Acetobacter garciniae sp. nov., an acetic acid bacterium isolated from fermented mangosteen peel in Thailand

Two isolates, MS16-SU-2T and MS18-SU-3, obtained from fermented mangosteen peel in vinegar were suggested to constitute a new species assignable to the genus Acetobacter based on the results of 16S rRNA gene sequencing. The two isolates showed the highest sequence similarity (98.58%) to Acetobacter tropicalis NBRC 16470T and Acetobacter senegalensis LMG 23690T. However, the calculated similarity values were lower than the threshold for species demarcation. The phylogenetic analysis showed that the branches of the two isolates were separated from other Acetobacter species, and the two isolates constituted a new species in the genus Acetobacter . The genomic DNA of isolate MS16-SU-2T was sequenced. The assembled genome of the isolate was analysed, and the results showed that the highest average nucleotide identity value of 75.9 % was with Acetobacter papayae JCM 25143T and the highest digital DNA–DNA hybridization value of 25.1 % was with Acetobacter fallax LMG 1636T, which were lower than the cutoff values for species delineation. The phylogenetic tree based on the genome sequences showed that the lineage of isolate MS16-SU-2T was most closely related to A. papayae JCM 25143T and Acetobacter suratthaniensis TBRC 1719T, but separated from the branches of these two species. In addition, the two isolates could be distinguished from the type strains of closely related species by their phenotypic characteristics and MALDI-TOF profiles. Therefore, the two isolates, MS16-SU-2T (=TBRC 12339T=LMG 32243T) and MS18-SU-3 (=TBRC 12305), can be assigned to an independent species within the genus Acetobacter , and the name of Acetobacter garciniae sp. nov. is proposed for the two isolates.

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.

Highly tunable TetR-dependent target gene expression in the acetic acid bacterium Gluconobacter oxydans

For the acetic acid bacterium (AAB) Gluconobacter oxydans only recently the first tight system for regulatable target gene expression became available based on the heterologous repressor-activator protein AraC from Escherichia coli and the target promoter ParaBAD. In this study, we tested pure repressor-based TetR- and LacI-dependent target gene expression in G. oxydans by applying the same plasmid backbone and construction principles that we have used successfully for the araC-ParaBAD system. When using a pBBR1MCS-5-based plasmid, the non-induced basal expression of the Tn10-based TetR-dependent expression system was extremely low. This allowed calculated induction ratios of up to more than 3500-fold with the fluorescence reporter protein mNeonGreen (mNG). The induction was highly homogeneous and tunable by varying the anhydrotetracycline concentration from 10 to 200 ng/mL. The already strong reporter gene expression could be doubled by inserting the ribosome binding site AGGAGA into the 3’ region of the Ptet sequence upstream from mNG. Alternative plasmid constructs used as controls revealed a strong influence of transcription terminators and antibiotics resistance gene of the plasmid backbone on the resulting expression performance. In contrast to the TetR-Ptet-system, pBBR1MCS-5-based LacI-dependent expression from PlacUV5 always exhibited some non-induced basal reporter expression and was therefore tunable only up to 40-fold induction by IPTG. The leakiness of PlacUV5 when not induced was independent of potential read-through from the lacI promoter. Protein-DNA binding simulations for pH 7, 6, 5, and 4 by computational modeling of LacI, TetR, and AraC with DNA suggested a decreased DNA binding of LacI when pH is below 6, the latter possibly causing the leakiness of LacI-dependent systems hitherto tested in AAB. In summary, the expression performance of the pBBR1MCS-5-based TetR-Ptet system makes this system highly suitable for applications in G. oxydans and possibly in other AAB. Key Points • A pBBR1MCS-5-based TetR-Ptet system was tunable up to more than 3500-fold induction. • A pBBR1MCS-5-based LacI-PlacUV5 system was leaky and tunable only up to 40-fold. • Modeling of protein-DNA binding suggested decreased DNA binding of LacI at pH < 6.

Cellulose-Synthesizing Machinery in Bacteria

Cellulose is produced by all plants and a number of other organisms, including bacteria. The most representative cellulose-producing bacterial species is Gluconacetobacter xylinus (G. xylinus), an acetic acid bacterium. Cellulose produced by G. xylinus, commonly referred to as bacterial cellulose (BC), has exceptional physicochemical properties resulting in its use in a variety of applications. All cellulose-producing organisms that synthesize cellulose microfibrils have membrane-localized protein complexes (also called terminal complexes or TCs) that contain the enzyme cellulose synthase and other proteins. The bacterium G. xylinus is a prolific cellulose producer and a model organism for studies on cellulose biosynthesis. The widths of cellulose fibers produced by Gluconacetobacter are 50‒100 nm, suggesting that cellulose-synthesizing complexes are nanomachines spinning a nanofiber. At least four different proteins (BcsA, BcsB, BcsC, and BcsD) are included in TC from Gluconacetobacter, and the proposed function of each is as follows: BcsA, synthesis of a glucan chain through glycosyl transfer from UDP-glucose; BcsB, complexes with BcsA for cellulose synthase activity; BcsC, formation of a pore in the outer membrane through which a glucan chain is extruded; BcsD, regulates aggregation of glucan chains through four tunnel-like structures. In this review, we discuss structures and functions of these four and a few other proteins that have a role in cellulose biosynthesis in bacteria.

Microbial Composition of SCOBY Starter Cultures Used by Commercial Kombucha Brewers in North America

Kombucha fermentation is initiated by transferring a solid-phase cellulosic pellicle into sweetened tea and allowing the microbes that it contains to initiate the fermentation. This pellicle, commonly referred to as a symbiotic culture of bacteria and yeast (SCOBY), floats to the surface of the fermenting tea and represents an interphase environment, where embedded microbes gain access to oxygen as well as nutrients in the tea. To date, various yeast and bacteria have been reported to exist within the SCOBY, with little consensus as to which species are essential and which are incidental to Kombucha production. In this study, we used high-throughput sequencing approaches to evaluate spatial homogeneity within a single commercial SCOBY and taxonomic diversity across a large number (n = 103) of SCOBY used by Kombucha brewers, predominantly in North America. Our results show that the most prevalent and abundant SCOBY taxa were the yeast genus Brettanomyces and the bacterial genus Komagataeibacter, through careful sampling of upper and lower SCOBY layers. This sampling procedure is critical to avoid over-representation of lactic acid bacteria. K-means clustering was used on metabarcoding data of all 103 SCOBY, delineating four SCOBY archetypes based upon differences in their microbial community structures. Fungal genera Zygosaccharomyces, Lachancea and Starmerella were identified as the major compensatory taxa for SCOBY with lower relative abundance of Brettanomyces. Interestingly, while Lactobacillacae was the major compensatory taxa where Komagataeibacter abundance was lower, phylogenic heat-tree analysis infers a possible antagonistic relationship between Starmerella and the acetic acid bacterium. Our results provide the basis for further investigation of how SCOBY archetype affects Kombucha fermentation, and fundamental studies of microbial community assembly in an interphase environment.

Chemical Constitution and Antimicrobial Activity of Kefir Fermented Beverage

Kefir beverage (KB) is a fermented milk initiated by kefir grains rich with starter probiotics. The KB produced in this study seemed to contain many chemical compounds elucidated by gas chromatography–mass spectrometry (GC-MS) and IR spectra. These compounds could be classified into different chemical groups such as alcohols, phenols, esters, fatty esters, unsaturated fatty esters, steroids, polyalkenes, heterocyclic compounds and aromatic aldehydes. Both KB and neutralized kefir beverage (NKB) inhibited some pathogenic bacteria including Escherichia coli ATCC11229 (E. coli), Listeria monocytogenes ATCC 4957 (L. monocytogenes), Bacillus cereus ATCC 14579 (B. cereus), Salmonella typhimurium ATCC 14028 (Sal. typhimurium) as well as some tested fungal strains such as Aspergillus flavus ATCC 16872 (A. flavus) and Aspergillus niger ATCC 20611 (A. niger), but the inhibitory activity of KB was more powerful than that obtained by NKB. It also appeared to contain four lactic acid bacteria species, one acetic acid bacterium and two yeast species. Finally, the KB inhibited distinctively both S. aureus and Sal. typhimurium bacteria in a brain heart infusion broth and in some Egyptian fruit juices, including those made with apples, guava, strawberries and tomatoes.

Identification of Gradient Promoters of Gluconobacter oxydans and Their Applications in the Biosynthesis of 2-Keto-L-Gulonic Acid

The acetic acid bacterium Gluconobacter oxydans is known for its unique incomplete oxidation and therefore widely applied in the industrial production of many compounds, e.g., 2-keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C. However, few molecular tools are available for metabolically engineering G. oxydans, which greatly limit the strain development. Promoters are one of vital components to control and regulate gene expression at the transcriptional level for boosting production. In this study, the low activity of SDH was found to hamper the high yield of 2-KLG, and enhancing the expression of SDH was achieved by screening the suitable promoters based on RNA sequencing data. We obtained 97 promoters from G. oxydans’s genome, including two strong shuttle promoters and six strongest promoters. Among these promoters, P3022 and P0943 revealed strong activities in both Escherichia coli and G. oxydans, and the activity of the strongest promoter (P2703) was about threefold that of the other reported strong promoters of G. oxydans. These promoters were used to overexpress SDH in G. oxydans WSH-003. The titer of 2-KLG reached 3.7 g/L when SDH was under the control of strong promoters P2057 and P2703. This study obtained a series of gradient promoters, including two strong shuttle promoters, and expanded the toolbox of available promoters for the application in metabolic engineering of G. oxydans for high-value products.

The FNR-type regulator GoxR of the obligatory aerobic acetic acid bacterium Gluconobacter oxydans affects expression of genes involved in respiration and redox metabolism

Gene expression in the obligately aerobic acetic acid bacterium Gluconobacter oxydans responds to oxygen limitation, but the regulators involved are unknown. In this study, we analyzed a transcriptional regulator named GoxR (GOX0974), which is the only member of the FNR family in this species. Evidence was obtained that GoxR contains an iron-sulfur cluster, suggesting that GoxR functions as an oxygen sensor similar to FNR. The direct target genes of GoxR were determined by combining several approaches including a transcriptome comparison of a ΔgoxR mutant with the wild type and detection of in vivo GoxR binding sites by ChAP-Seq. Prominent targets were the cioAB genes encoding a cytochrome bd oxidase with low O2 affinity, which were repressed by GoxR, and the pnt operon, which was activated by GoxR. The pnt operon encodes a transhydrogenase (pntA1A2B), an NADH-dependent oxidoreductase (GOX0313), and another oxidoreductase (GOX0314). Evidence was obtained for GoxR being active despite a high dissolved oxygen concentration in the medium. We suggest a model in which the very high respiration rates of G. oxydans due to the periplasmic oxidations cause an oxygen-limited cytoplasm and insufficient reoxidation of NAD(P)H in the respiratory chain, leading to an inhibited cytoplasmic carbohydrate degradation. GoxR-triggered induction of the pnt operon enhances fast interconversion of NADPH and NADH by the transhydrogenase and NADH reoxidation by the GOX0313 oxidoreductase via reduction of acetaldehyde formed by pyruvate decarboxylase to ethanol. In fact, small amounts of ethanol were formed by G. oxydans under oxygen-restricted conditions in a GoxR-dependent manner. IMPORTANCE Gluconobacter oxydans serves as cell factory for oxidative biotransformations based on membrane-bound dehydrogenases and as model organism for elucidating the metabolism of acetic acid bacteria. Surprisingly, to our knowledge none of the more than 100 transcriptional regulators encoded in the genome of G. oxydans has been studied experimentally up to now. In this work, we analyzed the function of a regulator named GoxR, which belongs to the FNR family. Members of this family serve as oxygen sensors by means of an oxygen-sensitive [4Fe-4S] cluster and typically regulate genes important for growth under anoxic conditions by anaerobic respiration or fermentation. Because G. oxydans has an obligatory aerobic respiratory mode of energy metabolism, it was tempting to elucidate the target genes regulated by GoxR. Our results show that GoxR affects the expression of genes that support the interconversion of NADPH and NADH and NADH reoxidation by reduction of acetaldehyde to ethanol.

Hybrid Living Capsules Autonomously Produced by Engineered Bacteria

Bacterial cellulose (BC) has excellent material properties and can be produced cheaply and sustainably through simple bacterial culture, but BC-producing bacteria lack the extensive genetic toolkits of model organisms such as Escherichia coli. Here, we describe a simple approach for producing highly programmable BC materials through incorporation of engineered E. coli. The acetic acid bacterium Gluconacetobacter hansenii was co-cultured with engineered E. coli in droplets of glucose-rich media to produce robust cellulose capsules, which were then colonized by the E. coli upon transfer to selective lysogeny broth media. We show that the encapsulated E. coli can produce engineered protein nanofibers within the cellulose matrix, yielding hybrid capsules capable of sequestering specific biomolecules from the environment and enzymatic catalysis. Furthermore, we produced capsules capable of altering their own bulk physical properties through enzyme-induced biomineralization. This novel system, based on autonomous biological fabrication, significantly expands the functionality of BC-based living materials.