Host-derived nutritional substrates fuel infection in invading bacteria, yet their potential as signaling molecules for host perception remains largely unexplored. Here, we report the functional characterization of HutC, a transcriptional repressor of hut genes for the utilization of histidine and its derivative, urocanate, in the human pathogenic bacterium Pseudomonas aeruginosa PAO1. Using electrophoretic mobility shift assay (EMSA) and DNase I footprinting combined with site-directed mutagenesis, we demonstrate that HutC specifically binds with high affinity to the promoters of the two hut operons. This analysis led to the identification of a noncanonical HutC-binding site in the hutF promoter of a non-pathogenic Pseudomonas strain, which is absent in P. aeruginosa. A genome-wide search of the PAO1 genome using a probability matrix of the canonical HutC-binding motif identified 172 candidate sites, many associated with bacterial pathogenesis. Their predicted low-affinity binding was experimentally validated by EMSA for six selected targets, including the aminoglycoside response regulator (arr). Deletion of hutC resulted in increased tobramycin-induced biofilm formation and impaired production of pyoverdine, an iron-scavenging siderophore. Moreover, the hutC mutant exhibited altered motility and significantly reduced virulence in the Caenorhabditis elegans infection model. Finally, transcriptome sequencing of three genetically distinct hutC mutants provided further support for the HutC-mediated global regulation. Together, these findings highlight the functional significance of low-affinity DNA binding by this transcription factor and support the hypothesis that HutC mediates P. aeruginosa virulence, with histidine and urocanate as effectors. Thus, HutC may represent a potential therapeutic target within the bacterial host-perception system.IMPORTANCEPseudomonas aeruginosa is a metabolically versatile environmental pathogen whose virulence relies on coordinated expression of catabolic genes, particularly the histidine utilization (hut) operon. Disruption of the hut operon reduces virulence, but the underlying mechanism remains rudimentary. Here, we genetically characterized the histidine-responsive transcriptional factor HutC in P. aeruginosa PAO1, alongside HutC in the non-pathogenic strain Pseudomonas fluorescens SBW25. Two important features emerged. First, HutC recognizes two distinct DNA-binding motifs with little sequence similarity; notably, a noncanonical-binding site was identified in the hutF promoter of SBW25 but was absent in PAO1. Second, HutC exhibits low-affinity binding to genes beyond histidine catabolism and contributes to the expression of multiple virulence traits. These findings identify HutC as a local regulator linking histidine catabolism with virulence and as a unique prokaryotic model for studying how noncanonical transcriptional factor-DNA interactions achieve binding specificity, a phenomenon so far investigated only in eukaryotes.

Soil organic carbon (SOC) exhibits substantial spatial variability across broad geographic regions due to interactions among climate, soil properties, and land management. In croplands, SOC accumulation is further shaped by cropping systems and residue management, yet the general patterns and mechanistic drivers of SOC formation across multiple carbon (C) pools and sources remain poorly understood. Here, we analyzed cropland soils along a Mollisol transect in Northeast China to elucidate the key mechanisms and pathways underlying SOC accumulation. The results showed that both microbial necromass and mineral-associated organic C (MAOC) declined with increasing mean annual temperature and collectively served as the predominant contributors to SOC in croplands under different cropping systems. Microbial necromass accumulation was primarily regulated by microbial properties (61% of explained variance) and soil organic matter (SOM) chemical recalcitrance (31%), whereas MAOC accumulation was mainly driven by microbial necromass (50% of explained variance) and mineral protection capacity (41%). Across the Mollisol transect, microbial necromass positively responded to microbial biomass C and fungal community dissimilarity, but decreased with enhanced SOM chemical recalcitrance, as indicated by elevated aromaticity, alkyl C/O-alkyl C ratio, and hydrophobicity index. Furthermore, MAOC accumulation followed a two-step pathway involving microbial necromass inputs through in vivo microbial turnover and subsequent mineral stabilization by association with poorly crystalline Fe/Al oxides and exchangeable Mg2+. Together, microbial and mineral C pumps jointly regulated SOC accumulation, with the mineral C pump exerting a dominant role. These findings highlight the importance of explicitly integrating coupled microbial and mineral C pumps mechanisms into SOC stabilization frameworks. We demonstrate that agricultural management practices that enhance microbial activity, necromass production, and mineral protection capacity can substantially increase SOC sequestration in cropland ecosystems.

This study aimed to determine the effect of various fermentation factors on levels of bioactive components, antioxidants, and antimicrobial activities of black tea Kombucha prepared with a starter culture obtained in New Zealand. Of the seven measured phenolic compounds, caffeine, gallic acid and theobromine were the predominant ones in the 17 fermented Kombucha samples developed using the Plackett-Burman design. All samples exhibited antioxidant activities for scavenging DPPH (20.78-91.28%) and ABTS+ (22.63-551.10 mu g TE/mL). The phenolic content and antioxidant activities were significantly influenced by the concentrations of black tea used (p < 0.05). Kombucha samples with the highest titratable acidity (1.03%) exhibited the strongest antimicrobial activities against Staphylococcus aureus MCTIC 4163, Bacillus cereus MU-A44 and Escherichia coli NCTIC 8196. Notably, the three Kombucha samples K15, K11 and K8 produced in this study contained high levels of bioactive compounds and exhibited strong antioxidant and antimicrobial activities.

Entomopathogenic bacteria express virulence genes in a temperature-dependent manner, but the underlying molecular mechanisms remain poorly understood. Here, we describe the roles of an unusual LytTR-containing transcription factor Yen6 in determining the virulence of Yersinia entomophaga (MH96). An initial transcriptome analysis in vivo revealed that yen6, located 1,372 bp upstream from genes encoding the Yen Toxin complex (Yen-Tc), exhibited significantly higher transcriptional activity at 37 °C compared to 25 °C (adjusted p-value < 0.001) during intrahemocoelic infection in the insect model Galleria‍ mellonella. Deletion of yen6 also significantly reduced MH96's virulence in G.‍‍ mellonella by up to three-fold. Next, comparing the transcriptome of wild-type MH96 and its derived Δyen6 mutant revealed genes for ribose utilization were downregulated whereas genes for fructose utilization and the RNA-binding protein YhbY were upregulated in the yen6-deficient mutant. Subsequent electromobility shift assays showed that purified Yen6His6 protein was capable of specifically binding to the promoter regions of the ribose and fructose utilization-related gene clusters, as well as yhbY. A 645-nucleotide-long 3' untranslated region, designated yen7_AS, was identified extending from the yen6 coding region. Notably, yen7_AS completely overlaps the yen7 gene on the opposing strand and may therefore act as a cis anti-sense RNA regulating yen7 expression, which encodes a putative transcriptional regulator of the Yen-Tc. This study highlights the critical role of the yen6-yen7 locus in MH96 virulence during G.‍ mellonella infection, including the complex mechanisms controlling insecticidal exoprotein production.

Maintenance of rod-shape in bacterial cells depends on the actin-like protein MreB. Deletion of mreB from Pseudomonas fluorescens SBW25 results in viable spherical cells of variable volume and reduced fitness. Using a combination of time-resolved microscopy and biochemical assay of peptidoglycan synthesis, we show that reduced fitness is a consequence of perturbed cell size homeostasis that arises primarily from differential growth of daughter cells. A 1,000-generation selection experiment resulted in rapid restoration of fitness with derived cells retaining spherical shape. Mutations in the peptidoglycan synthesis protein Pbp1A were identified as the main route for evolutionary rescue with genetic reconstructions demonstrating causality. Compensatory pbp1A mutations that targeted transpeptidase activity enhanced homogeneity of cell wall synthesis on lateral surfaces and restored cell size homeostasis. Mechanistic explanations require enhanced understanding of why deletion of mreB causes heterogeneity in cell wall synthesis. We conclude by presenting two testable hypotheses, one of which posits that heterogeneity stems from non-functional cell wall synthesis machinery, while the second posits that the machinery is functional, albeit stalled. Overall, our data provide support for the second hypothesis and draw attention to the importance of balance between transpeptidase and glycosyltransferase functions of peptidoglycan building enzymes for cell shape determination.

Acetyl-CoA synthetase (ACS) is a well-characterized enzyme that catalyzes the ATP-dependent ligation of acetate and coenzyme A to produce acetyl-CoA, a central metabolite coordinating energy metabolism, carbon flux distribution, and post-translational protein modification. Recently, ACS has emerged as a metabolic nexus with broad implications for plant-microbe interactions in agriculture. Beyond its canonical role in primary metabolism, ACS governs diverse physiological processes in beneficial plant-associated microorganisms, including rhizosphere colonization, stress adaptation, secondary metabolite biosynthesis, and morphological development-all of which enhance plant growth and resilience. In contrast, in phytopathogens, ACS is closely related to the expression of virulence factors. Thus, ACS exerts a dual influence, shaping both mutualistic and antagonistic microbial lifestyles in planta. This review synthesizes recent advances in the structural and catalytic diversity of ACS, delineates its ecological and functional roles in agriculturally relevant microorganisms, and explores the environmental and host-derived signals that regulates its expression and activity. Particular attention is given to the interplay between ACS-mediated carbon metabolism and protein acetylation, which together modulate microbial physiology and plant-associated behaviors. ACS is thereby positioned as a strategic metabolic hub, providing a framework for future research at the interface of microbial metabolism, environmental adaptation, and plant health.

Xisha melon is a selenium-rich watermelon variety cultivated in semi-arid regions of north-central China as a primary economic crop. However, continuous monocropping has led to a notable increase in fusarium wilt disease incidence, and efficient control strategies are yet to be established. This work aims to investigate the biotic and abiotic factors influencing the population dynamics of rhizosphere microbial communities during continuous monocropping. Soil samples were collected from the rhizosphere of healthy watermelon grown in fields after 1, 3, 5, 10, 18, and 23 years of continuous monocropping, along with samples from nearby uncultivated land as a reference control. The soils were characterized by analysis of chemical properties and high-throughput sequencing of ribosomal amplicons targeting bacteria and fungi. The sequence data were then subjected to vigorous statistical analysis, including structural equation modelling. The data consistently revealed a general trend of increasing bacterial diversity but decreasing fungal diversity during continuous monocropping. Specifically, concerning the Fusarium population, its relative abundance decreased from 4.41% in the uncultivated soil to 0.61% in rhizosphere soil after 23 years of continuous monocropping, with nitrate-nitrogen being the most influential determining factor. Bacillus was among the bacterial genera enriched by continuous cropping. Our data collectively reveal that continuous watermelon cropping led to significant shifts in both bacterial and fungal communities, resulting in a decline of the Fusarium population in the rhizosphere. Furthermore, our findings suggest the potential roles of nitrate fertilization in manipulating the soil microbiome for efficient control of the fusarium wilt disease.

The current study investigated the in vitro probiotic potential of yeast isolated from kombucha, a tea beverage fermented with a symbiotic culture of acetic acid bacteria and yeast. A total of 62 yeast strains were previously isolated from four different commercial kombucha samples sold in New Zealand. Fifteen representative isolates belonging to eight different species were evaluated for their growth under different conditions (temperature, low pH, concentrations of bile salts, and NaCl). Cell surface characteristics, functional and enzymatic activities of the selected strains were also studied in triplicate experiments. Results showed that six strains (Dekkera bruxellensis LBY1, Sachizosaccharomyces pombe LBY5, Hanseniaspora valbyensis DOY1, Brettanomyces anomalus DOY8, Pichia kudraivzevii GBY1, and Saccharomyces cerevisiae GBY2) were able to grow under low-acid conditions (at pH 2 and pH 3) and in the presence of bile salts. This suggests their potential to survive passage through the human gut. All 15 strains exhibited negative enzymatic activity reactions (haemolytic, gelatinase, phospholipase, and protease activities), and thus, they can be considered safe to consume. Notably, two of the fifteen strains (Pichia kudraivzevii GBY1 and Saccharomyces cerevisiae GBY2) exhibited desirable cell surface hydrophobicity (64.60–83.87%), auto-aggregation (>98%), co-aggregation, resistance to eight tested antibiotics (ampicillin, chloramphenicol, colistin sulphate, kanamycin, nalidixic acid, nitrofurantoin, streptomycin, and tetracycline), and high levels of antioxidant activities (>90%). Together, our data reveal the probiotic activities of two yeast strains GBY1 and GBY2 and their potential application in functional food production.

Woodchip bioreactors are an eco-friendly technology for removing nitrogen (N) pollution. However, there needs to be more clarity regarding the dissolved organic matter (DOM) characteristics and bacterial community succession mechanisms and their association with the N removal performance of bioreactors. The laboratory woodchip bioreactors were continuously operated for 360 days under three influent N level treatments, and the results showed that the average removal rate of TN was 45.80 g N/(m3·day) when the influent N level was 100 mg N/L, which was better than 10 mg N/L and 50 mg N/L. Dynamic succession of bacterial communities in response to influent N levels and DOM characteristics was an important driver of TN removal rates. Medium to high N levels enriched a copiotroph bacterial module (Module 1) detected by network analysis, including Phenylobacterium, Xanthobacteraceae, Burkholderiaceae, Pseudomonas, and Magnetospirillaceae, carrying N-cycle related genes for denitrification and ammonia assimilation by the rapid consumption of DOM. Such a process can increase carbon limitation to stimulate local organic carbon decomposition to enrich oligotrophs with fewer N-cycle potentials (Module 2). Together, this study reveals that the compositional change of DOM and bacterial community succession are closely related to N removal performance, providing an ecological basis for developing techniques for N-rich effluent treatment.

Kombucha is a fermented tea beverage containing live microorganisms, mostly beneficial strains of yeast and acetic acid bacteria (AAB), but empirical evidence is limited supporting the probiotic potential of kombucha. This study reports the in vitro probiotic potential of 36 AAB strains isolated from three Kombucha samples commercially available in New Zealand. Nine representative AAB strains, belonging to three species (Komagataeibacter rhaeticus, Acetobacter musti and Gluconobacter potus), were examined for their primary probiotic characteristics such as tolerance to bile salts, NaCl, and low pH, and temperature. Three non-cellulose forming strains (A. musti LOAAB1, G. potus LOAAB2 and G. potus GBAAB3) were assayed for their cell surface characteristics such as auto-aggregation, co-aggregation with pathogenic bacteria, and hydrophobicity. Antimicrobial, antioxidant activities and enzymatic activities were also investigated for the strains of interest. Results indicated that nine strains were able to grow under low pH in the presence of bile salts, suggesting their potential to survive in the human gut. Six K. rhaeticus strains produced cellulosic pellicles, a potential source of prebiotics for beneficial bacteria. The three AAB strains LOAAB1, LOAAB2 and GBAAB3 showed promising cell surface characteristics, such as auto-aggregation rates (>80 %), co-aggregation with four pathogenic bacteria (13.24–43.47 %), hydrophobicity (42.12 % to 50.20 %), and antioxidant activities (>90 %). All nine strains tested negative for enzymatic activities (haemolytic, proteolytic, phospholipase, and gelatinase), suggesting that they are safe to consume. Together, these data indicate the potential for the three AAB strains to be further investigated as probiotic sources with more in vivo tests for applications in the food and beverages industry.

The rhizosphere system of plants hosts a diverse consortium of bacteria that confer beneficial effects on plant, such as plant growth-promoting rhizobacteria (PGPR), biocontrol agents with disease-suppression activities, and symbiotic nitrogen fixing bacteria with the formation of root nodule. Efficient colonization in planta is of fundamental importance for promoting of these beneficial activities. However, the process of root colonization is complex, consisting of multiple stages, including chemotaxis, adhesion, aggregation, and biofilm formation. The secondary messenger, c-di-GMP (cyclic bis-(3 '-5 ') dimeric guanosine monophosphate), plays a key regulatory role in a variety of physiological processes. This paper reviews recent progress on the actions of c-di-GMP in plant beneficial bacteria, with a specific focus on its role in chemotaxis, biofilm formation, and nodulation.

Kombucha is a popular sparkling sugared tea, fermented by a symbiotic culture of acetic acid bacteria (AAB) and yeast. The demand for kombucha continues to increase worldwide, mainly due to its perceived health benefits and appealing sensory properties. This study isolated and characterised the dominant AAB and yeast from a starter culture and kombucha broth after 0, 1, 3, 5, 7, 9, 11, and 14 days of fermentation at ambient temperature (22 °C). Yeast and AAB were isolated from the Kombucha samples using glucose yeast extract mannitol ethanol acetic acid (GYMEA) and yeast extract glucose chloramphenicol (YGC) media, respectively. The phenotypic and taxonomic identification of AAB and yeast were determined by morphological and biochemical characterisation, followed by a sequence analysis of the ribosomal RNA gene (16S rRNA for AAB and ITS for yeast). The changes in the microbial composition were associated with variations in the physico-chemical characteristics of kombucha tea, such as pH, titratable acidity, and total soluble solids (TSS). During fermentation, the acidity increased and the TSS decreased. The yield, moisture content, and water activity of the cellulosic pellicles which had developed at the end of fermentation were attributed to the presence of AAB. The dominant AAB species in the cellulosic pellicles and kombucha broth were identified as Komagataeibacter rhaeticus. The yeast isolates belonged to Debaryomyces prosopidis and Zygosaccharomyces lentus.

A novel heterotrophic nitrification and aerobic denitrification (HN-AD) bacterium D1-1 was identified as Pseudomonas nicosulfuronedens D1-1. Strain D1-1 removed 97.24%, 97.25%, and 77.12% of 100 mg/L NH4+-N, NO3- -N, and NO2- -N, with corresponding maximum removal rates of 7.42, 8.69, and 7.15 mg & BULL;L-1 & BULL;h-1, respectively. Strain D1-1 bioaugmentation enhanced woodchip bioreactor performance with an average NO3- -N removal efficiency of 93.8%. Bioaugmentation enriched N cyclers along with increased bacterial diversity and predicted genes for denitrification, DNRA (dissimilatory nitrate reduction to ammonium), and ammonium oxidation. It also reduced local selection and network modularity from 4.336 to 0.934, resulting in predicted nitrogen (N) cycling genes shared by more modules. These observations suggested that bioaugmentation could enhance the functional redundancy to stabilize the NO3- -N removal performance. This study provides insights into the potential applications of HN-AD bacteria in bioremediation or other environmental engineering fields, relying on their ability to shape bacterial communities.

Microorganisms play essential roles in soil ecosystem functioning and maintenance, but methods are currently lacking for quantitative assessments of the mechanisms underlying microbial diversity patterns observed across disparate systems and scales. Here we established a quantitative model to incorporate pH into metabolic theory to capture and explain some of the unexplained variation in the relationship between temperature and soil bacterial diversity. We then tested and validated our newly developed models across multiple scales of ecological organization. At the species level, we modeled the diversification rate of the model bacterium Pseudomonas fluorescens evolving under laboratory media gradients varying in temperature and pH. At the community level, we modeled patterns of bacterial communities in paddy soils across a continental scale, which included natural gradients of pH and temperature. Last, we further extended our model at a global scale by integrating a meta-analysis comprising 870 soils collected worldwide from a wide range of ecosystems. Our results were robust in consistently predicting the distributional patterns of bacterial diversity across soil temperature and pH gradients—with model variation explaining from 7 to 66% of the variation in bacterial diversity, depending on the scale and system complexity. Together, our study represents a nexus point for the integration of soil bacterial diversity and quantitative models with the potential to be used at distinct spatiotemporal scales. By mechanistically representing pH into metabolic theory, our study enhances our capacity to explain and predict the patterns of bacterial diversity and functioning under current or future climate change scenarios.

Chemoreceptors play a crucial role in assisting bacterial sensing and response to environmental stimuli. Genome analysis of Azorhizobium caulinodans ORS571 revealed the presence of 43 putative chemoreceptors, but their biological functions remain largely unknown. In this study, we identified the chemoreceptor AmaP (methyl-accepting protein of A. caulinodans), characterized by the presence of the CHASE3 domain and exhibited a notable response to acetoin. Thus, we investigated the effect of acetoin sensing on its symbiotic association with the host. Our findings uncovered a compelling role for acetoin as a key player in enhancing various facets of A. caulinodans ORS571's performance including biofilm formation, colonization, and nodulation abilities. Notably, acetoin bolstered A. caulinodans ORS571's efficacy in promoting the growth of S. rostrata, even under moderate salt stress conditions. This study not only broadens our understanding of the AmaP protein with its distinctive CHASE3 domain but also highlights the promising potential of acetoin in fortifying the symbiotic relationship between A. caulinodans and Sesbania rostrata.

Legumes in the inverted repeat-lacking clade (IRLC) each produce a unique set of nodule-specific cysteine-rich (NCR) peptides, which act in concert to determine the terminal differentiation of nitrogen-fixing bacteroid. IRLC legumes differ greatly in their numbers of NCR and sequence diversity. This raises the significant question how bacteroid differentiation is collectively controlled by the specific NCR repertoire of an IRLC legume. Astragalus sinicus is an IRLC legume that forms indeterminate nodules with its microsymbiont Mesorhizobium huakuii 7653R. Here, we performed transcriptome analysis of root and nodule samples at 3, 7, 14, 28 days postinoculation with M. huakuii 7653R and its isogenic ∆ bacA mutant. BacA is a broad-specificity peptide transporter required for the host-derived NCRs to target rhizobial cells. A total of 167 NCRs were identified in the RNA transcripts. Comparative sequence and electrochemical analysis revealed that A. sinicus NCRs (AsNCRs) are dominated by a unique cationic group (termed subgroup C), whose mature portion is relatively long (>60 amino acids) and phylogenetically distinct and possessing six highly conserved cysteine residues. Subsequent functional characterization showed that a 7653R variant harboring AsNCR083 (a representative of subgroup C AsNCR) displayed significant growth inhibition in laboratory media and formed ineffective white nodules on A. sinicus with irregular symbiosomes. Finally, bacterial two-hybrid analysis led to the identification of GroEL1 and GroEL3 as the molecular targets of AsNCR067 and AsNCR076. Together, our data contribute to a systematic understanding of the NCR repertoire associated with the A. sinicus and M. huakuii symbiosis. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Legumes in the inverted repeat-lacking clade (IRLC) each produce a unique set of nodule-specific cysteine-rich (NCR) peptides, which act in concert to determine the terminal differentiation of nitrogen-fixing bacteroid. IRLC legumes differ greatly in NCR number and sequence diversity. This raises a significant question how bacteroid differentiation is collectively controlled by the specific NCR repertoire of an IRLC legume. Astragalus sinicus is an IRLC legume that forms indeterminate nodules with its microsymbiont Mesorhizobium huakuii 7653R. Here, we performed transcriptome analysis of root and nodule samples at 3, 7, 14, 28 days post inoculation with M. huakuii 7653R and its isogenic ∆bacA mutant. BacA is a broad-specificity peptide transporter required for the host-derived NCRs to target rhizobial cells. A total of 167 NCRs were identified in the RNA transcripts. Comparative sequence and electrochemical analysis revealed that A. sinicus NCRs (AsNCRs) are dominated by a unique cationic group (termed subgroup C), whose mature portion is relatively long (>60 amino acids) and phylogenetically distinct and possessing six highly conserved cysteine residues. Subsequent functional characterization showed that a 7653R variant harboring AsNCR03 (a representative of subgroup C AsNCR) displayed significant growth inhibition in laboratory media, and formed ineffective white nodules on A. sinicus with irregular symbiosomes. Finally, bacterial two-hybrid analysis led to the identification of GroEL1 and GroEL3 as the molecular targets of AsNCR067 and AsNCR076. Together, our data contribute to a systematic understanding of the NCR repertoire associated with the A. sinicus and M. huakuii symbiosis.

The demand for Kombucha, a sparkling sugared tea beverage fermented by a symbiotic culture of acetic acid bacteria (AAB) and yeast is increasing worldwide. Despite the popularity of the beverage which is mainly due to its perceived health benefits and appealing sensory properties, the microbial composition of the products at the time of consumption is unknown. Such information is important to both manufacturers and consumers. Therefore, this study characterised the dominant AAB and yeast present in six commercial Kombucha samples sold in New Zealand which comprised of three domestic and three imported samples. Acetic acid bacteria and yeast were isolated from the Kombucha samples using glucose yeast extract peptone mannitol (GYPM) and yeast extract glucose chloramphenicol (YGC) media, respectively. Phenotypic and taxonomic identification of AAB and yeast were achieved by morphological and biochemical characterisation, followed by sequence analysis of ribosomal RNA genes (16S rRNA for AAB and 26S rRNA for yeast). Viable AAB and yeast were only found in domestically produced Kombucha samples and not in the imported products. The dominant AAB species were identified as Acetobacter musti and Gluconobacter potus. The yeast isolates belonged to Dekkera bruxelensis, Schizosaccharomyces pombes, Hanseniaspora valbyensis, Brettanomyces anamalus, Pichia kudriavzevii, Starmerella vitis and Saccharomyces cerevisiae. The yeast communities were more complex and variable than the AAB communities in the analysed Kombucha samples.

AbstractAntimicrobial copper‐containing surface materials have a great potential of reducing the risks of healthcare‐associated infections (HAIs), but their increased use in hospital facilities may select copper‐resistant strains, causing concerns to antimicrobial resistance management. Here, we describe a long‐term bacterial evolution experiment wherein a non‐pathogenic Pseudomonas strain was subjected to daily transfer in laboratory media with and without copper‐mediated contact killing. The copper treatment sequentially involved two surface materials differing in Cu content and thus contact killing effectiveness: first on brass (Cu 63.5%) and then on pure copper (Cu 99.9%). A gradual increase in bacterial survival rate (or a decrease of killing effectiveness) was observed over time on the related copper surfaces. For the final evolved populations after 320 transfers, 37.8% cells of the copper‐evolved populations were able to survive 60 min on pure copper, whereas populations in the control lines remained sensitive with a survival rate of 0.09% under the same contact killing condition. Genome re‐sequencing revealed ~540 mutations accumulated in the copper lines but only 71, on average, in the control lines (variant frequency > 0.5). The mutagenic activities of Cu+ ions were confirmed by measuring spontaneous mutation rate in a laboratory medium supplemented with copper sulfate at a non‐inhibitory concentration. The copper‐evolved populations have acquired increased resistance to Cu+ ions and tobramycin (an aminoglycoside antibiotic), but showed decreased production of biofilm, exoprotein, and pyoverdine. Together, our data demonstrate the potential of bacteria to evolve prolonged survival on metallic copper, and the long‐term impacts should be considered with increased copper usage in hospital environments.

Kombucha is a sparkling sugared tea commonly prepared using a sugared tea infusion and fermented at ambient temperature for several days using a cellulose pellicle also called tea fungus that is comprised of acetic acid bacteria and yeast. Consumption of Kombucha has been reported as early as 220 B.C. with various reported potential health benefits and appealing sensory properties. During Kombucha fermentation, sucrose is hydrolysed by yeast cells into fructose and glucose, which are then metabolised to ethanol. The ethanol is then oxidised by acetic acid bacteria (AAB) to produce acetic acid which is responsible for the reduction of the pH and also contributes to the sour taste of Kombucha. Characterisation of the AAB and yeast in the Kombucha starter culture can provide a better understanding of the fermentation process. This knowledge can potentially aid in the production of higher quality products as these microorganisms affect the production of metabolites such as organic acids which are associated with potential health benefits, as well as sensory properties. This review presents recent advances in the isolation, enumeration, biochemical characteristics, conventional phenotypic identification system, and modern genetic identification techniques of AAB and yeast present in Kombucha to gain a better understanding of the microbial diversity of the beverage.