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Plasmid Sep 2021Gluconobacter oxydans is an obligate Gram-negative bacterium that belongs to the family Acetobacteraceae. It is one of the most frequently used microorganisms in...
Gluconobacter oxydans is an obligate Gram-negative bacterium that belongs to the family Acetobacteraceae. It is one of the most frequently used microorganisms in industrial biotechnology to produce chemicals related to incomplete oxidation. However, the fine-tuning of G. oxydans is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. Thus, a series of shuttle vectors for G. oxydans inspired by a series of wild-type plasmids in different G. oxydans strains were constructed. Fifteen shuttle vectors were employed to express mCherry in G. oxydans WSH-003 using the replication origin of these wild-type plasmids. Among them, the intensity of fluorescent proteins expressed by p15-K-mCherry was about 10 times that of fluorescent proteins expressed by p5-K-mCherry. Quantitative real-time polymerase chain reaction showed that the relative copy number of p15-K-mCherry reached 19 and had high stability. In contrast, some of the plasmids had a relative copy number of less than 10. The co-expression of multiple shuttle vectors revealed five shuttle vectors that could be transformed into G. oxydans WSH-003 and could express five different fluorescent proteins. The shuttle vectors will facilitate genetic operations for Gluconobacter strains to produce useful compounds more efficiently.
Topics: Biotechnology; Genetic Vectors; Gluconobacter oxydans; Plasmids
PubMed: 34256060
DOI: 10.1016/j.plasmid.2021.102588 -
Analysis of cellulose synthesis in a high-producing acetic acid bacterium Komagataeibacter hansenii.Applied Microbiology and Biotechnology May 2023Bacterial cellulose (BC) represents a renewable biomaterial with unique properties promising for biotechnology and biomedicine. Komagataeibacter hansenii ATCC 53,582 is...
Bacterial cellulose (BC) represents a renewable biomaterial with unique properties promising for biotechnology and biomedicine. Komagataeibacter hansenii ATCC 53,582 is a well-characterized high-yield producer of BC used in the industry. Its genome encodes three distinct cellulose synthases (CS), bcsAB1, bcsAB2, and bcsAB3, which together with genes for accessory proteins are organized in operons of different complexity. The genetic foundation of its high cellulose-producing phenotype was investigated by constructing chromosomal in-frame deletions of the CSs and of two predicted regulatory diguanylate cyclases (DGC), dgcA and dgcB. Proteomic characterization suggested that BcsAB1 was the decisive CS because of its high expression and its exclusive contribution to the formation of microcrystalline cellulose. BcsAB2 showed a lower expression level but contributes significantly to the tensile strength of BC and alters fiber diameter significantly as judged by scanning electron microscopy. Nevertheless, no distinct extracellular polymeric substance (EPS) from this operon was identified after static cultivation. Although transcription of bcsAB3 was observed, expression of the protein was below the detection limit of proteome analysis. Alike BcsAB2, deletion of BcsAB3 resulted in a visible reduction of the cellulose fiber diameter. The high abundance of BcsD and the accessory proteins CmcAx, CcpAx, and BglxA emphasizes their importance for the proper formation of the cellulosic network. Characterization of deletion mutants lacking the DGC genes dgcA and dgcB suggests a new regulatory mechanism of cellulose synthesis and cell motility in K. hansenii ATCC 53,582. Our findings form the basis for rational tailoring of the characteristics of BC. KEY POINTS: • BcsAB1 induces formation of microcrystalline cellulose fibers. • Modifications by BcsAB2 and BcsAB3 alter diameter of cellulose fibers. • Complex regulatory network of DGCs on cellulose pellicle formation and motility.
Topics: Acetic Acid; Extracellular Polymeric Substance Matrix; Proteomics; Acetobacteraceae; Cellulose
PubMed: 36930278
DOI: 10.1007/s00253-023-12461-z -
BMC Microbiology Dec 2022The microbiome in the insect reproductive tract is poorly understood. Our previous study demonstrated the presence of Lactobacillus spp. in female moths, but their...
BACKGROUND
The microbiome in the insect reproductive tract is poorly understood. Our previous study demonstrated the presence of Lactobacillus spp. in female moths, but their distribution and function remain unclear. Lactobacillus spp. are known as the 'healthy' vaginal microbiome in humans.
RESULTS
Here, we studied the microbiome in the reproductive system (RS) and gut of Spodoptera frugiperda using 16S rDNA sequences. The obtained 4315 bacterial OTUs were classified into 61 phyla and 642 genera, with Proteobacteria, Firmicutes and Bacteroidota being the top three dominant phyla and Enterococcus and Asaia being dominant genera in most samples. Mating dramatically increased the abundance of pathogens or pathogenic functions in the gut, while in the RS, the change range was trivial. Taxonomy assignment identified thirteen Lactobacillus spp. in S. frugiperda, with Lactobacillus crustorum and Lactobacillus murinus showing high abundance. Three species found in S. frugiperda, namely L. reuteri, L. plantarum and L. brevis, have also been identified as human 'healthy' vaginal bacterial species. Lactobacillus spp. showed higher abundance in the RS of virgin females and lower abundance in the RS of virgin males and the gut of virgin females. Mating reduced their abundance in the RS of females but increased their abundance in the RS of males, especially in males mated with multiple females. The RS of virgin females and of multiple mated males were very similar in terms of composition and abundance of Lactobacillus species, with Lactobacillus crustorum showing much higher abundance in both tissues, potentially due to sexual transmission.
CONCLUSIONS
Lactobacillus spp. showed high abundance and diversity in the RS of female moths. The higher abundance of Lactobacillus spp. in the RS of female moths and the similarity of Lactobacillus species in female moths with human 'healthy' vaginal Lactobacillus spp. suggest that these bacterial strains are also an important microbiome in the RS of female moths.
Topics: Male; Animals; Humans; Female; Gastrointestinal Microbiome; Moths; Lactobacillus; Vagina; Acetobacteraceae
PubMed: 36536275
DOI: 10.1186/s12866-022-02724-6 -
Food Research International (Ottawa,... Mar 2021Noni (Morinda citrifolia L.) has been recognized as an important herb for treating various physiological disorders worldwide. Fermented noni fruit juice, established as...
Noni (Morinda citrifolia L.) has been recognized as an important herb for treating various physiological disorders worldwide. Fermented noni fruit juice, established as a novel food in European Union, is the most important noni product. However, the structure, functions and enzyme profiles of microbiome during fermentation remain unclear. The metatranscriptomic was used to comprehensively explore the active microbial community and key metabolic function. Acetobacter sp., Acetobacter aceti and Gluconobacter sp. were the major microorganisms and appeared in succession during fermentation. According to principal components analysis (PCA) of metabolism-related unigenes by KEGG database, the fermentation process was divided into three stages and almost completed at the end of the second stage. Furthermore, carbohydrate-active enzymes (CAZymes) and the expression of key enzymes in major metabolic pathways were analyzed systematically. Analysis by HS-SPME-GC-MS and odor active value (OAV) revealed that butanoic acid and hexanoic acid were the main volatile compounds for the unpleasant odor of fermented noni fruit juice. The microbiome in the fermentation process lacked key enzymes that degrade butanoic acid and hexanoic acid, which imparted rancid and sweat odor. This study provides theoretical basis for product improvement and new product development, thus promoting the development of noni food industry.
Topics: Acetobacter; Fermentation; Fruit; Morinda
PubMed: 33641950
DOI: 10.1016/j.foodres.2020.109999 -
MicrobiologyOpen May 2019Komagataeibacter species are well-recognized bionanocellulose (BNC) producers. This bacterial genus, formerly assigned to Gluconacetobacter, is known for its phenotypic...
Komagataeibacter species are well-recognized bionanocellulose (BNC) producers. This bacterial genus, formerly assigned to Gluconacetobacter, is known for its phenotypic diversity manifested by strain-dependent carbon source preference, BNC production rate, pellicle structure, and strain stability. Here, we performed a comparative study of nineteen Komagataeibacter genomes, three of which were newly contributed in this work. We defined the core genome of the genus, clarified phylogenetic relationships among strains, and provided genetic evidence for the distinction between the two major clades, the K. xylinus and the K. hansenii. We found genomic traits, which likely contribute to the phenotypic diversity between the Komagataeibacter strains. These features include genome flexibility, carbohydrate uptake and regulation of its metabolism, exopolysaccharides synthesis, and the c-di-GMP signaling network. In addition, this work provides a comprehensive functional annotation of carbohydrate metabolism pathways, such as those related to glucose, glycerol, acetan, levan, and cellulose. Findings of this multi-genomic study expand understanding of the genetic variation within the Komagataeibacter genus and facilitate exploiting of its full potential for bionanocellulose production at the industrial scale.
Topics: Acetobacteraceae; Cellulose; Genes, Bacterial; Genetic Variation; Genome, Bacterial; Genomics; Nanoparticles; Phylogeny; Synteny
PubMed: 30365246
DOI: 10.1002/mbo3.731 -
Journal of Visualized Experiments : JoVE Apr 2021Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial diversity on...
Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial diversity on earth. Novel approaches are required to culture these uncultured bacteria so that investigators can effectively study their physiology and lifestyle using the powerful tools available in the laboratory. The Candidate Phyla Radiation (CPR) is one of the largest groups of uncultivated bacteria, comprising ~15% of the living diversity on earth. The first isolate of this group was a member of the Saccharibacteria phylum, 'Nanosynbacter lyticus' strain TM7x. TM7x is an unusually small bacterium that lives as a symbiont in direct contact with a bacterial host, Schaalia odontolytica, strain XH001. Taking advantage of the unusually small cell size and its lifestyle as a symbiotic organism, we developed a protocol to rapidly culture Saccharibacteria from dental plaque. This protocol will show how to filter a suspension of dental plaque through a 0.2 µm filter, then concentrate the collected Saccharibacteria cells and infect a culture of host organisms. The resulting coculture can be passaged as any normal bacterial culture and infection is confirmed by PCR. The resulting binary culture can be maintained in the laboratory and used for future experiments. While contamination is a possibility, the binary culture can be purified by either further filtering and reinfection of host, or by plating the binary culture and screening for infected colonies. We hope this protocol can be expanded to other sample types and environments, leading to the cultivation of many more species in the CPR.
Topics: Acetobacteraceae; Bacteria; Mouth; Symbiosis
PubMed: 33938896
DOI: 10.3791/62484 -
Archives of Microbiology May 2022A Gram-stain-negative, cocci-to-oval-shaped bacterial strain, designated XZZS9, was isolated from the rhizosphere soil of Pinus sylvestris var. mongolica and...
A Gram-stain-negative, cocci-to-oval-shaped bacterial strain, designated XZZS9, was isolated from the rhizosphere soil of Pinus sylvestris var. mongolica and characterized taxonomically using a polyphasic approach. Growth occurred at 20-35 °C (optimum, 28 °C), pH 6.0-11.0 (optimum, pH 7.0), and in 0-1% NaCl (optimum, 0%). Phylogenetic analysis based on 16S rRNA gene sequencing indicated that strain XZZS9 was related to members of the genus Roseococcus, with the highest sequence identity to Roseococcus microcysteis NIBR12 (96.9%). The major cellular fatty acids (> 5% of the total) were C ω7c and C cyclo ω8c. The major isoprenoid quinone was Q-9 and the polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, an unidentified glycophospholipid, and an unidentified phospholipid. Genome sequencing revealed that had a genome size of 4.79 Mbp with a G + C content of 69.5%. Comparative genomic analyses clearly separated strain XZZS9 from the known species of the genus Roseococcus based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values below the thresholds for species delineation. Genome annotations did not find pufL and pufM genes in strain XZZS9, suggesting a possible lack of photosynthetic reaction. Based on genotypic and phenotypic characteristics, strain XZZS9 represents a novel species of the genus Roseococcus, for which we propose the name Roseococcus pinisoli sp. nov. The type strain is XZZS9 (= KCTC 82435 = JCM 34402 = GDMCC 1.2158).
Topics: Acetobacteraceae; Bacterial Typing Techniques; Bacteriochlorophyll A; DNA, Bacterial; Fatty Acids; Nucleic Acid Hybridization; Phospholipids; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA
PubMed: 35507236
DOI: 10.1007/s00203-022-02920-y -
The Journal of Biological Chemistry Sep 2021Understanding the mechanism by which cellulases from bacteria, fungi, and protozoans catalyze the digestion of lignocellulose is important for developing cost-effective...
Understanding the mechanism by which cellulases from bacteria, fungi, and protozoans catalyze the digestion of lignocellulose is important for developing cost-effective strategies for bioethanol production. Cel7A from the fungus Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. Here, we used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled TrCel7A molecules binding to and moving processively along immobilized cellulose. Individual enzyme molecules were localized with a spatial precision of a few nanometers and followed for hundreds of seconds. Most enzyme molecules bound to cellulose in a static state and dissociated without detectable movement, whereas a minority of molecules moved processively for an average distance of 39 nm at an average speed of 3.2 nm/s. These data were integrated into a three-state model in which TrCel7A molecules can bind from solution into either static or processive states and can reversibly switch between states before dissociating. From these results, we conclude that the rate-limiting step for cellulose degradation by Cel7A is the transition out of the static state, either by dissociation from the cellulose surface or by initiation of a processive run. Thus, accelerating the transition of Cel7A out of its static state is a potential avenue for improving cellulase efficiency.
Topics: Acetobacteraceae; Cellulose; Cellulose 1,4-beta-Cellobiosidase; Fungal Proteins; Hydrolysis; Hypocreales; Microscopy, Atomic Force; Microscopy, Fluorescence; Quantum Dots; Substrate Specificity
PubMed: 34339742
DOI: 10.1016/j.jbc.2021.101029 -
Scientific Reports Aug 2017Bacterial cellulose (BC) is widely used in industries owing to its high purity and strength. Although Komagataeibacter nataicola is a representative species for BC...
Bacterial cellulose (BC) is widely used in industries owing to its high purity and strength. Although Komagataeibacter nataicola is a representative species for BC production, its intracellular metabolism leading to BC secretion is unclear. In the present study, a genome-scale metabolic network of cellulose-producing K. nataicola strain RZS01 was reconstructed to understand its metabolic behavior. This model iHZ771 comprised 771 genes, 2035 metabolites, and 2014 reactions. Constraint-based analysis was used to characterize and evaluate the critical intracellular pathways. The analysis revealed that a total of 71 and 30 genes are necessary for cellular growth in a minimal medium and complex medium, respectively. Glycerol was identified as the optimal carbon source for the highest BC production. The minimization of metabolic adjustment algorithm identified 8 genes as potential targets for over-production of BC. Overall, model iHZ771 proved to be a useful platform for understanding the physiology and BC production of K. nataicola.
Topics: Acetobacteraceae; Cellulose; Culture Media; Genome, Bacterial; Glycerol; Metabolic Networks and Pathways; Models, Biological
PubMed: 28801647
DOI: 10.1038/s41598-017-06918-1 -
Carbohydrate Polymers Apr 2019The synergy of the local delivery of nucleic acids using a hydrogel-based gene activated matrix (GAM) might support regenerative processes on a genetic level by...
The synergy of the local delivery of nucleic acids using a hydrogel-based gene activated matrix (GAM) might support regenerative processes on a genetic level by concurrently providing a cell-friendly microenvironment. To investigate bacterial nanocellulose (BNC) as GAM, two plasmids (pSV-β-Gal and pGL3) were incorporated by reswelling and injection techniques forming matrix and core-shell systems as determined by SEM and staining experiments. The release was found to be dependent on the type of BNC, the plasmid and the loading technique, and lasted over at least 20 days. No morphological or mechanical changes of the BNC due to the presence of plasmids were observed. Immobilized plasmids especially in the matrix systems were protected against enzymatic degradation by maintaining the high biocompatibility of BNC and transfection efficacy of the plasmids. These results indicate that BNC can be used as a promising and renewable carrier for the application as local gene delivery system.
Topics: Acetobacteraceae; Animals; CHO Cells; Cellulose; Chick Embryo; Cricetulus; DNA; Drug Carriers; Drug Liberation; Materials Testing; Nanostructures; Plasmids; Transfection
PubMed: 30732826
DOI: 10.1016/j.carbpol.2019.01.009