Phylogenomic and comparative analyses support the reclassification of several Komagataeibacter species as novel members of the Novacetimonas gen. nov. and bring new insights into the evolution of cellulose synthase genes.

The genus Komagataeibacter harbours bacteria presenting the ability to produce increased levels of crystalline nanocellulose, as well as strains used in the industrial production of fermented products and beverages. Still, most of the studies of this biotechnologically relevant genus were conducted based on limited phenotypic methodologies and taxonomical classifications. In this work, a detailed analysis of the currently described genus Komagataeibacter was conducted based on phylogenomic analysis, unveiling the phylogenomic relationships within the genus and allowing a detailed phylogenetic analysis of biotechnologically important genes such as those involved in cellulose biosynthesis (bcs genes). Phylogenomic and comparative genomic analysis revealed that several type strains formed an independent genomic group from those of other Komagataeibacter, prompting their reclassification as members of a novel genus, hereby termed Novacetimonas gen. nov. The results support the reclassification of Komagataeibacter hansenii, Komagataeibacter cocois, Komagataeibacter maltaceti and Komagataeibacter pomaceti as novel members of the genus Novacetimonas. The Novacetimonas hansenii species is the proposed representative of the novel genus. Importantly, phylogenetic analysis based on cellulose biosynthesis genes (bcsABCD, bcsAB2XYC2, bcsAB3C3, bcsAB4), showed that the evolutionary history of these genes is closely related to the strain's phylogenomic/taxonomic classification. Hence, the robust taxonomic classification of these bacteria will allow the better characterization and selection of strains for biotechnological applications.

Roseococcus microcysteis sp. nov., isolated from a Microcystis aeruginosa culture sample.

Strain NIBR12T (=KACC 22094T=HAMBI 3739T), a novel Gram-stain-negative, obligate aerobic, non-spore-forming, non-motile and coccobacillus-shaped bacterium, was isolated from a cyanobacterial sample culture (Microcysitis aeruginosa NIBRCYC000000452). The newly identified bacterial strain grew optimally in modified Reasoner's 2A medium under the following conditions: 0 % (w/v) NaCl, pH 7.5 and 35 °C. Phylogenetic analysis using the 16S rRNA gene sequence confirmed that strain NIBR12T belongs to the genus Roseococcus, with its closest neighbours being Roseococcus suduntuyensis SHETT (98.8%), Roseococcus thiosulfatophilus RB-3T (97.7%), "Sediminicoccus rosea" R-30T (95.7 %) and Rubritepida flocculans H-8T (95.0 %). Genomic comparison of strain NIBR12T with type species in the genus Roseococcus was conducted using digital DNA-DNA hybridization, average nucleotide identity and average amino acid identity analyses, resulting in values of ≤53.7, ≤93.7 and ≤96.1 %, respectively. The genomic DNA G+C content of strain NIBR12T was 70.9 mol%. The major fatty acids of strain NIBR12T were summed feature 8 (C18 : 1 ω7c and/or C18:1 ω6c) and summed feature 3 (C16 : 1 ω6c/C16 : 1 ω7c). Q-9 was its major respiratory quinone. Moreover, the major polar lipids of strain NIBR12T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. Based on our chemotaxonomic, genotypic and phenotype analyses, strain NIBR12T is identified as represeting a novel species of the genus Roseococcus, for which the name Roseococcus microcysteis sp. nov. is proposed.

Molecular detection and maternal transmission of a bacterial symbiont Asaia species in field-caught Anopheles mosquitoes from Cameroon.

BACKGROUND: Malaria control relies mainlyon insecticide-based tools. However, the effectiveness of these tools is threatened by widespread insecticide resistance in malaria vectors, highlighting the need for alternative control approaches. The endosymbiont Asaia has emerged as a promising candidate for paratransgenic control of malaria, but its biology and genetics still need to be further analyzed across Africa. Here, we investigated the prevalence of Asaia and its maternal transmission in the natural population of Anopheles mosquitoes in Cameroon. METHODS: Indoor-resting adult mosquitoes belonging to four species (An. coluzzii, An. arabiensis, An. funestus and An. gambiae) were collected from eight localities across Cameroon from July 2016 to February 2020. PCR was performed on the Asaia-specific 16S ribosomal RNA gene, and samples positive by PCR for Asaia were confirmed by Sanger sequencing and phylogenetic analysis. The vertical transmission of Asaia was investigated by screening F1 mosquitoes belonging to F0 Asaia-positive females. RESULTS: A total of 895 mosquitoes were screened. We found 43% (384) Asaia infection prevalence in four mosquito species. Phylogenetic analysis revealed that Asaia from Cameroon clustered together with the strains of Asaia isolated from other parts of the world. In addition, seven nucleotide sequence variants were found with low genetic diversity (π = 0.00241) and nucleotide sequence variant diversity (Hd = 0.481). Asaia was vertically transmitted with high frequency (range from 42.5 to 100%). CONCLUSIONS: This study provides field-based evidence of the presence of Asaia in Anopheles mosquitoes in Cameroon for exploitation as a symbiont in the control of malaria in sub-Saharan Africa.

Complete Genomes of Clade G6 Saccharibacteria Suggest a Divergent Ecological Niche and Lifestyle.

Saccharibacteria (formerly TM7) have reduced genomes and a small cell size and appear to have a parasitic lifestyle dependent on a bacterial host. Although there are at least 6 major clades of Saccharibacteria inhabiting the human oral cavity, complete genomes of oral Saccharibacteria were previously limited to the G1 clade. In this study, nanopore sequencing was used to obtain three complete genome sequences from clade G6. Phylogenetic analysis suggested the presence of at least 3 to 5 distinct species within G6, with two discrete taxa represented by the 3 complete genomes. G6 Saccharibacteria were highly divergent from the more-well-studied clade G1 and had the smallest genomes and lowest GC content of all Saccharibacteria. Pangenome analysis showed that although 97% of shared pan-Saccharibacteria core genes and 89% of G1-specific core genes had putative functions, only 50% of the 244 G6-specific core genes had putative functions, highlighting the novelty of this group. Compared to G1, G6 harbored divergent metabolic pathways. G6 genomes lacked an F1Fo ATPase, the pentose phosphate pathway, and several genes involved in nucleotide metabolism, which were all core genes for G1. G6 genomes were also unique compared to that of G1 in that they encoded d-lactate dehydrogenase, adenylate cyclase, limited glycerolipid metabolism, a homolog to a lipoarabinomannan biosynthesis enzyme, and the means to degrade starch. These differences at key metabolic steps suggest a distinct lifestyle and ecological niche for clade G6, possibly with alternative hosts and/or host dependencies, which would have significant ecological, evolutionary, and likely pathogenic implications. IMPORTANCESaccharibacteria are ultrasmall parasitic bacteria that are common members of the oral microbiota and have been increasingly linked to disease and inflammation. However, the lifestyle and impact on human health of Saccharibacteria remain poorly understood, especially for the clades with no complete genomes (G2 to G6) or cultured isolates (G2 and G4 to G6). Obtaining complete genomes is of particular importance for Saccharibacteria, because they lack many of the "essential" core genes used for determining draft genome completeness, and few references exist outside clade G1. In this study, complete genomes of 3 G6 strains, representing two candidate species, were obtained and analyzed. The G6 genomes were highly divergent from that of G1 and enigmatic, with 50% of the G6 core genes having no putative functions. The significant difference in encoded functional pathways is suggestive of a distinct lifestyle and ecological niche, probably with alternative hosts and/or host dependencies, which would have major implications in ecology, evolution, and pathogenesis.

Belnapia mucosa sp. nov. and Belnapia arida sp. nov., isolated from desert biocrust.

Two novel Gram-staining-negative, aerobic, cocci-shaped, non-motile, non-spore forming, pink-pigmented bacteria designated strains T6T and T18T, were isolated from a biocrust (biological soil crust) sample from the vicinity of the Tabernas Desert (Spain). Both strains were catalase-positive and oxidase-negative, and grew under mesophilic, neutrophilic and non-halophilic conditions. According to the 16S rRNA gene sequences, strains T6T and T18T showed similarities with Belnapia rosea CGMCC 1.10758T and Belnapia moabensis CP2CT (98.11 and 98.55% gene sequence similarity, respectively). The DNA G+C content was 69.80 and 68.96% for strains T6T and T18T, respectively; the average nucleotide identity by blast (ANIb) and digital DNA-DNA hybridization (dDDH) values confirmed their adscription to two novel species within the genus Belnapia. The predominant fatty acids were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c), C16 : 0, C18 : 1 2-OH and summed feature 3 (C16 : 1ω7c/C16 : 1ω6c). According to he results of the polyphasic study, strains T6T and T18T represent two novel species in the genus Belnapia (which currently includes only three species), for which names Belnapia mucosa sp. nov. (type strain T6T = CECT 30228T=DSM 112073T) and Belnapia arida sp. nov. (type strain T18T=CECT 30229T=DSM 112074T) are proposed, respectively.

The Komagataeibacter europaeus GqqA is the prototype of a novel bifunctional N-Acyl-homoserine lactone acylase with prephenate dehydratase activity.

Previously, we reported the isolation of a quorum quenching protein (QQ), designated GqqA, from Komagataeibacter europaeus CECT 8546 that is highly homologous to prephenate dehydratases (PDT) (Valera et al. in Microb Cell Fact 15, 88. https://doi.org/10.1186/s12934-016-0482-y , 2016). GqqA strongly interfered with N-acyl-homoserine lactone (AHL) quorum sensing signals from Gram-negative bacteria and affected biofilm formation in its native host strain Komagataeibacter europaeus. Here we present and discuss data identifying GqqA as a novel acylase. ESI-MS-MS data showed unambiguously that GqqA hydrolyzes the amide bond of the acyl side-chain of AHL molecules, but not the lactone ring. Consistent with this observation the protein sequence does not carry a conserved Zn2+ binding motif, known to be essential for metal-dependent lactonases, but in fact harboring the typical periplasmatic binding protein domain (PBP domain), acting as catalytic domain. We report structural details for the native structure at 2.5 Å resolution and for a truncated GqqA structure at 1.7 Å. The structures obtained highlight that GqqA acts as a dimer and complementary docking studies indicate that the lactone ring of the substrate binds within a cleft of the PBP domain and interacts with polar residues Y16, S17 and T174. The biochemical and phylogenetic analyses imply that GqqA represents the first member of a novel type of QQ family enzymes.

Coproduction of bacterial cellulose and pear vinegar by fermentation of pear peel and pomace.

Bacterial cellulose (BC)-derived materials are given significant attention due to their porous fibrous texture, high crystallinity and extraordinary physico-mechanical properties. The main reason for the restricted use of BC is its high production cost. To reduce the production cost, the suitability of pear residue for the production of BC and pear vinegar was investigated. Komagataeibacter rhaeticus and Komagataeibacter intermedius with high fermentation ability screened from the surface of vinegar film of millet fermentation were used to produce BC and pear vinegar simultaneously. Through response surface optimization, the maximum yield of BC from pear residue medium was 10.94 ± 0.42 g/L, which was higher than the synthesis medium generally used for Acetobacter strains. When pear residue medium was incubated at 30 °C for 7 days, the contents of total acid and soluble solids were greater than 0.3 g/100 mL and 3%, respectively, which met the standard requirements for fruit vinegar. The flavour components of pear vinegar were determined using gas chromatography-mass spectrometry. The pear vinegar showed similar flavour characteristics to conventional fruit vinegar. This research not only solved the utilization of agricultural resources but also avoided the discharge of waste liquid when producing BC. In addition, a more environmentally friendly and less expensive way to produce BC and pear vinegar was achieved.

Valorization of fruit processing waste to produce high value-added bacterial nanocellulose by a novel strain Komagataeibacter xylinus IITR DKH20.

To date, the production of bacterial nanocellulose (BNC) by standard methods has been well known, while the use of low-cost feedstock as an alternative medium still needs to be explored for BNC commercialization. This study explores the prospect for the use of the different aqueous extract of fruit peel wastes (aE-FPW) as a nutrient and carbon source for the production of BNC. Herein, this objective was accomplished by the use of a novel, high- yielding strain, isolated from rotten apple and further identified as Komagataeibacter xylinus IITR DKH20 using 16 s rRNA sequencing analysis. The physicochemical properties of BNC matrix collected from the various aE-FPW mediums were similar or advanced to those collected with the HS medium. Statistical optimization of BNC based on Central Composite Design was performed to study the effect of significant parameters and the results demonstrated that the BNC yield (11.44 g L-1) was increased by 4.5 fold after optimization.

Phylogenomics Reveals that Asaia Symbionts from Insects Underwent Convergent Genome Reduction, Preserving an Insecticide-Degrading Gene.

The mosquito microbiota is composed of several lineages of microorganisms whose ecological roles and evolutionary histories have yet to be investigated in depth. Among these microorganisms, Asaia bacteria play a prominent role, given their abundance in the gut, reproductive organs, and salivary glands of different mosquito species, while their presence has also been reported in several other insects. Notably, Asaia has great potential as a tool for the control of mosquito-borne diseases. Here, we present a wide phylogenomic analysis of Asaia strains isolated from different species of mosquito vectors and from different populations of the Mediterranean fruit fly (medfly), Ceratitis capitata, an insect pest of worldwide economic importance. We show that phylogenetically distant lineages of Asaia experienced independent genome reductions, despite following a common pattern, characterized by the early loss of genes involved in genome stability. This result highlights the role of specific metabolic pathways in the symbiotic relationship between Asaia and the insect host. Finally, we discovered that all but one of the Asaia strains included in the study possess the pyrethroid hydrolase gene. Phylogenetic analysis revealed that this gene is ancestral in Asaia, strongly suggesting that it played a role in the establishment of the symbiotic association between these bacteria and the mosquito hosts. We propose that this gene from the symbiont contributed to initial pyrethroid resistance in insects harboring Asaia, also considering the widespread production of pyrethrins by several plants.IMPORTANCE We have studied genome reduction within several strains of the insect symbiont Asaia isolated from different species/strains of mosquito and medfly. Phylogenetically distant strains of Asaia, despite following a common pattern involving the loss of genes related to genome stability, have undergone independent genome reductions, highlighting the peculiar role of specific metabolic pathways in the symbiotic relationship between Asaia and its host. We also show that the pyrethroid hydrolase gene is present in all the Asaia strains isolated except for the South American malaria vector Anopheles darlingi, for which resistance to pyrethroids has never been reported, suggesting a possible involvement of Asaia in determining resistance to insecticides.

Bombella favorum sp. nov. and Bombella mellum sp. nov., two novel species isolated from the honeycombs of Apis mellifera.

As part of a study investigating the microbiome of bee hives and honey, two novel strains (TMW 2.1880T and TMW 2.1889T) of acetic acid bacteria were isolated and subsequently taxonomically characterized by a polyphasic approach, which revealed that they cannot be assigned to known species. The isolates are Gram-stain-negative, aerobic, pellicle-forming, catalase-positive and oxidase-negative. Cells of TMW 2.1880T are non-motile, thin/short rods, and cells of TMW 2.1889T are motile and occur as rods and long filaments. Morphological, physiological and phylogenetic analyses revealed a distinct lineage within the genus Bombella. Strain TMW 2.1880T is most closely related to the type strain of Bombella intestini with a 16S rRNA gene sequence similarity of 99.5 %, and ANIb and in silico DDH values of 94.16 and 56.3 %, respectively. The genome of TMW 2.1880T has a size of 1.98 Mb and a G+C content of 55.3 mol%. Strain TMW 2.1889T is most closely related to the type strain of Bombella apis with a 16S rRNA gene sequence similarity of 99.5 %, and ANIb and in silico DDH values of 85.12 and 29.5 %, respectively. The genome of TMW 2.1889T has a size of 2.07 Mb and a G+C content of 60.4 mol%. Ubiquinone analysis revealed that both strains contained Q-10 as the main respiratory quinone. Major fatty acids for both strains were C16 : 0, C19 : 0 cyclo ω8c and summed feature 8, respectively, and additionally C14 : 0 2-OH only for TMW 2.1880T and C14 : 0 only for TMW 2.1889T. Based on polyphasic evidence, the two isolates from honeycombs of Apis mellifera represent two novel species of the genus Bombella, for which the names Bombella favorum sp. nov and Bombella mellum sp. nov. are proposed. The designated respective type strains are TMW 2.1880T (=LMG 31882T=CECT 30114T) and TMW 2.1889T (=LMG 31883T=CECT 30113T).

Metagenome Analysis of a Hydrocarbon-Degrading Bacterial Consortium Reveals the Specific Roles of BTEX Biodegraders.

Environmental contamination by petroleum hydrocarbons is of concern due to the carcinogenicity and neurotoxicity of these compounds. Successful bioremediation of organic contaminants requires bacterial populations with degradative capacity for these contaminants. Through successive enrichment of microorganisms from a petroleum-contaminated soil using diesel fuel as the sole carbon and energy source, we successfully isolated a bacterial consortium that can degrade diesel fuel hydrocarbons. Metagenome analysis revealed the specific roles of different microbial populations involved in the degradation of benzene, toluene, ethylbenzene and xylene (BTEX), and the metabolic pathways involved in these reactions. One hundred and five putative coding DNA sequences were identified as responsible for both the activation of BTEX and central metabolism (ring-cleavage) of catechol and alkylcatechols during BTEX degradation. The majority of the Coding DNA sequences (CDSs) were affiliated to Acidocella, which was also the dominant bacterial genus in the consortium. The inoculation of diesel fuel contaminated soils with the consortium resulted in approximately 70% hydrocarbon biodegradation, indicating the potential of the consortium for environmental remediation of petroleum hydrocarbons.

Oecophyllibacter saccharovorans gen. nov. sp. nov., a bacterial symbiont of the weaver ant Oecophylla smaragdina.

In this study, bacterial strains Ha5T, Ta1, and Jb2 were isolated from different colonies of weaver ant Oecophylla smaragdina. They were identified as bacterial symbionts of the ant belonging to family Acetobacteraceae and were distinguished as different strains based on distinctive random-amplified polymorphic DNA (RAPD) fingerprints. Cells of these bacterial strains were Gram-negative, rod-shaped, aerobic, non-motile, catalase-positive and oxidase-negative. They were able to grow at 15-37°C (optimum, 28-30°C) and in the presence of 0-1.5% (w/v) NaCl (optimum 0%). Their predominant cellular fatty acids were C18:1ω7c, C16:0, C19:0ω8c cyclo, C14:0, and C16:0 2-OH. Strains Ha5T, Ta1, and Jb2 shared highest 16S rRNA gene sequence similarity (94.56-94.63%) with Neokomagataea tanensis NBRC106556T of family Acetobacteraceae. Both 16S rRNA gene sequence-based phylogenetic analysis and core gene-based phylogenomic analysis placed them in a distinct lineage in family Acetobacteraceae. These bacterial strains shared higher than species level thresholds in multiple overall genome-relatedness indices which indicated that they belonged to the same species. In addition, they did not belong to any of the current taxa of Acetobacteraceae as they had low pairwise average nucleotide identity (< 71%), in silico DNA-DNA hybridization (< 38%) and average amino acid identity (< 67%) values with all the type members of the family. Based on these results, bacterial strains Ha5T, Ta1, and Jb2 represent a novel species of a novel genus in family Acetobacteaceae, for which we propose the name Oecophyllibacter saccharovorans gen. nov. sp. nov., and strain Ha5T as the type strain.

Lichenicola cladoniae gen. nov., sp. nov., a member of the family Acetobacteraceae isolated from an Antarctic lichen.

Two Gram-stain-negative, facultative anaerobic, chemoheterotrophic, pink-coloured, rod-shaped and non-motile bacterial strains, PAMC 26568 and PAMC 26569T, were isolated from an Antarctic lichen. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains PAMC 26568 and PAMC 26569T belong to the family Acetobacteraceae and the most closely related species are Gluconacetobacter takamatsuzukensis (96.1 %), Gluconacetobacter tumulisoli (95.9 %) and Gluconacetobacter sacchari (95.7 %). Phylogenomic and genomic relatedness analyses showed that strains PAMC 26568 and PAMC 26569T are clearly distinguished from other genera in the family Acetobacteraceae by average nucleotide identity values (<72.8 %) and the genome-to-genome distance values (<22.5 %). Genomic analysis revealed that strains PAMC 26568 and PAMC 26569T do not contain genes involved in atmospheric nitrogen fixation and utilization of sole carbon compounds such as methane and methanol. Instead, strains PAMC 26568 and PAMC 26569T possess genes to utilize nitrate and nitrite and certain monosaccharides and disaccharides. The major fatty acids (>10 %) are summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c; 40.3-40.4 %), C18 : 1 2OH (22.7-23.7 %) and summed feature 2 (C14 : 0 3OH and/or C16 : 1 iso I; 12.0 % in PAMC 26568). The major respiratory quinone is Q-10. The genomic DNA G+C content of PAMC 26568 and PAMC 26569T is 64.6 %. Their distinct phylogenetic position and some physiological characteristics distinguish strains PAMC 26568 and PAMC 26569T from other genera in the family Acetobacteraceae supporting the proposal of Lichenicola gen. nov., with the type species Lichenicola cladoniae sp. nov. (type strain, PAMC 26569T=KCCM 43315T=JCM 33604T).

Roseomonas algicola sp. nov., isolated from a green alga, Pediastrum duplex.

A Gram-stain-negative, strictly aerobic bacterium, designated strain PeD5T, was isolated from a green alga Pediastrum duplex from the Nakdong river of the Republic of Korea. Cells were non-motile cocci, catalase-negative and oxidase-positive. Growth of PeD5T was observed at 25-40 °C (optimum, 35 °C) and pH 5.0-10.0 (optimum, pH 7-8), and in the presence of 0-0.25% (w/v) NaCl (optimum, 0%). PeD5T contained C16:0, C18:1ω7c 11-methyl, summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c) and summed feature 8 (comprising C18:1ω7c and/or C18:1ω6c) as major cellular fatty acids (>5%) and ubiquinone-10 as the sole isoprenoid quinone. Phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified phospholipid and an unidentified aminolipid were detected as major polar lipids. The genomic DNA G+C content of PeD5T was 71.0 mol%. PeD5T was most closely related to Roseomonas stagni HS-69T with a 97.6% 16S rRNA sequence similarity and shared less than 96.3% 16S rRNA sequence similarities with type strains of other species. Phylogenetic analysis based on 16S rRNA gene sequences indicated that PeD5T formed a phyletic lineage with Roseomonas stagni HS-69T within the genus Roseomonas. On the basis of results of phenotypic, chemotaxonomic and molecular analysis, strain PeD5T clearly represents a novel species of the genus Roseomonas, for which the name Roseomonas algicola sp. nov. is proposed. The type strain is PeD5T (=KACC 19925T=JCM 33309T).

Rhodovarius crocodyli sp. nov., isolated from a crocodile pond.

Bacterial strain CCP-6T, isolated from a freshwater pond in Taiwan, was characterized using a polyphasic taxonomy approach. Phylogenetic analyses based on 16S rRNA gene sequences and an up-to-date bacterial core gene set (92 protein clusters) indicated that strain CCP-6T is affiliated with species in the genus Rhodovarius. Strain CCP-6T was most closely related to Rhodovarius lipocyclicus CCUG 44693T with a 98.9% 16S rRNA gene sequence similarity. Cells were Gram-stain-negative, aerobic, non-motile, rod-shaped and formed light pink-coloured colonies. Optimal growth occurred at 30 °C, pH 6 and in the absence of NaCl. The major fatty acids of strain CCP-6T were C18 : 1 ω7c, C16 : 0 and C19 : 0 cyclo ω8c. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidyldimethylethanolamine, phosphatidylmethylethanolamine, diphosphatidylglycerol, three unidentified aminophospholipids and an unidentified phospholipid. The predominant polyamine was spermidine. The major isoprenoid quinone was Q-10. The DNA G+C content of the genomic DNA was 69.3 mol%. Strain CCP-6T showed 85.8% average nucleotide identity and 14.5% digital DNA-DNA hybridization identity with Rhodovarius lipocyclicus CCUG 44693T. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain CCP-6T represents a novel species in the genus Rhodovarius, for which the name Rhodovarius crocodyli sp. nov. is proposed. The type strain is CCP-6T (=BCRC 81095T=LMG 30310T=KCTC 62188T).

Lichenicoccus roseus gen. nov., sp. nov., the first bacteriochlorophyll a-containing, psychrophilic and acidophilic Acetobacteraceae bacteriobiont of lichen Cladonia species.

Gram-negative, aerobic, chemo-organotrophic and bacteriochlorophyll a-containing bacterial strains, KEBCLARHB70RT, KAMCLST3051 and KAMCLST3152, were isolated from the thalli of Cladonia arbuscula and Cladonia stellaris lichens. Cells from the strains were coccoid and reproduced by binary division. They were motile at the early stages of growth and utilized sugars and alcohols. All strains were psychrophilic and acidophilic, capable of growth between pH 3.5 and 7.5 (optimum, pH 5.5), and at 4-30 °C (optimum, 10-15 °C). The major fatty acids were C18 : 1 ω7c and C18 : 0; the lipids were phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids, phosphatidylglycerol, glycolipids, diphosphatidylglycerol and polar lipids with an unknown structure. The quinone was Q-10. The DNA G+C content was 67.8 mol%. Comparative 16S rRNA gene analysis together with other data, supported that the strains, KEBCLARHB70RT, KAMCLST3051 and KAMCLST3152 belonged to the same species. Whole genome analysis of the strain KEBCLARHB70RT and average amino acid identity values confirmed its distinctive phylogenetic position within the family Acetobacteraceae. Phenotypic, ecological and genomic characteristics distinguished strains KEBCLARHB70RT, KAMCLST3051 and KAMCLST3152 from all genera in the family Acetobacteraceae. Therefore, we propose a novel genus and a novel species, Lichenicoccus roseus gen. nov., sp. nov., for these novel Acetobacteraceae members. Strain KEBCLARHB70RT (=KCTC 72321T=VKM B-3305T) has been designated as the type strain.

Bacterial nanocellulose from agro-industrial wastes: low-cost and enhanced production by Komagataeibacter saccharivorans MD1.

Bacterial nanocellulose (BNC) has been drawing enormous attention because of its versatile properties. Herein, we shed light on the BNC production by a novel bacterial isolate (MD1) utilizing various agro-industrial wastes. Using 16S rRNA nucleotide sequences, the isolate was identified as Komagataeibacter saccharivorans MD1. For the first time, BNC synthesis by K. saccharivorans MD1 was investigated utilizing wastes of palm date, fig, and sugarcane molasses along with glucose on the Hestrin-Schramm (HS) medium as a control. After incubation for 168 h, the highest BNC yield was perceived on the molasses medium recording 3.9 g/L with an initial concentration of (v/v) 10%. The physicochemical characteristics of the BNC sheets were inspected adopting field-emission scanning electron microscope (FESEM), atomic force microscopy (AFM), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) analysis. The FESEM characterization revealed no impact of the wastes on either fiber diameter or the branching scheme, whereas the AFM depicted a BNC film with minimal roughness was generated using date wastes. Furthermore, a high crystallinity index was estimated by XRD up to 94% for the date wastes-derived BNC, while the FTIR analyses exhibited very similar profiles for all BNC films. Additionally, mechanical characteristics and water holding capacity of the produced BNCs were studied. Our findings substantiated that expensive substrates could be exchanged by agro-industrial wastes for BNC production conserving its remarkable physical and microstructural properties.

Molecular characterization of RNase III protein of Asaia sp. for developing a robust RNAi-based paratransgensis tool to affect the sexual life-cycle of Plasmodium or Anopheles fitness.

BACKGROUND: According to scientific recommendations, paratransgenesis is one of the solutions for improving the effectiveness of the Global Malaria Eradication Programme. In paratransgenesis, symbiont microorganisms are used for distorting or blocking the parasite life-cycle, affecting the fitness and longevity of vectors or reducing the vectorial competence. It has been revealed recently that bacteria could be used as potent tools for double stranded RNA production and delivery to insects. Moreover, findings showed that RNase III mutant bacteria are more competent for this aim. Asaia spp. have been introduced as potent paratransgenesis candidates for combating malaria and, based on their specific features for this goal, could be considered as effective dsRNA production and delivery tools to Anopheles spp. Therefore, we decided to characterize the rnc gene and its related protein to provide the basic required information for creating an RNase III mutant Asaia bacterium. METHODS: Asaia bacteria were isolated from field-collected Anopheles stephensi mosquitoes. The rnc gene and its surrounding sequences were characterized by rapid amplification of genomic ends. RNase III recombinant protein was expressed in E. coli BL21 and biological activity of the purified recombinant protein was assayed. Furthermore, Asaia RNaseIII amino acid sequence was analyzed by in silico approaches such as homology modeling and docking to determine its structural properties. RESULTS: In this study, the structure of rnc gene and its related operon from Asaia sp. was determined. In addition, by performing superimposition and docking with specific substrate, the structural features of Asaia RNaseIII protein such as critical residues which are involved and essential for proper folding of active site, binding of magnesium ions and double stranded RNA molecule to protein and cleaving of dsRNA molecules, were determined. CONCLUSIONS: In this study, the basic and essential data for creating an RNase III mutant Asaia sp. strain, which is the first step of developing an efficient RNAi-based paratransgenesis tool, were acquired. Asaia sp. have been found in different medically-important vectors and these data are potentially very helpful for researchers studying paratransgenesis and vector-borne diseases and are interested in applying the RNAi technology in the field.

Genome sequencing and phylogenetic analysis of K1G4: a new Komagataeibacter strain producing bacterial cellulose from different carbon sources.

OBJECTIVE: The objective of this study was to evaluate the ability of a new Komagataeibacter xylinus strain in producing bacterial cellulose from glucose, mannitol and glycerol, and to assess the genome sequencing with special focus on bacterial cellulose related genes. RESULTS: Bacterial cellulose production during 9 days of cultivation was tested in glucose, mannitol and glycerol, respectively. Differences in the bacterial cellulose kinetic formation was observed, with a final yield of 9.47 g/L in mannitol, 8.30 g/L in glycerol and 7.57 g/L in glucose, respectively. The draft genome sequencing of K1G4 was produced, revealing a genome of 3.09 Mbp. Two structurally completed cellulose synthase operons and a third copy of the catalytic subunit of cellulose synthase were found. By using phylogenetic analysis, on the entire rRNA operon sequence, K1G4 was found to be closely related to Komagataeibacter xylinus LMG 1515T and K. xylinus K2G30. CONCLUSIONS: The different yields of bacterial cellulose produced on glucose, mannitol and glycerol can be correlated with the third copy of bcsAB operon harboured by K1G4, making it a versatile strain for industrial applications.

Komagataeibacter diospyri sp. nov., a novel species of thermotolerant bacterial nanocellulose-producing bacterium.

Thermotolerant bacterial nanocellulose-producing strains, designated MSKU 9T and MSKU 15, were isolated from persimmon and sapodilla fruits, respectively. These strains were aerobic, Gram-stain-negative, had rod-shaped cells, were non-motile and formed white-cream colonies. Phylogeny based on the 16S rRNA gene sequences revealed that MSKU 9T and MSKU 15 represented members of the genus Komagataeibacter and formed a monophyletic branch with K. swingsii JCM 17123T and K. europaeus DSM 6160T. The genomic analysis revealed that overall genomic relatedness index values of MSKU 9T with K. swingsii JCM 17123T and K. europaeus DSM 6160T were ~90 % average nucleotide identity (ANI) and ≤58.2 % digital DNA-DNA hybridization (dDDH), respectively. MSKU 9T and MSKU 15 can be differentiated from the closely related K. swingsii JCM 17123T by their growth on 30 % d-glucose and ability to utilize and to form acid from raffinose and sucrose as carbon sources, and from K. europaeus DSM 6160T by their ability to grow without acetic acid. The genomic DNA G+C contents of MSKU 9T and MSKU 15 were 60.4 and 60.2 mol%, respectively. The major fatty acids of MSKU 9T and MSKU 15 were summed feature 8 (C18 : 1 ω7c and/or C18  : 1ω6c). The respiratory quinone was determined to be Q10. On the basis of the results of the polyphasic taxonomic analysis, MSKU 9T (=TBRC 9844T=NBRC 113802T) represents a novel species of the genus Komagataeibacter, for which the name Komagataeibacter diospyri sp. nov. is proposed.