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Bacterial cellulose synthesis from defined media and waste products has attracted increasing interest in the circular economy context for sustainable productions. In this study, a glucose dehydrogenase-deficient Δgdh K2G30 strain of Komagataeibacter xylinus was obtained from the parental wild type through homologous recombination. Both strains were grown in defined substrates and cheese whey as an agri-food waste to assess the effect of gene silencing on bacterial cellulose synthesis and carbon source metabolism. Wild type K2G30 boasted higher bacterial cellulose yields when grown in ethanol-based medium and cheese whey, although showing an overall higher D-gluconic acid synthesis. Conversely, the mutant Δgdh strain preferred D-fructose, D-mannitol, and glycerol to boost bacterial cellulose production, while displaying higher substrate consumption rates and a lower D-gluconic acid synthesis. This study provides an in-depth investigation of two K. xylinus strains, unravelling their suitability for scale-up BC production., (© 2024. The Author(s).)

Nature is home to a variety of microorganisms that create materials under environmentally friendly conditions. While this offers an attractive approach for sustainable manufacturing, the production of materials by native microorganisms is usually slow and synthetic biology tools to engineer faster microorganisms are only available when prior knowledge of genotype-phenotype links is available. Here, we utilize a high-throughput directed evolution platform to enhance the fitness of whole microorganisms under selection pressure and identify genetic pathways to enhance the material production capabilities of native species. Using Komagataeibacter sucrofermentans as a model cellulose-producing microorganism, we show that our droplet-based microfluidic platform enables the directed evolution of these bacteria toward a small number of cellulose overproducers from an initial pool of 40,000 random mutants. Sequencing of the evolved strains reveals an unexpected link between the cellulose-forming ability of the bacteria and a gene encoding a protease complex responsible for protein turnover in the cell. The ability to enhance the fitness of microorganisms toward a specific phenotype and to unravel genotype-phenotype links makes this high-throughput directed evolution platform a promising tool for the development of new strains for the sustainable manufacturing of materials., Competing Interests: Competing interests statement:Two patent applications were submitted based on the results reported in this research.

Transovarial transmission is the most reliable way of passing on essential nutrient-providing endosymbionts from mothers to offspring. However, not all endosymbiotic microbes follow the complex path through the female host tissues to oocytes on their own. Here, we demonstrate an unusual transmission strategy adopted by one of the endosymbionts of the planthopper Trypetimorpha occidentalis (Hemiptera: Tropiduchidae) from Bulgaria. In this species, an Acetobacteraceae endosymbiont is transmitted transovarially within deep invaginations of cellular membranes of an ancient endosymbiont Sulcia -strikingly resembling recently described plant virus transmission. However, in males, Acetobacteraceae colonizes the same bacteriocytes as Sulcia but remains unenveloped. Then, the unusual endobacterial localization of Acetobacteraceae observed in females appears to be a unique adaptation to maternal transmission. Further, the symbiont's genomic features, including encoding essential amino acid biosynthetic pathways and its similarity to a recently described psyllid symbiont, suggest a unique combination of the ability to horizontally transmit among species and confer nutritional benefits. The close association with Acetobacteraceae symbiont correlates with the so-far-unreported level of genomic erosion of ancient nutritional symbionts of this planthopper. In Sulcia , this is reflected in substantial changes in genomic organization, reported for the first time in the symbiont renowned for its genomic stability. In Vidania , substantial gene loss resulted in one of the smallest genomes known, at 108.6 kb. Thus, the symbionts of T. occidentalis display a combination of unusual adaptations and genomic features that expand our understanding of how insect-microbe symbioses may transmit and evolve.IMPORTANCEReliable transmission across host generations is a major challenge for bacteria that associate with insects, and independently established symbionts have addressed this challenge in different ways. The facultatively endobacterial localization of Acetobacteraceae symbiont, enveloped by cells of ancient nutritional endosymbiont Sulcia in females but not males of the planthopper Trypetimorpha occidentalis , appears to be a unique adaptation to maternal transmission. Acetobacteraceae 's genomic features indicate its unusual evolutionary history, and the genomic erosion experienced by ancient nutritional symbionts demonstrates the apparent consequences of such close association. Combined, this multi-partite symbiosis expands our understanding of the diversity of strategies that insect symbioses form and some of their evolutionary consequences., Competing Interests: The authors declare no conflict of interest.

Bacterial cellulose (BC) is a renewable biomaterial that has attracted significant attention due to its excellent properties and wide applications. Komagataeibacter xylinus CGMCC 2955 is an important BC-producing strain. It primarily produces BC from glucose while simultaneously generating gluconic acid as a by-product, which acidifies the medium and inhibits BC synthesis. To enhance glucose uptake and BC synthesis, we reconstructed the phosphoenolpyruvate-dependent glucose phosphotransferase system (PTS Glc ) and strengthened glycolysis by introducing heterologous genes, resulting in a recombinant strain (GX08PTS03; Δgcd::ptsHIcrr E. coli ::ptsG E. coli ::pfkA E. coli ). Strain GX08PTS03 efficiently utilized glucose for BC production without accumulating gluconic acid. Subsequently, the fermentation process was systematically optimized. Under optimal conditions, strain GX08PTS03 produced 7.74 g/L of BC after 6 days of static fermentation, with a BC yield of 0.39 g/g glucose, which were 87.41 % and 77.27 % higher than those of the wild-type strain, respectively. The BC produced by strain GX08PTS03 exhibited a longer fiber diameter along with a lower porosity, significantly higher solid content, crystallinity, tensile strength, and Young's modulus. This study is novel in reporting that the engineered PTS Glc -based glucose metabolism could effectively enhance the production and properties of BC, providing a future outlook for the biopolymer industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bacterial cellulose (BC) is a biopolymer synthesized by bacteria, which possess excellent characteristics such as high water holding capacity, high crystallinity, and high purity. It is widely used in food, medical, cosmetics, and functional films. Komagataeibacter xylinus is a model strain used in BC synthesis research. In bacteria, motility-related genes are associated with BC synthesis, whereas in Komagataeibacter xylinus CGMCC 2955, the functions of motility-related genes and their effects on BC synthesis are not known. To address this gap, we used the λ Red recombinant system to individually knock out motA , motB , and mot2A respectively, and constructed the knockout strains K . x -Δ motA , K . x -Δ motB , and K . x -Δ mot2A . Additionally, both motA and motB were disrupted to construct the K . x -Δ motAB mutant. The results demonstrated that knockout strain K . x -Δ motAB exhibited the highest BC yield, reaching (5.05±0.26) g/L, which represented an increase of approximately 24% compared to wild-type strains. Furthermore, the BC synthesized by this strain exhibited the lowest porosity, 54.35%, and displayed superior mechanical properties with a Young's modulus of up to 5.21 GPa. As knocking out motA and motB genes in K . xylinus CGMCC 2955 did not reduce BC yield; instead, it promoted BC synthesis. Consequently, this research further deepened our understanding of the relationship between motility and BC synthesis in acetic acid bacteria. The knockouts of motA and motB genes resulted in reduced BC porosity and improved mechanical properties, provides a reference for BC synthesis and membrane structure regulation modification.

Importance: Acetobacteraceae are one of the best known and most extensively studied groups of bacteria, which nowadays encompasses a variety of taxa that are very different from the vinegar-producing species defining the family. Our paper presents the most detailed phylogeny of all current taxa classified as Acetobacteraceae , for which we propose a taxonomic revision. Several of such taxa inhabit some of the most extreme environments on the planet, from the deserts of Antarctica to the Sinai desert, as well as acidic niches in volcanic sites like the one we have been studying in Patagonia. Our work documents the progressive variation of the respiratory chain in early branching Acetobacteraceae into the different respiratory chains of acidophilic taxa such as Acidocella and acetous taxa such as Acetobacter . Remarkably, several genomes retain remnants of ancestral photosynthetic traits and functional bc 1 complexes. Thus, we propose that the common ancestor of Acetobacteraceae was photosynthetic., Competing Interests: The authors declare no conflict of interest.

Bacterial cellulose (BC) is a biopolymer principally synthetized by strains of the genus Komagataeibacter. However, high costs and low production yield make large-scale application difficult. The aim of this work was to evaluate the effects of successive batch culture before fermentation on the ability to increase the capacity of bacterial cellulose biosynthesis by a low-producing strain. The Komagataeibacter hansenii strain ATCC 23,769 was initially cultivated in fermentation broth for two periods of 35 or 56 days under static conditions. At the end of each period of time, they were transferred to new broth to be cultivated again (new batch culture cycle) for 35 or 56 days and carried out in parallel with a 10-day fermentation to determine the quantity of BC produced. As a result, a greater increase was observed after the end of the second and third batch cultures of 56 days (increases of 137% and 187% in relation to the nonbatch cultured strain, respectively). The produced samples presented higher crystallinity and thermal properties but lower water holding capacity. Through this work, it was concluded that the longer the batch culture time was, the greater the increase in the capacity of cellulose biosynthesis, which also depended on the number of successive batch culture cycles carried out., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

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., (© 2023. The Author(s).)

Bacterial nanocellulose (BC) is a highly versatile biopolymer currently pursued as a material of choice in varied themes of biomedical and material science research fields. With the aim to extend the biotechnological applications, the genetic tractability of the BC producers within the Komagataeibacter genus and its potential as an alternative host chassis in synthetic biology have been extensively studied. However, such studies have been largely focused on the model Komagataeibacter spp. Here, we present a novel K. intermedius strain capable of utilizing glucose, and glycerol sources for biomass and BC synthesis. Genome assembly identified one bacterial cellulose synthetase (bcs) operon containing the complete gene set encoding the BC biogenesis machinery (bcsI) and three additional copies (bcsII-IV). Investigations on the genetic tractability confirmed plasmid transformation, propagation of vectors with pBBR1 and p15A origin of replications and constitutive and inducible induction of recombinant protein in K. intermedius ENS15. This study provides the first report on the genetic tractability of K. intermedius, serving as starting point towards future genetic engineering of this strain., (© 2022. The Author(s).)

Acetic acid bacteria (AAB) are a group of gram-negative, obligate aerobic bacteria within the Acetobacteraceae family of the alphaproteobacteria class, which are distributed in a wide variety of different natural sources that are rich in sugar and alcohols, as well as in several traditionally fermented foods. Their versatile capabilities are not limited to producing acetic acid and brewing vinegar, as their names suggest. They can also be used for fixing nitrogen, yielding pigments and exopolysaccharides (EPS), and most typically, producing a variety of aldehydes, ketones and other organic acids from the incomplete oxidation of the corresponding alcohols and/or sugars (also referred to as oxidative fermentation). In order to gain more insight into these organisms, molecular biology techniques have been extensively applied in almost all aspects of AAB research, including their identification and classification, acid resistance mechanisms, oxidative fermentation, EPS production, thermotolerance and so on. In this review, we mainly focus on the application of molecular biological technologies in the advancement of research into AAB while presenting the progress of the latest studies using these techniques., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In this study, a novel bacterium designated F3b2 T was isolated from the gut sample of weaver ant Oecophylla smaragdina and characterised. Strain F3b2 T was a Gram-negative, aerobic, non-motile, ovoid-shaped bacterium and grows optimally at 28-30 °C. Its major respiratory quinone is ubiquinone 10 (Q-10) and the major fatty acids are C 18:1 ω7c, C 19:0 cyclo ω8c and C 16:0 , representing 85% of the total fatty acids. The 16S rRNA gene sequence of strain F3b2 T was highest in similarity to that of Oecophyllibacter saccharovorans DSM106907 T and Swingsia samuieinsis NBRC 107927 T at 94.35% and 91.96%, respectively. A 16S rRNA gene-based phylogenetic analysis and a core genes-based phylogenomic analysis placed strain F3b2 T in a distinct lineage in the family Acetobacteraceae. The phylogenetic placement was supported by lower than species delineation threshold average nucleotide identity (ANI) (≤ 70.2%), in silico DNA-DNA hybridization (DDH) (≤ 39.5%) and average amino acid identity (AAI) (≤ 63.5%) values between strain F3b2 T and closest neighbours. These overall genome relatedness indices also supported the assignment of strain F3b2 T to a novel genus within Acetobacteraceae. The genome of strain F3b2 T was 1.96 Mb with 60.4% G + C DNA content. Based on these results, strain F3b2 T represented a novel taxon of Acetobacteraceae, for which we proposed the name Formicincola oecophyllae gen. nov. sp. nov., and strain F3b2 T (= LMG 30590 T  = DSM 106908 T  = NBRC 113640 T  = KCTC 62951 T ) as the type strain., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

A novel Gram-stain-negative, non-motile, ellipsoidal-shaped, red-pigmented, facultatively aerobic strain designated NE82 T was isolated from mud sample from Jiugongli Lake in Inner Mongolia Autonomous Region, China. Optimal growth occurred at 28-33 °C (range 15-42 °C) and pH 7.0-7.5 (range 5.5-8.5) with 0% (w/v) NaCl (range 0-1.0%). Cells of strain NE82 T were 0.4-0.9 μm in diameter, catalase-positive and oxidase-negative. Q-10 was the sole respiratory quinone and the major cellular fatty acids (> 10%) in strain NE82 T were summed feature 8 (C 18:1 ω7c and C 18:1 ω6c). The polar lipids of strain NE82 T were phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, an unidentified aminophospholipid and four unidentified phospholipids. The G+C content of the genomic DNA was 72.0 mol%. Based on the 16S rRNA gene sequence, strain NE82 T showed the highest similarity (97.2%) to Roseicella frigidaeris DB1506 T within the family Acetobacteraceae, thus representing a novel species of the genus Roseicella, for which the name Roseicella aquatilis sp. nov. is proposed. The type strain is NE82 T (= KCTC 62412 T  = MCCC 1H00292 T )., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A Gram-stain-negative, cocci-to-oval-shaped bacterial strain, designated XZZS9 T , 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 T was related to members of the genus Roseococcus, with the highest sequence identity to Roseococcus microcysteis NIBR12 T (96.9%). The major cellular fatty acids (> 5% of the total) were C 18:1 ω7c and C 19:0 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 T 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 T , suggesting a possible lack of photosynthetic reaction. Based on genotypic and phenotypic characteristics, strain XZZS9 T represents a novel species of the genus Roseococcus, for which we propose the name Roseococcus pinisoli sp. nov. The type strain is XZZS9 T (= KCTC 82435 T  = JCM 34402 T  = GDMCC 1.2158 T )., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Acetobacteraceae is an economically important family of bacteria that is used for industrial fermentation in the food/feed sector and for the preparation of sorbose and bacterial cellulose. It comprises two major groups: acetous species (acetic acid bacteria) associated with flowers, fruits and insects, and acidophilic species, a phylogenetically basal and physiologically heterogeneous group inhabiting acid or hot springs, sludge, sewage and freshwater environments. Despite the biotechnological importance of the family Acetobacteraceae, the literature does not provide any information about its ability to produce specialized metabolites. We therefore constructed a phylogenomic tree based on concatenated protein sequences from 141 type strains of the family and predicted the presence of small-molecule biosynthetic gene clusters (BGCs) using the antiSMASH tool. This dual approach allowed us to associate certain biosynthetic pathways with particular taxonomic groups. We found that acidophilic and acetous species contain on average ~ 6.3 and ~ 3.4 BGCs per genome, respectively. All the Acetobacteraceae strains encoded proteins involved in hopanoid biosynthesis, with many also featuring genes encoding type-1 and type-3 polyketide and non-ribosomal peptide synthases, and enzymes for aryl polyene, lactone and ribosomal peptide biosynthesis. Our in silico analysis indicated that the family Acetobacteraceae is a potential source of many undiscovered bacterial metabolites and deserves more detailed experimental exploration., (© 2021. The Author(s).)

This work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the blood meals was achieved through recombinant DNA-tagged Asaia bacteria that successfully colonised Aedes and Anopheles mosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles, and neonicotinoids were enriched for compounds with systemic adulticide activity against Anopheles. Using a luminescent Plasmodium falciparum reporter strain, barcoded screens identified 48 drug-like transmission-blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects and identifies novel candidate small molecules for disease control., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: KJD holds stock in TropIQ Health Sciences.

A cellulose-producing bacterium Komagataeibacter rhaeticus K15 was isolated from kombucha tea, and its metabolic pathways and cellulose synthesis operon were analyzed by genome sequencing. Different from the reported K. rhaeticus, the K15 produced little gluconic acid (2.26 g/L) when glucose was the sole carbon source and has the capacity for high cellulose production (4.76 g/L) with other carbon sources. Furthermore, six nitrogen-fixing genes were found to be responsible for the survival of K15 on a nitrogen-free medium. Based on its fermentation characteristics, K15 was cultured in a kitchen waste medium as a strategy for green and sustainable bacterial cellulose production. The SEM, XRD, and FTIR results indicated that synthesized cellulose has a mean diameter of 40-50 nm nanofiber, good crystallinity, and the same chemical structure. The K15 strain provides a highly viable alternative strategy to reduce the costs of bacterial cellulose production using agro-industrial residues as nutrient sources., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 F 1 mosquitoes belonging to F 0 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., (© 2021. The Author(s).)

In the industrial production of high-acidity vinegar, the initial ethanol and acetic acid concentrations are limiting factors that will affect acetic acid fermentation. In this study, Komagataeibacter europaeus CGMCC 20445 was used for acetic acid shake flask fermentation at an initial ethanol concentration of 4.3% (v/v). We conducted transcriptome analysis of K. europaeus CGMCC 20445 samples under different acidity conditions to elucidate the changes in differentially expressed genes throughout the fermentation process. We also analyzed the expression of genes associated with acid-resistance mechanisms. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the differentially expressed genes were enriched in ribosomes, citrate cycle, butanoate metabolism, oxidative phosphorylation, pentose phosphate, and the fatty acid biosynthetic pathways. In addition, this study found that K. europaeus CGMCC 20445 regulates the gene expression levels of cell envelope proteins and stress-responsive proteins to adapt to the gradual increase in acidity during acetic acid fermentation. This study improved the understanding of the acid resistance mechanism of K. europaeus and provided relevant reference information for the further genetic engineering of this bacterium., Competing Interests: Conflict of interest The authors do not report any financial or personal connections with other persons or organizations, which might negatively affect the contents of this publication and/or claim authorship rights to this publication., (© 2021 Liting Wang et al.)

Honey bees are important pollinators of many major crops and add billions of dollars annually to the US economy through their services. Recent declines in the health of the honey bee have startled researchers and lay people alike as honey bees are agriculture's most important pollinator. One factor that may influence colony health is the microbial community. Although honey bee worker guts have a characteristic community of bee-specific microbes, the honey bee queen digestive tracts are colonized predominantly by a single acetic acid bacterium tentatively named ' Parasaccharibacter apium '. This bacterium is related to flower-associated microbes such as Saccharibacter floricola , and initial phylogenetic analyses placed it as sister to these environmental bacteria. We used a combination of phylogenetic and sequence identity methods to better resolve evolutionary relationships among ' P. apium ', strains in the genus Saccharibacter , and strains in the closely related genus Bombella . Interestingly, measures of genome-wide average nucleotide identity and aligned fraction, coupled with phylogenetic placement, indicate that many strains labelled as ' P. apium ' and Saccharibacter species are all the same species as Bombella apis . We propose reclassifying these strains as Bombella apis and outline the data supporting that classification below.

Vinegar is elaborated using a semi-continuous submerged culture of a complex microbiota of acetic acid bacteria. The genus Komagataeibacter provides much of the proteins of the metaproteome, being K. europaeus the main species working in this environment. In this work, the protein profile of the vinegar microbiota, obtained by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in samples from different cycle times of an acetification process using an alcohol medium, has been used to describe the functional metaproteome throughout the process. The analysis was focused on Komagataeibacter species which supplied about 90% of the metaproteome and particularly K. europaeus which accounts for more than 70%. According to these results, the natural behaviour of a microbial community in vinegar has been predicted at a quantitative proteomic level. The results revealed that most of the identified proteins involved in the metabolism of amino acids, biosynthesis of proteins, and energy production related-metabolic pathways increased their expression throughout the cycle loading phase and afterwards experimented a decrease coming into play other proteins acting against acetic acid stress. These findings may facilitate a better understanding of the microbiota's role and contributing to obtain a quality product., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

During acetic acid fermentation, acetic acid bacteria face oxygen depletion stress caused by the vigorous oxidation of ethanol to acetic acid. However, the molecular mechanisms underlying the response to oxygen depletion stress remain largely unknown. Here, we focused on an oxygen-sensing FNR homolog, FnrG, in Komagataeibacter medellinensis. Comparative transcriptomic analysis between the wild-type and fnrG-disrupted strains revealed that FnrG upregulated 8 genes (fold change >3). Recombinant FnrG bound to a specific DNA sequence only when FnrG was reconstituted anaerobically. An operon consisting of acetate kinase and xylulose-5-phosphate/fructose-6-phosphate phosphoketolase genes was found to be an FnrG regulon involved in cell survival under oxygen-limiting conditions. Moreover, a strain that overexpressed these 2 genes accumulated more acetic acid than the wild-type strain harboring an empty vector. Thus, these 2 genes could be new targets for the molecular breeding of acetic acid bacteria with high acetic acid productivity., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

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 F 1 F o 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. IMPORTANCE Saccharibacteria 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.

Acetic acid bacteria grow while producing acetic acid, resulting in acidification of the culture. Limited reports elucidate the effect of changes in intracellular pH on transcriptional factors. In the present study, the intracellular pH of Komagataeibacter europaeus was monitored with a pH-sensitive green fluorescent protein, showing that the intracellular pH decreased from 6.3 to 4.7 accompanied by acetic acid production during cell growth. The leucine-responsive regulatory protein of K. europaeus ( Ke Lrp) was used as a model to examine pH-dependent effects, and its properties were compared with those of the Escherichia coli ortholog ( Ec Lrp) at different pH levels. The DNA-binding activities of Ec Lrp and Ke Lrp with the target DNA ( Ec-ilvI and Ke-ilvI ) were examined by gel mobility shift assays under various pH conditions. Ec Lrp showed the highest affinity with the target at pH 8.0 ( K d [dissociation constant], 0.7 μM), decreasing to a minimum of 3.4 μM at pH 4.0. Conversely, Ke Lrp did not show significant differences in binding affinity between pH 4 and 7 ( K d , 1.0 to 1.5 μM), and the highest affinity was at pH 5.0 ( K d , 1.0 μM). Circular dichroism spectroscopy revealed that the α-helical content of Ke Lrp was the highest at pH 5.0 (49%) and was almost unchanged while being maintained at >45% over a range of pH levels examined, while that of Ec Lrp decreased from its maximum (49% at pH 7.0) to its minimum (36% at pH 4.0). These data indicate that Ke Lrp is stable and functions over a wide range of intracellular pH levels. IMPORTANCE Lrp is a highly conserved transcriptional regulator found in bacteria and archaea and regulates transcriptions of various genes. The intracellular pH of acetic acid bacteria (AAB) changes accompanied by acetic acid production during cell growth. The Lrp of AAB K. europaeus ( Ke Lrp) was structurally stable over a wide range of pH and maintained DNA-binding activity even at low pH compared with Lrp from E. coli living in a neutral environment. An in vitro experiment showed DNA-binding activity of Ke Lrp to the target varied with changes in pH. In AAB, change of the intracellular pH during a cell growth would be an important trigger in controlling the activity of Lrp in vivo .

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 Zn 2+ 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.

Asaia bacteria commonly comprise part of the microbiome of many mosquito species in the genera Anopheles and Aedes , including important vectors of infectious agents. Their close association with multiple organs and tissues of their mosquito hosts enhances the potential for paratransgenesis for the delivery of antimalaria or antivirus effectors. The molecular mechanisms involved in the interactions between Asaia and mosquito hosts, as well as Asaia and other bacterial members of the mosquito microbiome, remain underexplored. Here, we determined the genome sequence of Asaia strain W12 isolated from Anopheles stephensi mosquitoes, compared it to other Asaia species associated with plants or insects, and investigated the properties of the bacteria relevant to their symbiosis with mosquitoes. The assembled genome of strain W12 had a size of 3.94 MB, the largest among Asaia spp. studied so far. At least 3585 coding sequences were predicted. Insect-associated Asaia carried more glycoside hydrolase (GH)-encoding genes than those isolated from plants, showing their high plant biomass-degrading capacity in the insect gut. W12 had the most predicted regulatory protein components comparatively among the selected Asaia , indicating its capacity to adapt to frequent environmental changes in the mosquito gut. Two complete operons encoding cytochrome bo 3 -type ubiquinol terminal oxidases ( cyoABCD-1 and cyoABCD-2 ) were found in most Asaia genomes, possibly offering alternative terminal oxidases and allowing the flexible transition of respiratory pathways. Genes involved in the production of 2,3-butandiol and inositol have been found in Asaia sp. W12, possibly contributing to biofilm formation and stress tolerance.

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Biological systems assemble living materials that are autonomously patterned, can self-repair and can sense and respond to their environment. The field of engineered living materials aims to create novel materials with properties similar to those of natural biomaterials using genetically engineered organisms. Here, we describe an approach to fabricating functional bacterial cellulose-based living materials using a stable co-culture of Saccharomyces cerevisiae yeast and bacterial cellulose-producing Komagataeibacter rhaeticus bacteria. Yeast strains can be engineered to secrete enzymes into bacterial cellulose, generating autonomously grown catalytic materials and enabling DNA-encoded modification of bacterial cellulose bulk properties. Alternatively, engineered yeast can be incorporated within the growing cellulose matrix, creating living materials that can sense and respond to chemical and optical stimuli. This symbiotic culture of bacteria and yeast is a flexible platform for the production of bacterial cellulose-based engineered living materials with potential applications in biosensing and biocatalysis.

Acetic acid bacteria (AAB) are valuable biocatalysts for which there is growing interest in understanding their basics including physiology and biochemistry. This is accompanied by growing demands for metabolic engineering of AAB to take advantage of their properties and to improve their biomanufacturing efficiencies. Controlled expression of target genes is key to fundamental and applied microbiological research. In order to get an overview of expression systems and their applications in AAB, we carried out a comprehensive literature search using the Web of Science Core Collection database. The Acetobacteraceae family currently comprises 49 genera. We found overall 6097 publications related to one or more AAB genera since 1973, when the first successful recombinant DNA experiments in Escherichia coli have been published. The use of plasmids in AAB began in 1985 and till today was reported for only nine out of the 49 AAB genera currently described. We found at least five major expression plasmid lineages and a multitude of further expression plasmids, almost all enabling only constitutive target gene expression. Only recently, two regulatable expression systems became available for AAB, an N-acyl homoserine lactone (AHL)-inducible system for Komagataeibacter rhaeticus and an L-arabinose-inducible system for Gluconobacter oxydans. Thus, after 35 years of constitutive target gene expression in AAB, we now have the first regulatable expression systems for AAB in hand and further regulatable expression systems for AAB can be expected. KEY POINTS: • Literature search revealed developments and usage of expression systems in AAB. • Only recently 2 regulatable plasmid systems became available for only 2 AAB genera. • Further regulatable expression systems for AAB are in sight.

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., (Copyright © 2021 Comandatore et al.)

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.

In this study, bacterial strains Ha5 T , 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 C 18:1 ω7c, C 16:0 , C 19:0 ω8c cyclo, C 14:0 , and C 16:0 2-OH. Strains Ha5 T , Ta1, and Jb2 shared highest 16S rRNA gene sequence similarity (94.56-94.63%) with Neokomagataea tanensis NBRC106556 T 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 Ha5 T , 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 Ha5 T as the type strain.

Leishmaniases are severe vector-borne diseases affecting humans and animals, caused by Leishmania protozoans. Over one billion people and millions of dogs live in endemic areas for leishmaniases and are at risk of infection. Immune polarization plays a major role in determining the outcome of Leishmania infections: hosts displaying M1-polarized macrophages are protected, while those biased on the M2 side acquire a chronic infection that could develop into a deadly disease. The identification of the factors involved in M1 polarization is essential for the design of therapeutic and prophylactic interventions, including vaccines. Infection by the filarial nematode Dirofilaria immitis could be one of the factors that interfere with leishmaniasis in dogs. Indeed, filarial nematodes induce a partial skew of the immune response towards M1, likely caused by their bacterial endosymbionts, Wolbachia. Here we have examined the potential of Asaia WSP , a bacterium engineered for the expression of the Wolbachia surface protein (WSP), as an inductor of M1 macrophage activation and Leishmania killing. Macrophages stimulated with Asaia WSP displayed a strong leishmanicidal activity, comparable to that determined by the choice-drug amphotericin B. Additionally, Asaia WSP determined the expression of markers of classical macrophage activation, including M1 cytokines, ROS and NO, and an increase in phagocytosis activity. Asaia not expressing WSP also induced macrophage activation, although at a lower extent compared to Asaia WSP . In summary, the results of the present study confirm the immunostimulating properties of WSP highlighting a potential therapeutic efficacy against Leishmania parasites. Furthermore, Asaia was designed as a delivery system for WSP, thus developing a novel type of immunomodulating agent, worthy of being investigated for immuno-prophylaxis and -therapy of leishmaniases and other diseases that could be subverted by M1 macrophage activation., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Recent declines in the health of the honey bee have startled researchers and lay people alike as honey bees are agriculture's most important pollinator. Honey bees are important pollinators of many major crops and add billions of dollars annually to the US economy through their services. One factor that may influence colony health is the microbial community. Indeed, the honey bee worker digestive tract harbors a characteristic community of bee-specific microbes, and the composition of this community is known to impact honey bee health. However, the honey bee is a superorganism, a colony of eusocial insects with overlapping generations where nestmates cooperate, building a hive, gathering and storing food, and raising brood. In contrast to what is known regarding the honey bee worker gut microbiome, less is known of the microbes associated with developing brood, with food stores, and with the rest of the built hive environment. More recently, the microbe Bombella apis was identified as associated with nectar, with developing larvae, and with honey bee queens. This bacterium is related to flower-associated microbes such as Saccharibacter floricola and other species in the genus Saccharibacter, and initial phylogenetic analyses placed it as sister to these environmental bacteria. Here, we used comparative genomics of multiple honey bee-associated strains and the nectar-associated Saccharibacter to identify genomic changes that may be associated with the ecological transition to honey bee association. We identified several genomic differences in the honey bee-associated strains, including a complete CRISPR/Cas system. Many of the changes we note here are predicted to confer upon Bombella the ability to survive in royal jelly and defend themselves against mobile elements, including phages. Our results are a first step toward identifying potential function of this microbe in the honey bee superorganism., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Some animals, such as planaria, can regenerate complex anatomical structures in a process regulated by genetic and biophysical factors, but additional external inputs into regeneration remain to be uncovered. Microbial communities inhabiting metazoan organisms are important for metabolic, immune, and disease processes, but their instructive influence over host structures remains largely unexplored. Here, we show that Aquitalea sp. FJL05, an endogenous commensal bacterium of Dugesia japonica planarians, and one of the small molecules it produces, indole, can influence axial and head patterning during regeneration, leading to regeneration of permanently two-headed animals. Testing the impact of indole on planaria tissues via RNA sequencing, we find that indole alters the regenerative outcomes in planarians through changes in expression to patterning genes, including a downregulation of Wnt pathway genes. These data provide a unique example of the product of a commensal bacterium modulating transcription of patterning genes to affect the host's anatomical structure during regeneration., Competing Interests: Declaration of competing interest We declare that none of the authors have competing financial or non-financial interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The strains of the Komagataeibacter genus have been shown to be the most efficient bacterial nanocellulose producers. Although exploited for many decades, the studies of these species focused mainly on the optimisation of cellulose synthesis process through modification of culturing conditions in the industrially relevant settings. Molecular physiology of Komagataeibacter was poorly understood and only a few studies explored genetic engineering as a strategy for strain improvement. Only since recently the systemic information of the Komagataeibacter species has been accumulating in the form of omics datasets representing sequenced genomes, transcriptomes, proteomes and metabolomes. Genetic analyses of the mutants generated in the untargeted strain modification studies have drawn attention to other important proteins, beyond those of the core catalytic machinery of the cellulose synthase complex. Recently, modern molecular and synthetic biology tools have been developed which showed the potential for improving targeted strain engineering. Taking the advantage of the gathered knowledge should allow for better understanding of the genotype-phenotype relationship which is necessary for robust modelling of metabolism as well as selection and testing of new molecular engineering targets. In this review, we discuss the current progress in the area of Komagataeibacter systems biology and its impact on the research aimed at scaled-up cellulose synthesis as well as BNC functionalisation. Key points • The accumulated omics datasets advanced the systemic understanding of Komagataeibacter physiology at the molecular level. • Untargeted and targeted strain modification approaches have been applied to improve nanocellulose yield and properties. • The development of modern molecular and synthetic biology tools presents a potential for enhancing targeted strain engineering. • The accumulating omic information should improve modelling of Komagataeibacter's metabolism as well as selection and testing of new molecular engineering targets.

The first cultivated representative of the enigmatic phylum Saccharibacteria (formerly TM7) was isolated from humans and revealed an ultra-small cell size (200-300 nm), a reduced genome with limited biosynthetic capabilities, and a unique parasitic lifestyle. TM7x was the only cultivated member of the candidate phyla radiation (CPR), estimated to encompass 26% of the domain Bacteria. Here we report on divergent genomes from major lineages across the Saccharibacteria phylum in humans and mammals, as well as from ancient dental calculus. These lineages are present at high prevalence within hosts. Direct imaging reveals that all groups are ultra-small in size, likely feeding off commensal bacteria. Analyses suggest that multiple acquisition events in the past led to the current wide diversity, with convergent evolution of key functions allowing Saccharibacteria from the environment to adapt to mammals. Ultra-small, parasitic CPR bacteria represent a relatively unexplored paradigm of prokaryotic interactions within mammalian microbiomes., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bacterial cellulose(BC) is a kind of extracellular polymer synthesized by bacteria and it has very wide applications in many fields. However, the application of BC in a large commercial scale can still not be fulfilled due to the low yield and demanding for BC membranes with very different properties. To this end, a new BC-producer Komagataeibacter rhaeticus TJPU03 was isolated from rotten orange peel, which produced 8.28 ± 0.27 g/L(dry weight) in standard HS medium at the 10th day. The membrane is easier to be purified by one-step alkaline treatment and the produced BC(K-BC) membranes possess homogeneous, looser and more porous three-dimensional network composed by thinner cellulose fibrils. However, the wet K-BC possesses stronger mechanical properties and exhibits lower toxicity and higher cytocompatibility to mammalian cell. Owing to the more porous and homogeneous network, K-BC possesses high loading capacity of cell and protein drugs. Also, it exhibits sustained-controlled release ability for proteinaceous drug. The high yield of this strain and the special characteristics of K-BC predict this strain to be a very promising BC-producer and broad applications of K-BC in the fields of wound healing, scaffolds of tissue engineering, tissue repair and regeneration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In this study, a comprehensive genome-scale metabolic network of Komagataeibacter xylinus as the model microorganism was reconstructed based on genome annotation, for better understanding of metabolic growth and biosynthesis of bacterial cellulose (BC). The reconstructed network included 640 genes, 783 metabolic reactions and 865 metabolites. The model was completely successful to predict the lack of growth under anaerobic conditions. Model validation by the data for the growth of acetic acid bacteria with ethanol-limited chemostat cultures showed that there is a good agreement for the O 2 and CO 2 fluxes with actual growth conditions. Then the model was used to forecast the simultaneous production of BC and by-products. The obtained data showed that the rate of BC production is consistent with experimental data with an accuracy of 93.7%. Finally, the study of flux balance analysis (FBA) data showed that the pentose phosphate pathway and the TCA cycle play an important role in growth-promoting metabolism in K. xylinus and have a close relationship with BC biosynthesis. By integrating this model with various metabolic engineering and systems biology tools in the future, it is possible to overcome the common challenges in the large-scale BC production, such as low yield and productivity.

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 1515 T 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.

Wolbachia can reduce the capability of mosquitoes to transmit infectious diseases to humans and is currently exploited in campaigns for the control of arboviruses, like dengue and Zika. Under the assumption that Wolbachia-mediated activation of insect immunity plays a role in the reduction of mosquito vectorial capacity, we focused our attention on the Wolbachia surface protein (WSP), a potential inductor of innate immunity. We hypothesized that the heterologous expression of this protein in gut- and tissue-associated symbionts may reduce parasite transmission. We thus engineered the mosquito bacterial symbiont Asaia to express WSP (Asaia WSP ). Asaia WSP induced activation of the host immune response in Aedes aegypti and Anopheles stephensi mosquitoes, and inhibited the development of the heartworm parasite Dirofilaria immitis in Ae. aegypti. These results consolidate previous evidence on the immune-stimulating property of WSP and make Asaia WSP worth of further investigations as a potential tool for the control of mosquito-borne diseases.

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.

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.

Nonsense mutation in the bcsC gene occurred in the ethanol-adapted strain of Komagataeibacter oboediens MSKU 3, E3 strain, resulting in the loss of the function to produce BNC. In this study, we tried to restore the BNC-producing ability of E3 strain by the following adaptive mutation through repetitive static culture, and obtained four BNC-producing revertant strains, of which the bcsC gene had InDel mutations near the frameshift mutation region in E3 strain, resulting in several amino acid alterations compared with the BcsC of MSKU 3. Each revertant produced BNCs with different productivity on the static culture. Interestingly, one of the revertants, R37-9, produced BNC with a finer structure and narrower range of fibrils width, compared to others. The genome of R37-9 strain revealed only one amino acid substitution in the bcsC gene. Thus, we concluded that N713D mutation occurred in the bcsC gene is responsible for the finer fibrils structure., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Komagataeibacter hansenii HDM1-3 (K. hansenii HDM1-3) has been widely applied for producing bacterial cellulose (BC). The yield of BC has been frequently limited by the acidification during sugar metabolism, due to the generation of organic acids such as acetic acid. In this study, the acid resistance mechanism of K. hansenii HDM1-3 has been investigated from the aspect of metabolic adaptability of cell membrane fatty acids. Firstly, we observed that the survival rate of K. hansenii HDM1-3 was decreased with lowered pH values (adjusted with acetic acids), accompanied by increased leakage rate. Secondly, the cell membrane adaptability in response to acid stress was evaluated, including the variations of cell membrane fluidity and fatty acid composition. The proportion of unsaturated fatty acids was increased (especially, C18-1w9c and C19-Cyc), unsaturation degree and chain length of fatty acids were also increased. Thirdly, the potential molecular regulation mechanism was further elucidated. Under acid stress, the fatty acid synthesis pathway was involved in the structure and composition variations of fatty acids, which was proved by the activation of both fatty acid dehydrogenase (des) and cyclopropane fatty acid synthase (cfa) genes, as well as the addition of exogenous fatty acids. The fatty acid synthesis of K. hansenii HDM1-3 may be mediated by the activation of two-component sensor signaling pathways in response to the acid stress. The acid resistance mechanism of K. hansenii HDM1-3 adds to our knowledge of the acid stress adaptation, which may facilitate the development of new strategies for improving the industrial performance of this species under acid stress.

Enzymes from natural sources protect the environment via complex biological mechanisms, which aid in reductive immobilization of toxic metals including chromium. Nevertheless, progress was being made in elucidating high-resolution crystal structures of reductases and their binding with flavin mononucleotide (FMN) to understand the underlying mechanism of chromate reduction. Therefore, herein, we employed molecular dynamics (MD) simulations, principal component analysis (PCA), and binding free energy calculations to understand the dynamics behavior of these enzymes with FMN. Six representative chromate reductases in monomeric and dimeric forms were selected to study the mode, dynamics, and energetic component that drive the FMN binding process. As evidenced by MD simulation, FMN prefers to bind the cervix formed between the catalytic domain surrounded by strong conserved hydrogen bonding, electrostatic, and hydrophobic contacts. The slight movement and reorientation of FMN resulted in breakage of some crucial H-bonds and other nonbonded contacts, which were well compensated with newly formed H-bonds, electrostatic, and hydrophobic interactions. The critical residues aiding in tight anchoring of FMN within dimer were found to be strongly conserved in the bacterial system. The molecular mechanics combined with the Poisson-Boltzmann surface area binding free energy of the monomer portrayed that the van der Waals and electrostatic energy contribute significantly to the total free energy, where, the polar solvation energy opposes the binding of FMN. The proposed proximity relationships between enzyme and FMN binding site presented in this study will open up better avenues to engineer enzymes with optimized chromate reductase activity for sustainable bioremediation of heavy metals., (© 2019 Wiley Periodicals, Inc.)

Bacterial nanocellulose (BNC) produced by Komagataeibacter hansenii has received significant attention due to its unique supernetwork structure and properties. It is nevertheless necessary to modify bacterial nanocellulose to achieve materials with desired properties and thus with broader areas of application. The aim here was to influence the 3D structure of BNC by genetic modification of the cellulose producing K. hansenii strain ATCC 53582. Two genes encoding proteins with homology to the MotA and MotB proteins, which participate in motility and energy transfer, were selected for our studies. A disruption mutant of one or both genes and their respective complementation mutants were created. The phenotype analysis of the disruption mutants showed a reduction in motility, which resulted in higher compaction of nanocellulose fibers and improvement in their mechanical properties. The data strongly suggest that these genes play an important role in the formation of BNC membrane by Komagataeibacter species., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Bacterial cellulose is a strong and flexible biomaterial produced at high yields by Acetobacter species and has applications in health care, biotechnology and electronics. Naturally, bacterial cellulose grows as a large unstructured polymer network around the bacteria that produce it, and tools to enable these bacteria to respond to different locations are required to grow more complex structured materials. Here, we introduce engineered cell-to-cell communication into a bacterial cellulose-producing strain of Komagataeibacter rhaeticus to enable different cells to detect their proximity within growing material and trigger differential gene expression in response. Using synthetic biology tools, we engineer Sender and Receiver strains of K. rhaeticus to produce and respond to the diffusible signalling molecule, acyl-homoserine lactone. We demonstrate that communication can occur both within and between growing pellicles and use this in a boundary detection experiment, where spliced and joined pellicles sense and reveal their original boundary. This work sets the basis for synthetic cell-to-cell communication within bacterial cellulose and is an important step forward for pattern formation within engineered living materials., (© 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Komagataeibacter medellinensis ID13488 (formerly Gluconacetobacter medellinensis ID13488) is able to produce crystalline bacterial cellulose (BC) under high acidic growth conditions. These abilities make this strain desirable for industrial BC production from acidic residues (e.g. wastes generated from cider production). To explore the molecular bases of the BC biosynthesis in this bacterium, the genome has been sequenced revealing a sequence of 3.4 Mb containing three putative plasmids of 38.1 kb (pKM01), 4.3 kb (pKM02) and 3.3 Kb (pKM03). Genome comparison analyses of K. medellinensis ID13488 with other cellulose-producing related strains resulted in the identification of the bcs genes involved in the cellulose biosynthesis. Genes arrangement and composition of four bcs clusters (bcs1, bcs2, bcs3 and bcs4) was studied by RT-PCR, and their organization in four operons transcribed as four independent polycistronic mRNAs was determined. qRT-PCR experiments demonstrated that mostly bcs1 and bcs4 are expressed under BC production conditions, suggesting that these operons direct the synthesis of BC. Genomic differences with the close related strain K. medellinensis NBRC 3288 unable to produce BC were also described and discussed., (© 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Bacterial nanocellulose (BNC) synthesized by Komagataeibacter hansenii is a polymer that recently gained an attention of tissue engineers, since its features make it a suitable material for scaffolds production. Nevertheless, it is still necessary to modify BNC to improve its properties in order to make it more suitable for biomedical use. One approach to address this issue is to genetically engineer K. hansenii cells towards synthesis of BNC with modified features. One of possible ways to achieve that is to influence the bacterial movement or cell morphology. In this paper, we described for the first time, K. hansenii ATCC 23769 motA+ and motB+ overexpression mutants, which displayed elongated cell phenotype, increased motility, and productivity. Moreover, the mutant cells produced thicker ribbons of cellulose arranged in looser network when compared to the wild-type strain. In this paper, we present a novel development in obtaining BNC membranes with improved properties using genetic engineering tools.

Background: Symbiotic relationships between animals and bacteria have profound impacts on the evolutionary trajectories of each partner. Animals and gut bacteria engage in a variety of relationships, occasionally persisting over evolutionary timescales. Ants are a diverse group of animals that engage in many types of associations with taxonomically distinct groups of bacterial associates. Here, we bring into culture and characterize two closely-related strains of gut associated Acetobacteraceae (AAB) of the red carpenter ant, Camponotus chromaiodes., Results: Genome sequencing, assembly, and annotation of both strains delineate stark patterns of genomic erosion and sequence divergence in gut associated AAB. We found widespread horizontal gene transfer (HGT) in these bacterial associates and report elevated gene acquisition associated with energy production and conversion, amino acid and coenzyme transport and metabolism, defense mechanisms, and lysine export. Both strains have acquired the complete NADH-quinone oxidoreductase complex, plausibly from an Enterobacteriaceae origin, likely facilitating energy production under diverse conditions. Conservation of several lysine biosynthetic and salvage pathways and accumulation of lysine export genes via HGT implicate L-lysine supplementation by both strains as a potential functional benefit for the host. These trends are contrasted by genome-wide erosion of several amino acid biosynthetic pathways and pathways in central metabolism. We perform phylogenomic analyses on both strains as well as several free living and host associated AAB. Based on their monophyly and deep divergence from other AAB, these C. chromaiodes gut associates may represent a novel genus. Together, our results demonstrate how extensive horizontal transfer between gut associates along with genome-wide deletions leads to mosaic metabolic pathways. More broadly, these patterns demonstrate that HGT and genomic erosion shape metabolic capabilities of persistent gut associates and influence their genomic evolution., Conclusions: Using comparative genomics, our study reveals substantial changes in genomic content in persistent associates of the insect gastrointestinal tract and provides evidence for the evolutionary pressures inherent to this environment. We describe patterns of genomic erosion and horizontal acquisition that result in mosaic metabolic pathways. Accordingly, the phylogenetic position of both strains of these associates form a divergent, monophyletic clade sister to gut associates of honey bees and more distantly to Gluconobacter.