The phylogeny of Acetobacteraceae: photosynthetic traits and deranged respiratory enzymes.

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.

Analysis of cellulose synthesis in a high-producing acetic acid bacterium Komagataeibacter hansenii.

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.

Bacterial cellulose production by Komagataeibacter hansenii can be improved by successive batch culture.

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.

Characterization, genome analysis and genetic tractability studies of a new nanocellulose producing Komagataeibacter intermedius isolate.

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.

Formicincola oecophyllae gen. nov. sp. nov., a novel member of the family Acetobacteraceae isolated from the weaver ant Oecophylla smaragdina.

In this study, a novel bacterium designated F3b2T was isolated from the gut sample of weaver ant Oecophylla smaragdina and characterised. Strain F3b2T 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 C18:1 ω7c, C19:0 cyclo ω8c and C16:0, representing 85% of the total fatty acids. The 16S rRNA gene sequence of strain F3b2T was highest in similarity to that of Oecophyllibacter saccharovorans DSM106907T and Swingsia samuieinsis NBRC 107927T at 94.35% and 91.96%, respectively. A 16S rRNA gene-based phylogenetic analysis and a core genes-based phylogenomic analysis placed strain F3b2T 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 F3b2T and closest neighbours. These overall genome relatedness indices also supported the assignment of strain F3b2T to a novel genus within Acetobacteraceae. The genome of strain F3b2T was 1.96 Mb with 60.4% G + C DNA content. Based on these results, strain F3b2T represented a novel taxon of Acetobacteraceae, for which we proposed the name Formicincola oecophyllae gen. nov. sp. nov., and strain F3b2T (= LMG 30590T = DSM 106908T = NBRC 113640T = KCTC 62951T) as the type strain.

Roseicella aquatilis sp. nov., isolated from freshwater lake.

A novel Gram-stain-negative, non-motile, ellipsoidal-shaped, red-pigmented, facultatively aerobic strain designated NE82T 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 NE82T 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 NE82T were summed feature 8 (C18:1 ω7c and C18:1 ω6c). The polar lipids of strain NE82T 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 NE82T showed the highest similarity (97.2%) to Roseicella frigidaeris DB1506T 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 NE82T (= KCTC 62412T = MCCC 1H00292T).

Roseococcus pinisoli sp. nov., lacking pufL and pufM bacteriochlorophyll a: synthesizing genes.

A Gram-stain-negative, cocci-to-oval-shaped bacterial strain, designated XZZS9T, 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 XZZS9T was related to members of the genus Roseococcus, with the highest sequence identity to Roseococcus microcysteis NIBR12T (96.9%). The major cellular fatty acids (> 5% of the total) were C18:1 ω7c and C19:0 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 XZZS9T 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 XZZS9T, suggesting a possible lack of photosynthetic reaction. Based on genotypic and phenotypic characteristics, strain XZZS9T represents a novel species of the genus Roseococcus, for which we propose the name Roseococcus pinisoli sp. nov. The type strain is XZZS9T (= KCTC 82435T = JCM 34402T = GDMCC 1.2158T).

Molecular biology: Fantastic toolkits to improve knowledge and application of acetic acid bacteria.

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.

Genome analysis suggests the bacterial family Acetobacteraceae is a source of undiscovered specialized metabolites.

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.

Barcoded Asaia bacteria enable mosquito in vivo screens and identify novel systemic insecticides and inhibitors of malaria transmission.

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.

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.

Transcriptome Analysis of Komagataeibacter europaeus CGMCC 20445 Responses to Different Acidity Levels During Acetic Acid Fermentation.

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.

Reclassification of seven honey bee symbiont strains as Bombella apis.

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.

Bio-conversion of kitchen waste into bacterial cellulose using a new multiple carbon utilizing Komagataeibacter rhaeticus: Fermentation profiles and genome-wide analysis.

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.

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.

Leucine-Responsive Regulatory Protein in Acetic Acid Bacteria Is Stable and Functions at a Wide Range of Intracellular pH Levels.

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 (KeLrp) was used as a model to examine pH-dependent effects, and its properties were compared with those of the Escherichia coli ortholog (EcLrp) at different pH levels. The DNA-binding activities of EcLrp and KeLrp with the target DNA (Ec-ilvI and Ke-ilvI) were examined by gel mobility shift assays under various pH conditions. EcLrp showed the highest affinity with the target at pH 8.0 (Kd [dissociation constant], 0.7 µM), decreasing to a minimum of 3.4 µM at pH 4.0. Conversely, KeLrp did not show significant differences in binding affinity between pH 4 and 7 (Kd, 1.0 to 1.5 µM), and the highest affinity was at pH 5.0 (Kd, 1.0 µM). Circular dichroism spectroscopy revealed that the α-helical content of KeLrp 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 EcLrp decreased from its maximum (49% at pH 7.0) to its minimum (36% at pH 4.0). These data indicate that KeLrp 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 (KeLrp) 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 KeLrp 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.

Genome Features of Asaia sp. W12 Isolated from the Mosquito Anopheles stephensi Reveal Symbiotic Traits.

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 bo3-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.

Involvement of an FNR-like oxygen sensor in Komagataeibacter medellinensis for survival under oxygen depletion.

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 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.

Functional metaproteomic analysis of alcohol vinegar microbiota during an acetification process: A quantitative proteomic approach.

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.