-587-7m8 infection retaining a limited spread range. These modified AAV13 variants hold promising potential for applications in gene therapy and neuroscience research.

Identification of hub genes and therapeutic siRNAs to develop novel adjunctive therapy for Duchenne muscular dystrophy.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is a devastating X-linked neuromuscular disorder caused by various defects in the dystrophin gene and still no universal therapy. This study aims to identify the hub genes unrelated to excessive immune response but responsible for DMD progression and explore therapeutic siRNAs, thereby providing a novel treatment. METHODS: Top ten hub genes for DMD were identified from GSE38417 dataset by using GEO2R and PPI networks based on Cytoscape analysis. The hub genes unrelated to excessive immune response were identified by GeneCards, and their expression was further verified in mdx and C57 mice at 2 and 4 months (M) by (RT-q) PCR and western blotting. Therapeutic siRNAs were deemed as those that could normalize the expression of the validated hub genes in transfected C2C12 cells. RESULTS: 855 up-regulated and 324 down-regulated DEGs were screened from GSE38417 dataset. Five of the top 10 hub genes were considered as the candidate genes unrelated to excessive immune response, and three of these candidates were consistently and significantly up-regulated in mdx mice at 2 M and 4 M when compared with age-matched C57 mice, including Col1a2, Fbn1 and Fn1. Furthermore, the three validated up-regulated candidate genes can be significantly down-regulated by three rational designed siRNA (p < 0.0001), respectively. CONCLUSION: COL1A2, FBN1 and FN1 may be novel biomarkers for DMD, and the siRNAs designed in our study were help to develop adjunctive therapy for Duchenne muscular dystrophy.

Novel inner filter effect-based near-infrared electrochemiluminescence sensor mediated by well-matched AgBr-nitrogen-doped Ti3C2 MXene and nonmetallic plasmon WO3•H2O.

The development of innovative and efficient strategy is of paramount importance for near-infrared (NIR) electrochemiluminescence (ECL) sensing, which can substantially promote ECL detection in a wide range of situations. Herein, the inner filter effect (IFE) strategy was designed to construct an ultrasensitive NIR ECL biosensor based on the well-matched AgBr nanocrystals (NCs) decorated nitrogen-doped Ti3C2 MXene nanocomposites (AgBr-N-Ti3C2) and hydrated defective tungsten oxide nanosheets (dWO3•H2O). Specifically, the AgBr-N-Ti3C2 nanocomposites displayed extremely effective NIR ECL emission because N-doping could accelerate electron transfer and boost the red-shift of the ECL spectrum. The nonmetallic plasmon dWO3•H2O was used as an absorber due to its facile tuning of the spectra overlap and higher molar extinction coefficients. Time-resolved emission decay curves proved that the decreased ECL intensity was ascribed to the IFE-based steady quenching mechanism. With the support of tetracycline (TC) aptamer and the complementary DNA chain, the fabricated NIR ECL-IFE biosensor performed a wide linear range of 100 nM âˆ¼ 10 fM with a low detection limit of 2.2 fM (S/N = 3), and it exhibited excellent stability, sensitivity, and reproducibility, so as to be applied to real samples. This strategy opens a new avenue to constructing an efficient NIR ECL-IFE system and shows excellent practical potential in actual sample analysis.

Hydrovoltaic Effect Coupling with Capacitor Amplification: A Mode for Sensitive Self-Powered Electrochemical Sensing.

Self-powered electrochemical sensors, which can function without external electricity, are incredibly valuable in the realm of sensing. However, most of the present testing methods are normally confined to high environmental requirements, restricted lighting conditions, and temperature differences. Herein, an innovative self-powered electrochemical sensor was successfully developed based on hydrovoltaic effect coupling with capacitor amplification. Due to the combined merits from the two-dimensional transition metal carbides and nitrides (MXene)-polyaniline (PANI) with high surface potential and good hydrophilicity, and the capacitor amplification strategy, the device could harvest electric energy from water evaporation and displayed a high short circuit current value. Under optimal conditions, the proposed self-powered electrochemical sensor presented excellent sensitivity and high specificity for enrofloxacin (ENR) detection in the concentration range from 1 fM to 1 nM with a detection limit of 0.585 fM. Such a proposed sensor also has the advantages of environmental friendliness and ease of use, which is an ideal choice for accurately and precisely detecting ENR in real samples. The mode of such electrochemical detection outlined in this technical note implements a breakthrough in designing self-powered electrochemical sensors, providing a rational basis for development of a diversified sensing platform.

Ultrasensitive self-powered photoelectrochemical sensing for enrofloxacin detection by coupling piezoelectric effect with nonmetallic surface plasmon resonance based on ZnO nanorod arrays/WO3-x.

Exploring efficient strategy for high-efficiency photoelectric conversion is quite important to design sensitive self-powered photoelectrochemical (PEC) sensing platform. This work designed a high performance self-powered PEC sensing platform by the integration of piezoelectric effect with localized surface plasmon resonance (LSPR) effect based on ZnO-WO3-x heterostructures. Due to the fluid eddy induced piezoelectric effect by magnetic stirring, the piezoelectric semiconductor ZnO nanorod arrays (ZnO NRs) can facilitate the transfer of electrons and holes by generating piezoelectric potentials under external forces, thereby contributing to the performance of self-powered PEC platforms. Such working mechanism of the piezoelectric effect was studied by using the COMSOL software. Moreover, the introduction of defect engineered WO3 (WO3-x) can further broaden the light absorption and promote the charge transfer owing to the nonmetallic surface plasmon resonance effect. Remarkably, due to the synergizing piezoelectric and plasmonic effect, the photocurrent and maximum power output of ZnO-WO3-x heterostructures were enhanced by 3.3-fold and 5.5-fold than that of bare ZnO, respectively. After the immobilization of the enrofloxacin (ENR) aptamer, the self-powered sensor demonstrated an excellent linearity (1 × 10-14 M to 1 × 10-9 M) with a low detection limit of 1.8 × 10-15 M (S/N = 3). This work undoubtedly holds great promise to provide the innovative inspiration for the formation of high-performance self-powered sensing platform, which opens up a new horizon of potential in food safety and environmental monitoring.

Depth profiles of geochemical features, geochemical activities and biodiversity of microbial communities in marine sediments from the Shenhu area, the northern South China Sea.

The biogeochemical processes, anaerobic oxidation of methane (AOM) and methanogenesis, control methane emission and create distinct geochemical profiles with depth in marine sediments. Correlating the capacities and biodiversity of the microbial communities in marine sediments remains challenging. We therefore investigated the geochemical constituents and the capabilities and diversity of microbial communities in sediments at different depths in two cores from the Shenhu area in the northern South China Sea, which is characterized by underlying gas hydrates. The geochemical features, sulfate concentration decreased linearly and the acid volatile sulfur accumulated from 4 m below the seafloor (mbsf) to the bottom, indicating significant sulfate reduction. However, the methane concentration was relatively low and showed irregular trends, indicating that our study cores did not reach the sulfate-methane transition zone (SMTZ). Nevertheless, incubation experiments showed that the microbial groups in sediments performed AOM and methanogenesis in the region where sulfate decreased linearly above the SMTZ. We mapped the diversity and abundance of microbial communities in sediments with depth using high-throughput sequencing. A small proportion of known methanogens (<0.3%) may have been responsible for the methanogenesis during incubation. No classical archaeal anaerobic methanotroph (ANME) sequences were detected across all samples; only a small amount of SEEP-SRB1 were detected, and their abundance did not increase with increasing depth. Thus, unknown or unconventional phylotypes may have participated in AOM during the incubation, and the dominant phylum Bathyarchaeota or the small number of detected methanogens are the most likely performers of AOM.

Differences in bioavailability of canonical and non-canonical D-amino acids for marine microbes.

Dissolved organic matter (DOM) accounts for >95% of total marine organic matter, and >95% of marine DOM is refractory to biodegradation. The recalcitrancy of DOM determines its residence time and thus is of great concern regarding to carbon sequestration in the ocean. However, the recalcitrancy of DOM not only varies among different compounds but also within different conformations of a same molecule such as L-amino acids (L-AAs) and D-amino acids (D-AAs). While the former is labile, the latter is refractory and used as a proxy for estimation of bacterial refractory DOM in the ocean. However, some D-AAs are also reported to be bioavailable. To clarify the controversy, we examined the bioavailability of two types of D-AAs: canonical D-AAs, which mainly present as bacterial cell wall components, and non-canonical D-AAs (NCDAAs), which are secreted by various bacteria as signaling molecules in bacterial physiology. Bioassay experiments were conducted with nine marine bacterial strains and a natural microbial community. D-AAs were poorly utilized by the strains as sole carbon or nitrogen sources compared with L-AAs, in addition, NCDAAs were barely used compared with canonical D-AAs. In comparison, the microbial community consumed all three canonical D-AAs (D-alanine, D-aspartic acid and D-glutamic acid) as efficiently as their corresponding L-AAs when supplied separately; however, L-AAs were preferentially used over D-AAs when both forms were provided simultaneously. Remarkably, two NCDAAs, D-methionine and D-leucine, were poorly utilized regardless of the presence of the L-enantiomers. It was found for the first time that NCDAAs are relatively more refractory than canonical D-AAs to microbial utilization. This novel recognition of difference in recalcitrancy between NCDAAs and canonical D-AAs lays the foundation for a better understanding of carbon cycling and more accurate estimation of carbon storage in the ocean.

Improvement of ST0452 N-Acetylglucosamine-1-Phosphate Uridyltransferase Activity by the Cooperative Effect of Two Single Mutations Identified through Structure-Based Protein Engineering.

We showed previously that the Y97N mutant of the ST0452 protein, isolated from Sulfolobus tokodaii, exhibited over 4 times higher N-acetylglucosamine-1-phosphate (GlcNAc-1-P) uridyltransferase (UTase) activity, compared with that of the wild-type ST0452 protein. We determined the three-dimensional structure of the Y97N protein to explore the detailed mechanism underlying this increased activity. The overall structure was almost identical to that of the wild-type ST0452 protein (PDB ID 2GGO), with residue 97 (Asn) interacting with the O-5 atom of N-acetylglucosamine (GlcNAc) in the complex without metal ions. The same interaction was observed for Escherichia coli GlmU in the absence of metal ions. These observations indicated that the three-dimensional structure of the Y97N protein was not changed by this substitution but the interactions with the substrate were slightly modified, which might cause the activity to increase. The crystal structure of the Y97N protein also showed that positions 146 (Glu) and 80 (Thr) formed interactions with GlcNAc, and an engineering strategy was applied to these residues to increase activity. All proteins substituted at position 146 had drastically decreased activities, whereas several proteins substituted at position 80 showed higher GlcNAc-1-P UTase activity, compared to that of the wild-type protein. The substituted amino acids at positions 80 and 97 might result in optimized interactions with the substrate; therefore, we predicted that the combination of these two substitutions might cooperatively increase GlcNAc-1-P UTase activity. Of the four double mutant ST0452 proteins generated, T80S/Y97N showed 6.5-times-higher activity, compared to that of the wild-type ST0452 protein, revealing that these two substituted residues functioned cooperatively to increase GlcNAc-1-P UTase activity.IMPORTANCE We demonstrated that the enzymatic activity of a thermostable protein was over 4 times higher than that of the wild-type protein following substitution of a single amino acid, without affecting its thermostability. The three-dimensional structure of the improved mutant protein complexed with substrate was determined. The same overall structure and interaction between the substituted residue and the GlcNAc substrate as observed in the well-characterized bacterial enzyme suggested that the substitution of Tyr at position 97 by Asn might slightly change the interaction. This subtle change in the interaction might potentially increase the GlcNAc-1-P UTase activity of the mutant protein. These observations indicated that a drastic change in the structure of a natural thermostable enzyme is not necessary to increase its activity; a subtle change in the interaction with the substrate might be sufficient. Cooperative effects were observed in the appropriate double mutant protein. This work provides useful information for the future engineering of natural enzymes.

Identification of a Direct Biosynthetic Pathway for UDP-N-Acetylgalactosamine from Glucosamine-6-Phosphate in Thermophilic Crenarchaeon Sulfolobus tokodaii.

Most organisms, from Bacteria to Eukarya, synthesize UDP-N-acetylglucosamine (UDP-GlcNAc) from fructose-6-phosphate via a four-step reaction, and UDP-N-acetylgalactosamine (UDP-GalNAc) can only be synthesized from UDP-GlcNAc by UDP-GlcNAc 4-epimerase. In Archaea, the bacterial-type UDP-GlcNAc biosynthetic pathway was reported for Methanococcales. However, the complete biosynthetic pathways for UDP-GlcNAc and UDP-GalNAc present in one archaeal species are unidentified. Previous experimental analyses on enzymatic activities of the ST0452 protein, identified from the thermophilic crenarchaeon Sulfolobus tokodaii, predicted the presence of both a bacterial-type UDP-GlcNAc and an independent UDP-GalNAc biosynthetic pathway in this archaeon. In the present work, functional analyses revealed that the recombinant ST2186 protein possessed an glutamine:fructose-6-phosphate amidotransferase activity and that the recombinant ST0242 protein possessed a phosphoglucosamine-mutase activity. Along with the acetyltransferase and uridyltransferase activities of the ST0452 protein, the activities of the ST2186 and ST0242 proteins confirmed the presence of a bacterial-type UDP-GlcNAc biosynthetic pathway in S. tokodaii In contrast, the UDP-GlcNAc 4-epimerase homologue gene was not detected within the genomic data. Thus, it was expected that galactosamine-1-phosphate or galactosamine-6-phosphate (GalN-6-P) was provided by conversion of glucosamine-1-phosphate or glucosamine-6-phosphate (GlcN-6-P). A novel epimerase converting GlcN-6-P to GalN-6-P was detected in a cell extract of S. tokodaii, and the N-terminal sequence of the purified protein indicated that the novel epimerase was encoded by the ST2245 gene. Along with the ST0242 phosphogalactosamine-mutase activity, this observation confirmed the presence of a novel UDP-GalNAc biosynthetic pathway from GlcN-6-P in S. tokodaii Discovery of the novel pathway provides a new insight into the evolution of nucleotide sugar metabolic pathways.IMPORTANCE In this work, a novel protein capable of directly converting glucosamine-6-phosphate to galactosamine-6-phosphate was successfully purified from a cell extract of the thermophilic crenarchaeon Sulfolobus tokodaii Confirmation of this novel activity using the recombinant protein indicates that S. tokodaii possesses a novel UDP-GalNAc biosynthetic pathway derived from glucosamine-6-phosphate. The distributions of this and related genes indicate the presence of three different types of UDP-GalNAc biosynthetic pathways: a direct pathway using a novel enzyme and two conversion pathways from UDP-GlcNAc using known enzymes. Additionally, Crenarchaeota species lacking all three pathways were found, predicting the presence of one more unknown pathway. Identification of these novel proteins and pathways provides important insights into the evolution of nucleotide sugar biosynthesis, as well as being potentially important industrially.

A Novel Exopolysaccharide with Metal Adsorption Capacity Produced by a Marine Bacterium Alteromonas sp. JL2810.

Most marine bacteria can produce exopolysaccharides (EPS). However, very few structures of EPS produced by marine bacteria have been determined. The characterization of EPS structure is important for the elucidation of their biological functions and ecological roles. In this study, the structure of EPS produced by a marine bacterium, Alteromonas sp. JL2810, was characterized, and the biosorption of the EPS for heavy metals Cu2+, Ni2+, and Cr6+ was also investigated. Nuclear magnetic resonance (NMR) analysis indicated that the JL2810 EPS have a novel structure consisting of the repeating unit of [-3)-α-Rhap-(1→3)-α-Manp-(1→4)-α-3OAc-GalAp-(1→]. The biosorption of the EPS for heavy metals was affected by a medium pH; the maximum biosorption capacities for Cu2+ and Ni2+ were 140.8 ± 8.2 mg/g and 226.3 ± 3.3 mg/g at pH 5.0; however, for Cr6+ it was 215.2 ± 5.1 mg/g at pH 5.5. Infrared spectrometry analysis demonstrated that the groups of O-H, C=O, and C-O-C were the main function groups for the adsorption of JL2810 EPS with the heavy metals. The adsorption equilibrium of JL2810 EPS for Ni2+ was further analyzed, and the equilibrium data could be better represented by the Langmuir isotherm model. The novel EPS could be potentially used in industrial applications as a novel bio-resource for the removal of heavy metals.

Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position.

The ST0452 protein is a bifunctional protein exhibiting sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) and amino-sugar-1-phosphate acetyltransferase activities and was isolated from the thermophilic archaeon Sulfolobus tokodaii Based on the previous observation that five single mutations increased ST0452 sugar-1-P NTase activity, nine double-mutant ST0452 proteins were generated with the intent of obtaining enzymes exhibiting a further increase in catalysis, but all showed less than 15% of the wild-type N-acetyl-d-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity. The Y97A mutant exhibited the highest activity of the single-mutant proteins, and thus site saturation mutagenesis of the 97th position (Tyr) was conducted. Six mutants showed both increased GlcNAc-1-P UTase and glucose-1-phosphate uridyltransferase activities, eight mutants showed only enhanced GlcNAc-1-P UTase activity, and six exhibited higher GlcNAc-1-P UTase activity than that of the Y97A mutant. Kinetic analyses of three typical mutants indicated that the increase in sugar-1-P NTase activity was mainly due to an increase in the apparent kcat value. We hypothesized that changing the 97th position (Tyr) to a smaller amino acid with similar electronic properties would increase activity, and thus the Tyr at the corresponding 103rd position of the Escherichia coli GlmU (EcGlmU) enzyme was replaced with the same residues. The Y103N mutant EcGlmU showed increased GlcNAc-1-P UTase activity, revealing that the Tyr at the 97th position of the ST0452 protein (103rd position in EcGlmU) plays an important role in catalysis. The present results provide useful information regarding how to improve the activity of natural enzymes and how to generate powerful enzymes for the industrial production of sugar nucleotides. IMPORTANCE: It is typically difficult to increase enzymatic activity by introducing substitutions into a natural enzyme. However, it was previously found that the ST0452 protein, a thermostable enzyme from the thermophilic archaeon Sulfolobus tokodaii, exhibited increased activity following single amino acid substitutions of Ala. In this study, ST0452 proteins exhibiting a further increase in activity were created using a site saturation mutagenesis strategy at the 97th position. Kinetic analyses showed that the increased activities of the mutant proteins were principally due to increased apparent kcat values. These mutant proteins might suggest clues regarding the mechanism underlying the reaction process and provide very important information for the design of synthetic improved enzymes, and they can be used as powerful biocatalysts for the production of sugar nucleotide molecules. Moreover, this work generated useful proteins for three-dimensional structural analysis clarifying the processes underlying the regulation and mechanism of enzymatic activity.

Complete Genome Sequence of the d-Amino Acid Catabolism Bacterium Phaeobacter sp. Strain JL2886, Isolated from Deep Seawater of the South China Sea.

Phaeobacter sp. strain JL2886, isolated from deep seawater of the South China Sea, can catabolize d-amino acids. Here, we report the complete genome sequence of Phaeobacter sp. JL2886. It comprises ~4.06 Mbp, with a G+C content of 61.52%. A total of 3,913 protein-coding genes and 10 genes related to d-amino acid catabolism were obtained.

The Fate of Marine Bacterial Exopolysaccharide in Natural Marine Microbial Communities.

Most marine bacteria produce exopolysaccharides (EPS), and bacterial EPS represent an important source of dissolved organic carbon in marine ecosystems. It was proposed that bacterial EPS rich in uronic acid is resistant to mineralization by microbes and thus has a long residence time in global oceans. To confirm this hypothesis, bacterial EPS rich in galacturonic acid was isolated from Alteromonas sp. JL2810. The EPS was used to amend natural seawater to investigate the bioavailability of this EPS by native populations, in the presence and absence of ammonium and phosphate amendment. The data indicated that the bacterial EPS could not be completely consumed during the cultivation period and that the bioavailability of EPS was not only determined by its intrinsic properties, but was also determined by other factors such as the availability of inorganic nutrients. During the experiment, the humic-like component of fluorescent dissolved organic matter (FDOM) was freshly produced. Bacterial community structure analysis indicated that the class Flavobacteria of the phylum Bacteroidetes was the major contributor for the utilization of EPS. This report is the first to indicate that Flavobacteria are a major contributor to bacterial EPS degradation. The fraction of EPS that could not be completely utilized and the FDOM (e.g., humic acid-like substances) produced de novo may be refractory and may contribute to the carbon storage in the oceans.

Identification and characterization of a thermostable bifunctional enzyme with phosphomannose isomerase and sugar-1-phosphate nucleotidylyltransferase activities from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3.

Mannosylglycerate is known as a compatible solute, and plays important roles for salinity adaptation and high temperature stability of microorganisms. In the gene cluster for the mannosylglycerate biosynthetic pathway predicted from the genomic data of Pyrococcus horikoshii OT3, the PH0925 protein was found as a putative bifunctional enzyme with phosphomannose isomerase (PMI) and mannose-1-phosphate guanylyltransferase (Man-1-P GTase) activities, which can synthesize GDP-mannose when accompanied by a phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme (PH0923). The recombinant PH0925 protein, expressed in E. coli, exhibited both expected PMI and Man-1-P GTase activities, as well as absolute thermostability; 95 °C was the optimum reaction temperature. According to the guanylyltransferase activity (GTase) of the PH0925 protein, it was found that the protein can catalyze glucose-1-phosphate (Glc-1-P) and glucosamine-1-phosphate (GlcN-1-P) in addition to Man-1-P. The analyses of C-terminus-truncated forms of the PH0925 protein indicated that sugar-1-phosphate nucleotidylyltransferase (Sugar-1-P NTase) activity was located in the region from the N-terminus to the 345th residue, and that the C-terminal 114 residue region of the PH0925 protein inhibited the Man-1-P GTase activity. Conversely, the PMI activity was abolished by deletion of the C-terminal 14 residues. This is the first report of a thermostable enzyme with both PMI and multiple Sugar-1-P NTase activities.

Characterization of the amino acid residues mediating the unique amino-sugar-1-phosphate acetyltransferase activity of the archaeal ST0452 protein.

The ST0452 protein from the thermophilic archaean Sulfolobus tokodaii has been identified as an enzyme with multiple sugar-1-phosphate nucleotidylyltransferase and amino-sugar-1-phosphate acetyltransferase (amino-sugar-1-P AcTase) activities. Analysis of the protein showed that in addition to glucosamine-1-phosphate (GlcN-1-P) AcTase activity, it possesses unique galactosamine-1-phosphate (GalN-1-P) AcTase activity not detected in any other proteins. Comparison of the crystal structures of the ST0452 protein and GlmU from Escherichia coli (EcGlmU), which possesses only GlcN-1-P AcTase activity, showed that the overall sequence identity between these two proteins is less than 25 %, but the amino acid residues predicted to comprise the catalytic center of EcGlmU are conserved in the ST0452 protein. To understand the molecular mechanism by which the ST0452 amino-sugar-1-P AcTase activity recognizes two independent substrates, several ST0452 substitution and truncation mutant proteins were constructed and analyzed. We found that His308 is essential for both GalN-1-P and GlcN-1-P AcTase activities, whereas Tyr311 and Asn331 are important only for the GalN-1-P AcTase activity. In addition, deletion of the C-terminal 5 or 11 residues showed that the 11-residue C-terminal region exerts a modest stimulatory effect on GalN-1-P AcTase activity but dramatically suppresses GlcN-1-P AcTase activity. This region also appears to make an important contribution to the thermostability of the entire ST0452 protein. Systematic deletions from the C-terminus also demonstrated that the C-terminal region with the ß-helix structure has an important role mediating the trimerization of the ST0452 protein. This is the first report of an analysis of a thermostable archaeal enzyme exhibiting multiple amino-sugar-1-P AcTase activities.

Draft Genome Sequence of the Novel Exopolysaccharide-Producing Bacterium Altibacter lentus Strain JLT2010T, Isolated from Deep Seawater of the South China Sea.

Altibacter lentus strain JLT2010(T) is the type strain of the recently identified novel genus and species of the family Flavobacteriaceae and was first isolated from deep seawater of the South China Sea. It can produce exopolysaccharide. Here we report the first draft genome of JLT2010(T) (3,160,033 bp, with GC content of 42.12%) and major findings from its annotation. It is the first reported genome in the genus Altibacter.

Effects of D-amino acids on the EPS production and cell aggregation of Alteromonas macleodii strain JL2069.

Exopolysaccharide (EPS) is produced by many marine bacteria and is important for cell aggregation in the ocean. D-amino acids are important components in bacteria and are recently recognized as signal molecules for regulation of bacterial growth. In this study, the effects of D-amino acids on EPS production, cell aggregation, and metabolic activity were investigated using an EPS-producing bacterium Alteromonas macleodii strain JL2069. EPS produced by JL2069 was inhibited by 1 mM of D-Ala and D-Ser, but not by D-Glu. The formation of particulate organic matter (POM) was promoted by the three amino acids. A new technique of microcalorimetry analysis indicated that the metabolic activity of the JL2069 cells was inhibited by these D-amino acids. Our results suggested that D-amino acids may reduce the bacterial metabolism by changing bacterial lifestyle from planktonic to cell aggregation growth which occurs independent of the production of EPS.

Altuibacter lentus gen. nov., sp. nov., a novel member of family Flavobacteriaceae isolated from deep seawater of the South China Sea.

A novel chemoheterotrophic, aerobic, Gram-negative, rod-shaped, yellow-pigmented, bacterial strain JLT2010(T) was isolated from deep seawater of the South China Sea. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain JLT2010(T) belongs to the family Flavobacteriaceae and is most closely related to Ulvibacter antarcticus IMCC3101(T) with 95.7 % similarity. Some phenotypic characteristics such as the absence of flexirubin-type pigments, growth at 37 °C, hydrolysis of casein differentiated strain JLT2010(T) from the genus Ulvibacter as well as other genera in the family Flavobacteriaceae. The DNA G+C content of the strain JLT2010(T) was found to be 35.7 mol% and the major respiratory quinone was found to be MK-6. On the basis of phenotypic and phylogenetic features, JLT2010(T) is classified as a novel genus and species within the family Flavobacteriaceae, for which the name Altuibacter lentus gen. nov., sp. nov. is proposed. The type strain is JLT2010(T) (=JCM 18884(T) = CGMCC 1.12167(T)).

Bioconversion of L-arabinose and other carbohydrates from plant cell walls to alpha-glucan by a soil bacterium, Sporosarcina sp. N52.

A Gram-positive bacterium, N52, that produces intracellular glucan from l-arabinose, was isolated from soil and identified as Sporosarcina sp. according to rRNA gene sequence analysis and physiological/biochemical characterizations. Glucan production by N52 increased significantly in the exponential phase of aerobic liquid culture and was maintained at the highest level during the stationary phase, reaching 37.0% of the cell dry weight. The glucan was also produced from other tested sugars originating from plant cell walls and was composed exclusively of alpha-1,4- and alpha-1,6-glucosidic linkages. When distillery waste was treated with N52 for 72 h, the total organic carbon (TOC), chemical oxygen demand and biochemical oxygen demand were reduced by 42.6%, 45.9% and 82.5%, respectively. Bacterial cells accumulated 31.9% of glucan per cell dry weight, fixing 16.0% of the TOC in the soluble fraction. Thus, this strain could provide us with a new process for waste management, including the bioconversion of organic materials to the valuable byproduct, alpha-glucan.

Identification of novel acetyltransferase activity on the thermostable protein ST0452 from Sulfolobus tokodaii strain 7.

A 401-residue-long protein, ST0452, has been identified from a thermophilic archaeon, Sulfolobus tokodaii strain 7, as a glucose-1-phosphate thymidylyltransferase (Glc-1-P TTase) homolog with a 170-residue-long extra C-terminus portion. Functional analyses of the ST0452 protein have confirmed that the protein possessed dual sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) activities. The 24 repeats of a signature motif sequence which has been found in bacterial acetyltransferases, (L/I/V)-(G/A/E/D)-XX-(S/T/A/V)-X, were detected at the C terminus of the ST0452 protein. This observation prompted our group to investigate the acetyltransferase activity of the ST0452 protein. Detection of the release of coenzyme A (CoA) from acetyl-CoA and the production of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) from glucosamine-1-phosphate (GlcN-1-P) and UTP in the presence of the ST0452 protein revealed that this protein possesses the GlcN-1-P-specific acetyltransferase activity. In addition, analyses of substrate specificity showed that acetyltransferase activity of the ST0452 protein is capable of catalyzing the change of galactosamine-1-phosphate (GalN-1-P) to N-acetyl-d-galactosamine-1-phosphate (GalNAc-1-P) as well as GlcN-1-P and that its sugar-1-P NTase activity is capable of producing UDP-GalNAc from GalNAc-1-P and UTP. This is the first report of a thermostable bifunctional enzyme with GalN-1-P acetyltransferase and GalNAc-1-P uridyltransferase activities. The observation reveals that the bacteria-type UDP-GlcNAc biosynthetic pathway from fructose-6-phospate is utilized in this archaeon and represents a novel biosynthetic pathway for producing UDP-GalNAc from GalN-1-P in this microorganism.