Environmental Microbiology Reports

Bacteria of the genus Achromatium are known for their large cell sizes and intracellular calcium carbonate deposits. Achromatium inhabit freshwater, brackish, and marine sediments where they accumulate to high abundances at the oxic-anoxic interface. These bacteria alter their vertical position in the sediment along with daily fluctuations in oxygen concentrations. Yet, the mechanism behind their migration in the sediment remains unknown. In this study, we used chemotaxis assays and time-lapse microphotography to analyze the motility and chemotactic behavior of Achromatium oxaliferum. Microscopic observations revealed that rolling and gliding were the main forms of locomotion exhibited by Achromatium. In absence of any stimulant, the movement appeared to be mostly random and changes in direction frequently occurred. Chemotaxis assays showed a negative chemotaxis of Achromatium to oxygen, sulfide, and nitrate, as evidenced by the change from undirected to directed locomotion against the respective chemical gradient. For periods of more than 1 hour, Achromatium cells moved continuously towards regions of low concentration. We further investigated whether the genetic repertoire of Achromatium corresponds to our observations. Based on lab experiments and bioinformatic analyses we conclude that Achomatium motility is propelled by type IV pili guided by a plethora of chemo- and photoreceptors. We conclude that Achromatium uses negative chemo- and phototaxis to confine their distribution in aquatic sediments between opposing oxygen and sulfide gradients. This allows Achromatium to dynamically adjust its position in redox gradients, and thus is likely to have a major contribution to its success in the global colonization of diverse aquatic sediments.

Bacterial interactions-whether positive or negative - are crucial for the functioning of microbial communities. Though bacterial interactions are mainly expected to be negative, the sign and strength of interactions are predicted to be context dependent, with interactions typically being more positive in more stressful and nutrient-poor conditions. However, systematic studies investigating how the environment affects interactions between multiple taxa are lacking. Here, we determine if interactions between a panel of natural soil isolates change in response to the environment in which they are grown, with two different artificial media used (one simple and one complex) and a more ecologically relevant soil wash. To maximise natural variation in interactions, we collected multiple isolates from multiple sites: co-occurring (sympatric) isolates were predicted to show more negative interactions than allopatric isolates because of greater overlap in resource use. Pairwise interactions were in general negative, but more negative when grown in a complex lab-derived medium (Tryptic Soy Broth). Mutually beneficial interactions were most common in a simple resource medium (M9 minimal media) and exploitative interactions were most frequent in a soil broth. These patterns were independent of whether species originated from the same or a different site. The study supports the prediction that nutrient rich environments promote more negative interactions, and that measuring interactions of soil isolates in standard lab media is likely to misrepresent interactions occurring in natural environments.

Rock-inhabiting fungi thrive in subaerial oligotrophic environments such as desert rocks, solar panels and marble monuments where organic carbon and nitrogen are scarce. We tested whether the rock-inhabiting fungus Knufia petricola showed a preference regarding nitrogen ([Formula] or [Formula]) and carbon (glucose or sucrose) sources and whether it was sensitive towards carbon and nitrogen limitation. As this fungus produces the carbon-rich, nitrogen-free 1,8-dihydroxynaphthalene (DHN) melanin, we tested whether a melanin-deficient mutant would be less sensitive to carbon limitation. The carbon and nitrogen concentrations were the primary predictors of growth, with a broad optimum partially explained by an optimal fungal C:N ratio. Limiting carbon or nitrogen supply decreased biomass formation, CO2 production and biofilm thickness but promoted substratum penetration through filamentous growth. The nitrogen content of the biomass was flexible within limits, increasing upon increasing nitrogen supply or decreasing carbon supply. The carbon use efficiency was fairly constant, whereas melanization correlated with a higher nitrogen content of the biomass despite melanin being nitrogen-free. In conclusion, in vitro, K. petricola switches to explorative growth under nutrient limitations, like fast-growing fungi, revealing universal fungal resource-acquisition patterns. Graphical abstract text and imageCarbon and nitrogen availability affect biofilm growth and morphology of the extremotolerant fungus Knufia petricola Abolfazl Dehkohneh, Julia Schumacher, Bastiaan J. R. Cockx, Karin Keil, Tessa Camenzind, Jan-Ulrich Kreft, Anna A. Gorbushina, Ruben Gerrits Growth of the rock-inhabiting fungus Knufia petricola was studied by varying carbon and nitrogen sources and concentrations. Overall, growth was best predicted by the carbon and nitrogen concentrations. Carbon and nitrogen limitation promoted substratum penetration through filamentous growth. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/712823v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@6d98bdorg.highwire.dtl.DTLVardef@146aac5org.highwire.dtl.DTLVardef@757fa8org.highwire.dtl.DTLVardef@ff709_HPS_FORMAT_FIGEXP M_FIG C_FIG

The study of microbial volatile organic compounds (mVOCs) is a growing area of research, with applications ranging from agriculture to human health. The majority of the mVOC data are from in vitro liquid cultures, while few analyses of bacterial and fungal volatilomes on solid media cultures exist. Studies comparing liquid versus solid cultures of bacteria and fungi show significant changes to the soluble metabolites that are produced, suggesting that large differences would be observed for mVOCs based on the culture conditions. To test this idea, we characterized the volatilomes of Chromobacterium violaceum (strain ATCC(R) 12472) and C. vaccinii (strain MWU328), and those of their isogenic cviR- quorum sensing mutants cultured on solid versus liquid Kings Medium B media. VOCs were sampled using thin-film solid-phase microextraction (TF-SPME) and analyzed by two-dimensional gas chromatography-time-of-flight mass spectrometry (GCxGC-TOFMS). Of the three variables examined - Chromobacterium species, media type, and quorum sensing ability - growth on liquid versus solid media caused the most significant differences in the volatilomes. Bacterial species and quorum sensing ability were also influential, but to a lesser degree. Our findings indicate the importance of growth conditions in microbial volatilomics, and therefore, more consideration should be given to how microorganisms are cultured for volatilome analyses. ImportanceThe purpose of this work is to elucidate the differences in the volatile metabolic profiles of Chromobacterium spp. by exploring them through the lens of three variables: growth conditions, species, and the ability to quorum sense. Work on organismal metabolic differences stemming from factors such as liquid versus solid media types remains broadly overlooked. Understanding these effects will allow future researchers to design more robust experiments that better translate to native microbial ecosystems such as rhizosphere and phyllosphere, where volatile compounds may influence plant-pathogen or plant-saprobe interactions.

Phosphorus is a vital nutrient required for the functioning of living organisms. In aquatic environments, dissolved inorganic phosphate is considered its most bioavailable form. However, phosphate can be scarce, which has the potential to limit microbial metabolism and ecosystem functioning. To overcome phosphate scarcity, microbes produce alkaline phosphatase (AP) to access dissolved organic phosphorus (DOP). Here, we conducted a year-long study of alkaline phosphatase activity (APA) at the Ellen Browning Scripps Memorial Pier, a nutrient-rich coastal site. APA was observed throughout the year despite phosphate-replete conditions, suggesting that the role of APs in microbial nutrition is not completely understood. We tested the hypothesis that APA may promote acquisition of organic carbon liberated from DOP hydrolysis by growing the heterotrophic marine bacterium Ruegeria pomeroyi on three DOP compounds as sole carbon sources and assessing APA. Controlling for carbon concentration, all DOP sources supported growth, but at lower levels than glucose, with the highest growth observed on glucose-6-phosphate (G6P), followed by adenosine monophosphate (AMP) and adenosine triphosphate (ATP). Moreover, cell-specific APA was significantly enhanced in carbon-deplete conditions and during growth on G6P, relative to cultures grown on replete glucose or nucleotides. These findings suggest alkaline phosphatases (APs) are part of a generic carbon stress response and likely play a role in acquiring certain forms of organic carbon by R. pomeroyi, with implications for other taxa. Overall, this study helps advance the current state of knowledge regarding microbial phosphorus cycling and carbon utilization in aquatic environments.

The plant-beneficial bacterium Pseudomonas protegens CHA0 (CHA0) is widely studied for the biological control of soil-borne plant diseases. Beyond its root-colonising capabilities, CHA0 can also infect and kill insect larvae and thus exhibits a multi-host lifestyle shared with other plant- and insect-colonising bacteria. To better understand the robustness of this multi-host lifestyle, we subjected CHA0 to ten consecutive passages through larvae of the pest insect Plutella xylostella via repeated cycles of insect colonisation and killing forcing it into an insect-only lifestyle. Overall, serial passaging did not result in consistent changes in insect killing speed, larval or root colonisation, plant protection efficiency, microbial antagonism or in vitro growth. This suggests that its multi-host lifestyle was conserved following serial passage. Nonetheless, a few independently passaged lines showed an increase in larval killing speed, which in one case might be linked to choline uptake. To disentangle changes specific to the insect host from those arising due to the experimental system itself, we conducted parallel serial passages through the same system while omitting the insect host. In some of these lines, exposure to the background of the system led to changes in microbial antagonism and in in vitro growth, which likely are associated with mutations in regions encoding for regulatory systems. Our findings indicate that P. protegens CHA0 remains phenotypically stable in complex environments such as an insect host, suggesting that the multi-host lifestyle might also be conserved when applied in the field and supporting CHA0s potential for reliable biocontrol performance against both plant diseases and insect pests. Author summaryControlling insect pests with living organisms, known as biological control, offers an environmentally friendly alternative to chemical pesticides. The plant-beneficial bacterium Pseudomonas protegens CHA0 is a promising biocontrol candidate that not only colonizes plant roots but also infects and kills certain insect larvae. This ability to colonize different hosts appears to be a conserved trait also observed in other bacteria. To better understand the robustness of this multi-host lifestyle, we repeatedly exposed CHA0 to larvae of the insect pest Plutella xylostella and assessed the resulting physiological and genetic changes. Surprisingly, after ten cycles, CHA0 largely retained its insect-killing and plant-protective traits. Although a few populations showed minor changes, including slightly faster insect killing and traits associated with aspects of the experimental system, these changes were limited in scope. Overall, our findings suggest that P. protegens CHA0 does not change rapidly in complex environments such as an insect host, supporting its potential for reliable biocontrol performance in the field.

The interactions between microalgae and the bacteria living in the phycosphere are pivotal to the role they play in aquatic ecosystems. This study examines how two representatives of common phycosphere bacteria, Yoonia sp. TsM2_T14_4 (Rhodobacteraceae) and Maribacter sp. IgM3_T14_3 (Flavobacteriaceae), interact with three microalgal hosts: Isochrysis galbana, Tetraselmis suecica, and Conticribra weissflogii (formerly Thalassiosira weissflogii) using dual transcriptomic analyses of both bacteria and microalgae. Bacterial transcriptomes differed significantly depending on microalgal host, with notable changes in carbohydrate metabolism among other COG categories. Yoonia sp. expressed genes involved in anoxygenic photosynthesis in co-culture with I. galbana, presumably due to its inability to utilize carbohydrates from this algal host, whereas Maribacter sp. expressed polysaccharide degradation genes in co-culture with C. weissflogii along with T9SS genes, which can be employed to secrete these hydrolytic enzymes. Specifically, a putative glucan endo-1,3-beta-D-glucosidase was highly expressed, an enzyme that can hydrolyze laminarin and curdlan. Maribacter sp. IgM3_T14_3 could utilize laminarin as a sole carbon source in laboratory settings, a polysaccharide commonly found in marine environments and produced by C. weissflogii. Surprisingly, microalgal transcriptomes remained largely unaltered in the presence of either of the bacteria compared to transcriptomes of axenic algal cultures. These findings highlight the adaptability of phycosphere bacteria to different microalgal hosts. Furthermore, it also indicates a commensalism between microalgae, Yoonia sp. and Maribacter sp., in which the bacteria adapt to and benefit from microalgal host exudates, whereas under the conditions employed here the microalgae are unaffected by the presence of these bacterial symbionts. ImportanceMicroalgae are the key players in marine ecosystems, capturing carbon dioxide through photosynthesis and releasing carbohydrates into their immediate environment, the so-called phycosphere. Certain bacterial taxa are consistently found within the phycosphere, where they interact with their microalgal host in a variety of ways. However, the impact of these bacteria on the microalgae is not fully understood despite their ecological relevance. This study uses a dual transcriptomic approach to investigate the impact of such core phycosphere bacteria on microalgal hosts and vice versa to uncover the reason behind their success in the phycosphere and possible roles in marine ecosystems.

Plant roots host defined microbial communities that differ from those found in the surrounding soil and these communities shift dynamically in response to plant development and environmental changes. Whilst it is widely accepted that root exudates play a key role in the assembly and dynamics of root-associated microbial communities, the underlying mechanisms are not well understood. This is partly due to a lack of controlled experimental systems that monitor both exudate- and microbiome-dynamics simultaneously. Here, we compared two microcosm systems commonly used in either root microbiome (clay particle-based) or root exudate studies (glass bead-based) for their suitability to simultaneously monitor both aspects. We evaluated these systems based on plant performance, bacterial growth, and time-resolved community and exudate profiling. In both systems, we reveal an exudate effect, characterised by higher bacterial diversity and Pseudomonas abundances in proximity to plant roots. While clay particles impeded exudate recovery, even when plants were removed from microcosms for exudate collection, the glass bead set-up allowed us to uncover dynamic exudate shifts during bacterial community establishment. This highlighted a transient increase of glucosinolates upon root colonisation by initially dominant Pseudomonas species. Overall, the comparison proved only the glass bead-based semi-hydroponic system to be suitable for the paralleled study of exudate and root microbiome dynamics.

Despite the successful cultivation of many microbes from rich bacterial communities inhabiting alkaline soda lakes, members of the bacterial phylum Verrucomicrobiota have so far been detected only through metagenomics. Here, we used alginate as a selective substrate to enrich and isolate two strains of haloalkaliphilic Verrucomicrobiota. The isolates share identical 16S rRNA gene sequences representing a new genus lineage, and, together with other metagenome assembled genomes, a new family within Opitutales. Cells of strains AB-alg1T (from soda lakes) and AB-alg4 (from soda solonchak soils) are small and motile cocci forming submerged colonies in soft alginate agar. They are saccharolytic heterotrophs growing aerobically on polysaccharides (alginate, starch and inulin) and sugars (glucose, fructose, mannose, sucrose, melezitose, maltose and cellobiose). They also grow anaerobically by fermentation of alginate and D-mannose and by coupling incomplete denitrification to oxidation of alginate. Both isolates are obligately alkaliphilic and moderately salt-tolerant. The dominant membrane phospholipids include phosphatidylcholines and diphosphatidylglycerols (cardiolipins). The genome of AB-alg1T features polysaccharide lyases of the PL6, 7, 15, 17, 38, and 39 families for depolymerization of alginate. Based on distinct phenotype and phylogeny, we propose classification of strains AB-alg1T (JCM 35393T=UQM 41574T) and AB-alg4 as Verruconatronum alginivorum gen. nov., sp. nov. within a new family Verruconatronumaceae. ImportanceThe presented isolates are the first isolated representatives of an environmental family of Opitutales, part of the core microbiome of alkaline soda lakes. These bacteria feed on polysaccharides. We present the key enzymatic machinery for the polysaccharide breakdown. These enzymes are high-pH tolerant and have potential for industry applications, for example in washing powders and biomass waste recycling.

Auxotrophy, the inability of bacteria to synthesize one or multiple essential metabolites (e.g. amino acids, vitamins, metabolites) is thought to be common among bacteria. However, studies often rely either on bioinformatic tools to predict auxotrophies from genome data or on experiments with low numbers of strains. Here, we combine experimental and bioinformatic approaches to assess amino acid auxotrophy levels among 315 co-isolated natural Pseudomonas strains from pond and soil habitats. Both approaches revealed that Pseudomonas isolates are predominantly prototrophs. We identified one single histidine auxotroph and five non-specific auxotrophs featuring complex growth phenotypes incompatible with single amino acid auxotrophies. While different bioinformatic pipelines vary in the extent to which auxotrophy is over- or underestimated, none of the pipelines could resolve the basis of non-specific auxotrophies. Our analysis further revealed the existence of multiple alternative biosynthesis pathways for methionine, proline, and phenylalanine, with significant enrichments of specific pathways among soil or pond strains. We conclude that combining experiments with bioinformatics is a powerful approach to assess the metabolic potential of environmental bacteria. Moreover, taxa like Pseudomonas can be predominantly prototrophic possibly owing to their generalist lifestyle, thus calling for nuanced ecological concepts predicting auxotrophy levels based on lifestyle and habitat.

BackgroundLeptospirosis is a zoonotic disease caused by pathogenic Leptospira spp., which persist in soil and water environments for extended periods of time. The mechanisms enabling this environmental survival remain elusive. Free-living amoebae (FLA) are widespread protozoa that act as reservoirs or "Trojan horses" for numerous bacterial pathogens, protecting them from stress and contributing to their persistence. Whether pathogenic Leptospira exploit similar interactions with FLA has not been resolved. Methodology/Principal FindingsUsing live confocal microscopy, flow cytometry, and gentamicin protection assays, we investigated the interactions between pathogenic (Leptospira interrogans) and saprophytic (Leptospira biflexa) leptospires with three FLA species: Acanthamoeba castellanii, Dictyostelium discoideum, and Hartmannella vermiformis. While rapid internalization was observed, entry was only partially dependent on actin-driven processes and was enhanced by the presence of live bacteria. Following internalization, bacteria persisted for at least 48h as indicated by colony-forming assays. However, no evidence of intracellular replication was detected. The number of fluorescently labeled leptospires progressively declined over time, providing further evidence of leptospires survival without multiplication. Finally, analysis of environmental soils in New Caledonia showed co-occurrence of FLA and Leptospira. Soil-derived FLA also internalized pathogenic Leptospira in vitro, showing that these interactions extend to natural isolates. Conclusions/SignificanceOur results demonstrate that free-living amoebae internalize both pathogenic and saprophytic leptospires and allow their transient persistence without replication. By providing protection and prolonging viability in soil environments, FLA may contribute to the ecological maintenance of Leptospira. These findings pinpoint FLA as potential environmental reservoirs that could play a role in shaping leptospires survival strategies relevant for transmission and host infection. Author SummaryFor bacteria living in soils and freshwater environments, survival depends on their ability to adapt to complex ecological landscapes populated by numerous predators and competitors. In such habitats, interactions with other microorganisms are unavoidable and may shape long-term survival strategies. Pathogenic Leptospira, the bacteria responsible for leptospirosis, can persist for long periods outside their hosts, yet the ecological mechanisms supporting this environmental survival remain poorly understood. In soil and freshwater ecosystems, microscopic predators known as free-living amoebae commonly feed on bacteria. However, several bacterial pathogens can survive inside these amoebae and use them as temporary shelters. Because ancestral Leptospira were soil-dwelling saprophytes, interactions with amoebae likely represent an ancient ecological relationship in which successful survival strategies may have evolved and remain conserved in present-day pathogenic species. With this perspective in mind, we used microscopy approaches and bacterial viability assays to investigate whether Leptospira interacts with amoebae. We found that several amoeba species rapidly engulf both pathogenic and non-pathogenic Leptospira. Once internalized, the bacteria remained viable for up to two days but did not multiply. We also detected both amoebae and Leptospira in the same soil samples and showed that environmental amoebae could internalize the bacteria. These findings suggest that amoebae may act as temporary shelters for Leptospira, helping them persist in soils and water and potentially contributing to the environmental stage of leptospirosis transmission.

Favoured by global changes, freshwater cyanobacterial harmful blooms generate major ecological, economical and public health challenges. Microcystis, one of the most widespread cyanobacterial genera, grows within a phycosphere where specialised interactions with its microbiome occur, and are suspected to influence bloom appearance and its potential toxicity. Using a combination of metagenomic, metabolomic and metabolic modelling, we characterised the phycospheres of twelve Microcystis strains isolated from a French pond. The distribution of metabolic reactions within Microcystis was consistent with their genospecies, whereas the metabolic landscape at the community level diverged from cyanobacterial phylogeny indicating functional decoupling between cyanobacteria and their associated microbiomes. Phycosphere-associated bacteria substantially expand the metabolic repertoire of the system, while maintaining functional redundancy within and across communities. On the other hand, metabolomic profiles were largely driven by cyanobacterial metabolic outputs. Metabolic modelling, together with the identification of toxic specialised metabolites produced by specific biosynthetic gene clusters, further highlighted differences in metabolic potential among phycospheres. Together, these findings deepen the understanding of Microcystis phycosphere functioning, demonstrate the value of multi-omics systems biology approaches, and underscore the ecological relevance of interspecies and inter-phycosphere metabolic interactions as a structuring process in bloom-associated microbiomes.

The genus Exiguobacterium comprises Gram-positive, non-spore-forming, facultative anaerobic bacteria known for their remarkable adaptability to extreme environments, including soils, hot springs, glaciers, and the gastrointestinal tracts of certain organisms. Despite their unique adaptations for surviving in extreme environments, their pathogenicity is well documented. Here, we analyzed the phenotypical traits of two Mexican strains of Exiguobacterium--JVH47, isolated from contaminated urban sediments in Mexico City, and P4526, from the less human-impacted Cuatro Cienegas Basin. Furthermore, strains were related via comparative genomics using publicly available genomes. Phenotypic characterization demonstrated that both strains thrive across a wide range of temperatures (20-50 {degrees}C), pH (7-11), and salinity (up to 7% NaCl). Although sensitive to erythromycin, the JVH47 strain exhibited higher erythromycin resistance and harbored antibiotic resistance genes. This study underscores the ecological versatility of Exiguobacterium and its potential role as a reservoir for antibiotic resistance genes. While rarely associated with human infections, its ability to survive in extreme conditions and form biofilms raises concerns for immunocompromised individuals. These findings highlight the need for careful consideration of Exiguobacterium in biotechnological applications and its implications under the One Health framework.

Sourdough starters contain simple microbial communities typically consisting of a few bacterial species and one or two yeast species. The yeast Maudiozyma humilis and the lactic acid bacterium Fructilactobacillus sanfranciscensis often co-occur in sourdough starters, and have been presumed to exist in a trophic relationship supported by glucose cross-feeding. However, previous research has highlighted a lack of evidence showing that yeast strains consume the glucose that F. sanfranciscensis produces. We have investigated the interaction between sourdough isolates of M. humilis and F. sanfranciscensis in a synthetic wheat sourdough medium, allowing us to control substrate composition and use flow cytometry to enumerate living and dead cells. M. humilis fitness was found to be lower in co-culture with F. sanfranciscensis than when grown alone. Analysis of spent medium composition highlighted the reliance of M. humilis on glucose rather than maltose for growth. Comparisons of predicted and measured co-culture metabolite content also revealed that F. sanfranciscensis consumed less maltose in co-culture than when grown alone. For the first time, we examined potential amino acid cross-feeding between M. humilis and F. sanfranciscensis, and found that within the pairing, F. sanfranciscensis was the main producer of amino acids. Our findings suggest that the M. humilis-F. sanfranciscensis interaction is likely to be neutral, or even competitive, with the strain identity of F. sanfranciscensis playing a defining role in the observed dominance of the bacteria and spent medium metabolite composition. ImportanceThe association of the yeast Maudiozyma humilis and the bacterium Fructilactobacillus sanfranciscensis in sourdough starters is well-documented, and together this pairing makes key functional and organoleptic contributions to the final bread product. Their relationship has historically been thought to be stabilised by cross-feeding of glucose to M. humilis. However, this theory has been drawn into question by recent research which found no evidence that M. humilis consumes the glucose produced by F. sanfranciscensis. Our understanding of cooperation, coexistence, and competition in microbial consortia affects approaches to ecosystem management in a broad variety of applied fields. The significance of our research is in demonstrating that this pairing does not interact mutualistically within a specified setting, providing support for neutral or competitive interactions as drivers of ecological stability. Research areas:

BackgroundGlobally, seagrass ecosystems are threatened by anthropogenic activities that are leading to increased levels of eutrophication, coastal pollution and thermal conditions. Consequently, there is a growing need to develop new approaches that work to mitigate these stressors and enhance restoration efforts in seagrass meadows. One promising strategy is to identify, isolate and characterize microbial consortia that are likely to support seagrass productivity. However, our current understanding of key microbial functions that support plant growth in marine systems is limited. Based on evidence from terrestrial plant-microbe systems, seagrass-associated bacteria are expected to provide the plant with nitrogen and phosphorus resources while detoxifying sulfur and producing phytohormones. Here, we sequenced 61 bacterial cultures isolated from the rhizosphere, rhizoplane, and endosphere of the seagrass, Zostera marina to identify a consortium of six putative plant growth promoting (PGP) candidates. ResultsOur cultivation approach using plant-based media allowed us to isolate 201 bacteria from Z. marina, which reflected 18% of the total microbial diversity of the starting inoculum. Genomic and phenotypic analyses of the 61-sequenced pure-cultures revealed that most of the sequenced taxa were able to mobilize nitrogen primarily through catabolic pathways, including denitrification (51%), dissimilatory nitrate reduction to ammonia (71%), and C-N bond cleavage (83%). Six of the isolates, which represent new lineages of Agarivorans, coded for the nitrogenase gene cassette. Additionally, 52% of the genomes had genes for sulfur and/or thiosulfate oxidation, 88.5% for phosphorus solubilization, and 60.5% for IAA production. Genomic analysis also revealed that some pathways, including denitrification and dissimilatory nitrite to ammonia DNRA, required cross-species cooperation as no one taxa contained all the genes needed to complete these metabolic pathways. Based on draft genome models and results from phenotypic assays, isolates Streptomyces sp. (Iso23 and Iso384), Mesobacillus sp (Iso127), Roseibuim sp. (Iso195), Peribacillus sp. (Iso49), and Agarivorans sp. (Iso311) represent a minimal microbial community that is likely to promote seagrass growth and enhance restoration efforts. ConclusionOur work provides a detailed genomic and phenotypic analysis of bacteria isolated from Z. marina and identifies a minimal microbial community with complementary PGP traits. Isolating, identifying and characterizing bacteria that promote seagrass growth is critical towards enhancing restoration efforts of seagrass meadows.

Bacterial adaptation to fluctuations in salinity includes the intracellular accumulation of organic compounds called compatible solutes (CS) such as the amino acid derivatives ectoine and 5-hydroxyectoine. These compounds also play a less appreciated role as readily available nutrients, scavenged from dissolved organic matter in both marine and terrestrial environments. Vibrio diabolicus is a marine bacterium originally isolated from deep-sea hydrothermal vents and later shown to have worldwide distribution. In this work, we demonstrated the biosynthesis and uptake of CS ectoine and glycine betaine under high osmotic stress conditions, but not in unstressed V. diabolicus cells. A region on chromosome 1 of V. diabolicus strain 3098 encoded homologues of genes for ectoine and 5-hydroxyectoine catabolism (eutDE, ssd_atf_eutBCA), regulation (asnC, enuR), and transport (ectoine TRAP-type uehP, uehQM). Our data showed that ectoine was used as a high energy yielding sole carbon source and the eutD gene was essential for ectoine consumption. Phylogenetics based on EutD (DoeA) and gene neighborhood analyses showed that a catabolism cluster was present in Proteobacteria, Thermosulfobacteriota, Bacillota, Actinomycetota, and Archaea. The cluster had a limited phylogenetic distribution in Gammaproteobacteria and Betaproteobacteria and was widespread in Alphaproteobacteria. Phylogenetic reconstruction was consistent with vertical inheritance with gene loss with repeated horizontal acquisitions of the pathway across lineages. The ectoine catabolism pathway was vertically inherited in Halomonadaceae and Vibrionaceae, with patterns of gene and pathway loss. Betaproteobacteria Burkholderia, Caballeronia, and Paraburkholderia EutD proteins clustered together and EutD from most Pseudomonas species shared a most recent common with this group. EutD from Alphaproteobacteria branched in eight divergent clusters with long branch lengths but showed a remarkable conservation of synteny. Catabolism and transporter genes in this group were contiguous and contained either a TRAP-type UehPQM or an ABC-type EhuABCD ectoine transporter. Gram-positive bacteria and Archaea were not previously shown to consume ectoines, however, we identified putative ectoine catabolism clusters among Bacilli, Clostridia, Actinomycetes, and Halobacteria. IMPORTANCEEctoine is a well-established CS used to overcome osmotic stress, produced by a wide range of bacteria. The demonstration of ectoine biosynthesis and catabolism in V. diabolicus showed that it is conditionally utilized as an osmoprotectant or a nutrient source depending on environmental cues. The conservation of large syntenic blocks of ectoine catabolism, transport, and regulatory genes suggested strong selective pressure to maintain this trait. EutD (DoeA) phylogeny patterns largely followed taxonomy with evidence of horizontal transfer in specific clades, and showed ectoine consumption has a broad taxonomic spread and is lineage enriched. In our dataset, Alphaproteobacteria contained the largest diversity of EutD lineages; Gammaproteobacteria from marine environments formed a strong secondary group; and EutD from Betaproteobacteria were the least diverse. Many species that contained EutD are associated with saline, marine, and plant-associated niches, where ectoine can be scavenged as a nutrient source. The identification of a putative ectoine catabolism pathway in Gram-positive bacteria and Archaea needs to be experimentally confirmed and suggests undiscovered diversity to be revealed by future genome sequencing.

The role of microbial strains in regulating natural stresses and their impact on plant health is well-established. However, the role of microbial tolerance mechanisms in plant response to unnatural or anthropogenic stresses is less understood. Examination of these interactions impact our deeper understanding of plant-microbe interactions and our ability to enhance beneficial functions. In this study we use the model plant Brachypodium distachyon and its prominent root colonizer Paraburkholderia graminis OAS925 to investigate mechanisms of tolerance to alcohol and salt stress. We examined the ability of OAS925 to reduce root growth inhibition during exposure to short chain alcohols and salt. We also examined the tolerance mechanism for OAS925 towards these stresses using RB-TnSeq fitness assays. The most prominent tolerance systems in OAS925 are genes specifically involved in membrane transport (such as the Mla operon), efflux systems (e.g., RND efflux systems), signaling and regulation (PrtR/PrtI, NtrY/NtrX, and EnvZ/OmpR), and oxidative stress response (GshB). Our findings provide a model where bacterial membrane integrity, active solvent efflux, and stress signaling are crucial not only for bacterial survival but also for maintaining the root colonization and biofilm formation that confer protection to the host plant.

Myrmecocystus honeypot ants rely on specialized workers, repletes, to store dissolved carbohydrates in their crops long term. The repletes store this liquid, which does not spoil in their crops, for many months at a time. When resources are scarce, repletes redistribute the stored nutrients to their colony members via trophallaxis. While we suspect that the gut microbiome of honeypot ants may aid in spoilage prevention, before we can investigate this, we must first characterize it. Here, we used 16S rRNA gene sequencing to determine the microbial community composition across six Myrmecocystus honeypot ant species, sampling multiple colonies, castes, and organs. We found that microbiome community composition was strongly shaped by species, with variation between colonies in M. arenarius, M. depilis, and M. mexicanus. Organ level differences were observed in the crop and midgut in M. mexicanus. Caste differences were observed in M. flaviceps and M. mexicanus. Replete crops of M. mexicanus and M. depilis were enriched in Fructilactobacillus, other lactic acid bacteria, and acetic acid bacteria, whereas halophiles were more prominent in the gut of species such as M. flaviceps and M. wheeleri. In this study we demonstrate that Myrmecocystus ants host species-specific gut microbiomes and identify an association between lactic acid bacteria, acetic acid bacteria, and halophiles within replete crops. While much work remains in understanding the roles of the microbes in the symbiosis with their host ants, the dominance of these particular taxonomic groups suggests an association with a high sugar environment and a potential microbial role in preventing spoilage of the crop contents.

Three novel bioluminescent bacterial strains, VNB-15T, VNB-16 and SChm4, were isolated from water of the Black Sea (Russia) and intestines of the Black Sea horse mackerel. Cells of the isolated strains are motile Gram negative slightly curved rods with single polar flagellum. The temperature range for growth was 10-35{degrees}C, the optimum being 20-25{degrees}C. The pH range for growth was 6.0-9.0, the optimum being 7.0-8.0. The bacteria were able to grow in the presence of 0.5 to 5.0% NaCl (w/v), the optimum being 1.0-4.0% (w/v). Phylogenetic analysis based on comparison of 16S rRNA sequences shows these strains to have kinship with the species Vibrio jasicida, Vibrio hyugaensis, Vibrio alginolyticus, Vibrio campbelli, Vibrio rotiferianus, Vibrio harveyi and Vibrio owensii with sequence similarity from 99.6 to 98.0%. Phylogenetic analysis based on comparison of the sequences of genes gyrB, recA, pyrH, gapA, rpoA, mreB, ftsZ, topA shows that the strains VNB-15T, VNB-16 and SChm4 to form a cluster within the V. harveyi clade and belong to a new species of the Vibrio genus. Comparison of the complete genomic sequence of VNB-15T with typical strains of nearby species also indicates that VNB-15T belongs to a separate species (maximum similarity 98% with V. hyugaensis and 96% with V. jasicida). VNB-15T differs from closely related species by its ability to utilize glucose, mannitol, inositol, sorbitol, rhamnose and sucrose, and to form lysine decarboxylase, ornithine decarboxylase, lipase, acid phosphatase, -glucosidase, {beta}-glucosidase and N-acetyl-{beta}-D-glucosaminidase enzymes. Based on phylogenetic analysis and phenotypic characteristics, Vibrio aquamarinus sp. nov. is proposed. The type strain is VNB-15T (= VKPM B-11245T = DSM 26054 T). RepositoriesThe GenBank accession numbers for the gapA, 16S rRNA, gyrB, pyrH, rpoA, recA, mreB, ftsZ, topA genes sequences of strain VNB-15T are JQ319116-JQ319121, KX242381, KX 242384, KX242387, respectively. The GenBank accession numbers for the gapA, gyrB, pyrH, recA, rpoA,16S rRNA, mreB, ftsZ, topA genes sequences of strain VNB-16 are KP221561-KP221566, KX242382, KX 242385, KX242388 respectively. The GenBank accession numbers for the 16S rRNA, gapA gyrB, rpoA, recA, pyrH, mreB, ftsZ, topA genes sequences of strain SChm4 are KX242375-KX242380, KX242383, KX 242386, KX242389, respectively. The GenBank/EMBL/DDBJ accession numbers for the housekeeping gene sequences used in this study are detailed in supplementary Table S1, Figures S1-S8. The genome of Vibrio aquamarinus sp. nov. VNB-15T, comprising two chromosomes and a plasmid, has been assembled and deposited in the NCBI database under the submission number SUB14585067

Mixotrophy is emerging as a default nutritional strategy in phytoplankton but research seems so far isolated and mostly focussing on single phytoplankton groups or strains. Here we combined data from 24 oceanic and 22 freshwater strains - as well as results from other studies - to analyze phytoplanktons ability to utilize dissolved organic compounds and highlight potential influencing factors. The results emphasize that mixotrophy is ubiquitous in phytoplankton across functional groups and taxa isolated from various habitats, and not strictly dependent on light or nutrient deficiencies. Several factors such as taxonomic affiliation, temperature and growth phase can affect mixotrophic behavior but no consistent patterns have emerged regarding their effects. Hence, mixotrophic traits remain so far unpredictable. There is some indication that the strains origin - potentially through adaptation to habitat DOM availability - might predetermine phytoplanktons mixotrophic skills. For example, freshwater strains used overall more compounds than oceanic strains in our study and Ostreococcus exhibited a different use pattern depending on its origin. Nevertheless, many aspects of mixotrophy in phytoplankton - e.g. metabolic pathways - remain cryptic. By summarizing available knowledge and knowledge gaps, the present synthesis provides a guideline for upcoming research further exploring mixotrophy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/703725v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@12ac311org.highwire.dtl.DTLVardef@6c98deorg.highwire.dtl.DTLVardef@1a837ddorg.highwire.dtl.DTLVardef@eb9b53_HPS_FORMAT_FIGEXP M_FIG C_FIG