Flagellar interference with plasmid uptake in biofilms: a joint experimental and modeling study.

Plasmid conjugation is a key facilitator of horizontal gene transfer (HGT), and plasmids encoding antibiotic resistance drive the increasing prevalence of antibiotic resistance. In natural, engineered, and clinical environments, bacteria often grow in protective biofilms. Therefore, a better understanding of plasmid transfer in biofilms is needed. Our aim was to investigate plasmid transfer in a biofilm-adapted wrinkly colony mutant of Xanthomonas retroflexus (XRw) with enhanced matrix production and reduced motility. We found that XRw biofilms had an increased uptake of the broad host-range IncP-1ϵ plasmid pKJK5 compared to the wild type (WT). Proteomics revealed fewer flagellar-associated proteins in XRw, suggesting that flagella were responsible for reducing plasmid uptake. This was confirmed by the higher plasmid uptake of non-flagellated fliM mutants of the X. retroflexus wrinkly mutant as well as the wild type. Moreover, testing several flagellar mutants of Pseudomonas putida suggested that the flagellar effect was more general. We identified seven mechanisms with the potential to explain the flagellar effect and simulated them in an individual-based model. Two mechanisms could thus be eliminated (increased distances between cells and increased lag times due to flagella). Another mechanism identified as viable in the modeling was eliminated by further experiments. The possibility of steric hindrance of pilus movement and binding by flagella, reducing the frequency of contact and thus plasmid uptake, proved viable, and the three other viable mechanisms had a reduced probability of plasmid transfer in common. Our findings highlight the important yet complex effects of flagella during bacterial conjugation in biofilms.IMPORTANCEBiofilms are the dominant form of microbial life and bacteria living in biofilms are markedly different from their planktonic counterparts, yet the impact of the biofilm lifestyle on horizontal gene transfer (HGT) is still poorly understood. Horizontal gene transfer by conjugative plasmids is a major driver in bacterial evolution and adaptation, as exemplified by the troubling spread of antibiotic resistance. To either limit or promote plasmid prevalence and dissemination, we need a better understanding of plasmid transfer between bacterial cells, especially in biofilms. Here, we identified a new factor impacting the transfer of plasmids, flagella, which are required for many types of bacterial motility. We show that their absence or altered activity can lead to enhanced plasmid uptake in two bacterial species, Xanthomonas retroflexus and Pseudomonas putida. Moreover, we demonstrate the utility of mathematical modeling to eliminate hypothetical mechanisms.

Biofilm cultivation facilitates coexistence and adaptive evolution in an industrial bacterial community.

The majority of ecological, industrial and medical impacts of bacteria result from diverse communities containing multiple species. This diversity presents a significant challenge as co-cultivation of multiple bacterial species frequently leads to species being outcompeted and, with this, the possibility to manipulate, evolve and improve bacterial communities is lost. Ecological theory predicts that a solution to this problem will be to grow species in structured environments, which reduces the likelihood of competitive exclusion. Here, we explored the ability of cultivation in a structured environment to facilitate coexistence, evolution, and adaptation in an industrially important community: Lactococcus lactis and Leuconostoc mesenteroides frequently used as dairy starter cultures. As commonly occurs, passaging of these two species together in a liquid culture model led to the loss of one species in 6 of 20 lineages (30%). By contrast, when we co-cultured the two species as biofilms on beads, a stable coexistence was observed in all lineages studied for over 100 generations. Moreover, we show that the co-culture drove evolution of new high-yield variants, which compared to the ancestor grew more slowly, yielded more cells and had enhanced capability of biofilm formation. Importantly, we also show that these high-yield biofilm strains did not evolve when each species was passaged in monoculture in the biofilm model. Therefore, both co-culture and the biofilm model were conditional for these high-yield strains to evolve. Our study underlines the power of ecological thinking-namely, the importance of structured environments for coexistence-to facilitate cultivation, evolution, and adaptation of industrially important bacterial communities.

Metabolic Profiling of Interspecies Interactions During Sessile Bacterial Cultivation Reveals Growth and Sporulation Induction in Paenibacillus amylolyticus in Response to Xanthomonas retroflexus.

The toolbox available for microbiologists to study interspecies interactions is rapidly growing, and with continuously more advanced instruments, we are able to expand our knowledge on establishment and function of microbial communities. However, unravelling molecular interspecies interactions in complex biological systems remains a challenge, and interactions are therefore often studied in simplified communities. Here we perform an in-depth characterization of an observed interspecies interaction between two co-isolated bacteria, Xanthomonas retroflexus and Paenibacillus amylolyticus. Using microsensor measurements for mapping the chemical environment, we show how X. retroflexus promoted an alkalization of its local environment through degradation of amino acids and release of ammonia. When the two species were grown in proximity, the modified local environment induced a morphological change and growth of P. amylolyticus followed by sporulation. 2D spatial metabolomics enabled visualization and mapping of the degradation of oligopeptide structures by X. retroflexus and morphological changes of P. amylolyticus through e.g. the release of membrane-associated metabolites. Proteome analysis and microscopy were used to validate the shift from vegetative growth towards sporulation. In summary, we demonstrate how environmental profiling by combined application of microsensor, microscopy, metabolomics and proteomics approaches can reveal growth and sporulation promoting effects resulting from interspecies interactions.

Biofilms can act as plasmid reserves in the absence of plasmid specific selection.

Plasmids facilitate rapid bacterial adaptation by shuttling a wide variety of beneficial traits across microbial communities. However, under non-selective conditions, maintaining a plasmid can be costly to the host cell. Nonetheless, plasmids are ubiquitous in nature where bacteria adopt their dominant mode of life - biofilms. Here, we demonstrate that biofilms can act as spatiotemporal reserves for plasmids, allowing them to persist even under non-selective conditions. However, under these conditions, spatial stratification of plasmid-carrying cells may promote the dispersal of cells without plasmids, and biofilms may thus act as plasmid sinks.

Impact of Tellurite on the Metabolism of Paenibacillus pabuli AL109b With Flagellin Production Explaining High Reduction Capacity.

Tellurium (Te) is a metalloid with scarce and scattered abundance but with an increased interest in human activity for its uses in emerging technologies. As is seen for other metals and metalloids, the result of mining activity and improper disposal of high-tech devices will lead to niches with increased abundance of Te. This metalloid will be more available to bacteria and represent an increasing selective pressure. This environmental problem may constitute an opportunity to search for microorganisms with genetic and molecular mechanisms of microbial resistance to Te toxic anions. Organisms from Te-contaminated niches could provide tools for Te remediation and fabrication of Te-containing structures with added value. The objective of this study was to determine the ability of a high metal-resistant Paenibacillus pabuli strain ALJ109b, isolated from high metal content mining residues, to reduce tellurite ion, and to evaluate the formation of metallic tellurium by cellular reduction, isolate the protein responsible, and determine the metabolic response to tellurite during growth. P. pabuli ALJ109b demonstrated to be resistant to Te (IV) at concentrations higher than reported for its genus. It can efficiently remove soluble Te (IV) from solution, over 20% in 8 h of growth, and reduce it to elemental Te, forming monodisperse nanostructures, verified by scattering electron microscopy. Cultivation of P. pabuli ALJ109b in the presence of Te (IV) affected the general protein expression pattern, and hence the metabolism, as demonstrated by high-throughput proteomic analysis. The Te (IV)-induced metabolic shift is characterized by an activation of ROS response. Flagellin from P. pabuli ALJ109b demonstrates high Te (0) forming activity in neutral to basic conditions in a range of temperatures from 20°C to 37°C. In conclusion, the first metabolic characterization of a strain of P. pabuli response to Te (IV) reveals a highly resistant strain with a unique Te (IV) proteomic response. This strain, and its flagellin, display, all the features of potential tools for Te nanoparticle production.

Metagenomic analysis of a keratin-degrading bacterial consortium provides insight into the keratinolytic mechanisms.

Keratin is an insoluble fibrous protein from natural environments, which can be recycled to value-added products by keratinolytic microorganisms. A microbial consortium with efficient keratinolytic activity was previously enriched from soil, but the genetic basis behind its remarkable degradation properties was not investigated yet. To identify the metabolic pathways involved in keratinolysis and clarify the observed synergy among community members, shotgun metagenomic sequencing was performed to reconstruct metagenome-assembled genomes. More than 90% genera of the enriched bacterial consortium were affiliated to Chryseobacterium, Stenotrophomonas, and Pseudomonas. Metabolic potential and putative keratinases were predicted from the metagenomic annotation, providing the genetic basis of keratin degradation. Furthermore, metabolic pathways associated with keratinolytic processes such as amino acid metabolism, disulfide reduction and urea cycle were investigated from seven high-quality metagenome-assembled genomes, revealing the potential metabolic cooperation related to keratin degradation. This knowledge deepens the understanding of microbial keratinolytic mechanisms at play in a complex community, pinpointing the significance of synergistic interactions, which could be further used to optimize industrial keratin degradation processes.

The T-shirt microbiome is distinct between individuals and shaped by washing and fabric type.

Activity of the microbial population in clothing causes unpleasant odor and textile deterioration. However, little is known about how the textile microbial community is shaped. In this study, we developed a method for extracting DNA from small amounts of detergent-washed clothing, and applied it to both worn and unworn, washed and unwashed cotton and polyester samples of the axillary region of T-shirts from 10 male subjects. The combined application of 16S rRNA gene amplicon sequencing and quantitative PCR allowed us to estimate the absolute abundances of bacteria in the samples. We found that the T-shirt microbiome was highly individual, both in composition, diversity and microbial biomass. Fabric type was influential where Acinetobacter was more abundant in cotton. Intriguingly, unworn cotton T-shirts had a native microbiome dominated by Acinetobacter, whereas unworn polyester had no detectable bacterial microbiome. The native textile microbiome did not seem to have any effect on the microbial composition emerging from wearing the garment. Surprisingly, washing in mild detergent had only minor effects on the composition and biomass of the microbial community, and only few Amplicon Sequence Variants (ASV)s were found to decrease in abundance after washing. Individual variations between test subjects shaped the microbial community more than the type of fabric or wash with detergent. The individuality of T-shirt microbiomes and specificity of the washing procedure suggests that personalized laundry regimes could be applied to increase efficient removal of undesired bacteria.

Community-intrinsic properties enhance keratin degradation from bacterial consortia.

Although organic matter may accumulate sometimes (e.g. lignocellulose in peat bog), most natural biodegradation processes are completed until full mineralization. Such transformations are often achieved by the concerted action of communities of interacting microbes, involving different species each performing specific tasks. These interactions can give rise to novel "community-intrinsic" properties, through e.g. activation of so-called "silent genetic pathways" or synergistic interplay between microbial activities and functions. Here we studied the microbial community-based degradation of keratin, a recalcitrant biological material, by four soil isolates, which have previously been shown to display synergistic interactions during biofilm formation; Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus. We observed enhanced keratin weight loss in cultures with X. retroflexus, both in dual and four-species co-cultures, as compared to expected keratin degradation by X. retroflexus alone. Additional community intrinsic properties included accelerated keratin degradation rates and increased biofilm formation on keratin particles. Comparison of secretome profiles of X. retroflexus mono-cultures to co-cultures revealed that certain proteases (e.g. serine protease S08) were significantly more abundant in mono-cultures, whereas co-cultures had an increased abundance of proteins related to maintaining the redox environment, e.g. glutathione peroxidase. Hence, one of the mechanisms related to the community intrinsic properties, leading to enhanced degradation from co-cultures, might be related to a switch from sulfitolytic to proteolytic functions between mono- and co-cultures, respectively.

Heavy metal exposure causes changes in the metabolic health-associated gut microbiome and metabolites.

BACKGROUND: Exposure to arsenic and cadmium is common. Epidemiological and animal studies have suggested that exposure to these two heavy metals can cause metabolic health problems, including type 2 diabetes (T2DM). It has been hypothesized that T2DM could be mediated through the gut microbiome and the metabolites it produces. Although many studies have investigated the association between the gut microbiome and T2DM, few have focused on the connection to arsenic and cadmium. RESULTS: We applied 16S rRNA gene amplicon sequencing and untargeted LC-MS/MS metabolomics to examine the changes in the gut microbiome and metabolite profiles of exposed mice to relevant levels of cadmium and arsenic in the drinking water over two weeks. Cadmium chloride (Cd) exposure significantly changed the mice gut microbiome and resulted in a significantly lower microbial diversity whereas sodium arsenite (As) caused a non-significant decrease in microbial diversity. For Cd and As treatment respectively, we identified 5 and 2 phyla with significant changes and 42 and 24 genera. Bacterial genera that were observed to decline upon both treatments, included several butyrate-producers. Both As and Cd treatment perturbed the metabolome significantly, with 50 ppm Cd compound exposure having the greatest effect when compared to 50 ppm As compound exposure. Two unidentified features were differentially abundant in the As group, while 33 features changed in the Cd group. Differential abundance analysis of all bile acid associated molecular components showed differences under both treatments. Finally, integrative network analysis via bipartite correlation networks suggested that several genera, including the metabolically important Blautia, Eisenbergiella, Clostridium_XlVa, etc. declined in numbers of metabolite interactions. CONCLUSIONS: These results demonstrated that As and Cd exposure caused significant changes to the gut microbiome and metabolome by affecting bile acids, amino acids and taxa associated with metabolic health.

Construction of Simplified Microbial Consortia to Degrade Recalcitrant Materials Based on Enrichment and Dilution-to-Extinction Cultures.

The capacity of microbes to degrade recalcitrant materials has been extensively explored for environmental remediation and industrial production. Significant achievements have been made with single strains, but focus is now going toward the use of microbial consortia owning to their functional stability and efficiency. However, assembly of simplified microbial consortia (SMC) from complex environmental communities is still far from trivial due to large diversity and the effect of biotic interactions. Here we propose a strategy, based on enrichment and dilution-to-extinction cultures, to construct SMC with reduced diversity for degradation of keratinous materials. Serial dilutions were performed on a keratinolytic microbial consortium pre-enriched from a soil sample, monitoring the dilution effect on community growth and enzymatic activities. An appropriate dilution regime (10-9) was selected to construct a SMC library from the enriched microbial consortium. Further sequencing analysis and keratinolytic activity assays demonstrated that obtained SMC displayed actual reduced microbial diversity, together with various taxonomic composition, and biodegradation capabilities. More importantly, several SMC possessed equivalent levels of keratinolytic efficiency compared to the initial consortium, showing that simplification can be achieved without loss of function and efficiency. This methodology is also applicable to other types of recalcitrant material degradation involving microbial consortia, thus considerably broadening its application scope.

Enrichment and characterization of an environmental microbial consortium displaying efficient keratinolytic activity.

Keratin refers to a group of insoluble and recalcitrant protein materials. Slaughterhouses produce large amount of keratinous byproducts, which are either disposed or poorly valorized through costly thermochemical processes for animal feed formulation. Learning from nature, keratinolytic microbial consortia stand as a cost-efficient and environmental friendly way to valorize this recalcitrant resource. Directed selection was applied to enrich soil-born microbial consortia, using sequential batch cultivations in keratin medium, while measuring enzymes activity and monitoring consortia compositions via 16S rRNA gene amplicon sequencing. A promising microbial consortium KMCG6, featuring mainly members of Bacteroidetes and Proteobacteria, was obtained. It possessed keratinolytic activity with <25% residual substrate remaining, which also displayed a high degradation reproducibility level after long-term cryopreservation. This work represents an advance in the field of α-keratin degradation with potential for practical applications.

In Vitro Community Synergy between Bacterial Soil Isolates Can Be Facilitated by pH Stabilization of the Environment.

The composition and development of naturally occurring microbial communities are defined by a complex interplay between the community and the surrounding environment and by interactions between community members. Intriguingly, these interactions can in some cases cause synergies, where the community is able to outperform its single-species constituents. However, the underlying mechanisms driving community interactions are often unknown and difficult to identify due to high community complexity. Here, we show how opposite pH drift induced by specific community members leads to pH stabilization of the microenvironment, acting as a positive interspecies interaction, driving in vitro community synergy in a model consortium of four coisolated soil bacteria, Microbacterium oxydans, Xanthomonas retroflexus, Stenotrophomonas rhizophila, and Paenibacillus amylolyticus We use microsensor pH measurements to show how individual species change the local pH microenvironment and how cocultivation leads to a stabilized pH regime over time. Specifically, in vitro acid production from P. amylolyticus and alkali production primarily from X. retroflexus led to an overall pH stabilization of the local environment over time, which in turn resulted in enhanced community growth. This specific type of interspecies interaction was found to be highly dependent on medium type and concentration; however, similar pH drift from the individual species could be observed across medium variants.IMPORTANCE Understanding interspecies interactions in bacterial communities is important for unraveling species dynamics in naturally occurring communities. These dynamics are fundamental for identifying evolutionary drivers and for the development of efficient biotechnological industry applications. Recently, pH interplay among community members has been identified as a factor affecting community development, and pH stabilization has been demonstrated to result in enhanced community growth. The use of model communities in which the effect of changing pH level can be attributed to specific species contributes to the investigation of community developmental drivers. This contributes to assessment of the extent of emergent behavior and members' contributions to community development. Here, we show that pH stabilization of the microenvironment in vitro in a synthetic coisolated model community results in synergistic growth. This observation adds to the growing diversity of community interactions leading to enhanced community growth and hints toward pH as a strong driver for community development in diverse environments.

Enhanced bacterial mutualism through an evolved biofilm phenotype.

Microbial communities primarily consist of multiple species that affect one another's fitness both directly and indirectly. This study showed that the cocultivation of Paenibacillus amylolyticus and Xanthomonas retroflexus exhibited facultative mutualistic interactions in a static environment, during the course of which a new adapted phenotypic variant of X. retroflexus appeared. Although the emergence of this variant was not directly linked to the presence of P. amylolyticus, its establishment in the coculture enhanced the productivity of both species due to mutations that stimulated biofilm formation. The mutations were detected in genes encoding a diguanylate cyclase predicted to synthesise cyclic-di-GMP. Examinations of the biofilm formed in cocultures of P. amylolyticus and the new variant of X. retroflexus revealed a distinct spatial organisation: P. amylolyticus only resided in biofilms in association with X. retroflexus and occupied the outer layers. The X. retroflexus variant therefore facilitated increased P. amylolyticus growth as it produced more biofilm biomass. The increase in X. retroflexus biomass was thus not at the expense of P. amylolyticus, demonstrating that interspecies interactions can shape diversification in a mutualistic coculture and reinforce these interactions, ultimately resulting in enhanced communal performance.

Disease-induced assemblage of a plant-beneficial bacterial consortium.

Disease suppressive soils typically develop after a disease outbreak due to the subsequent assembly of protective microbiota in the rhizosphere. The role of the plant immune system in the assemblage of a protective rhizosphere microbiome is largely unknown. In this study, we demonstrate that Arabidopsis thaliana specifically promotes three bacterial species in the rhizosphere upon foliar defense activation by the downy mildew pathogen Hyaloperonospora arabidopsidis. The promoted bacteria were isolated and found to interact synergistically in biofilm formation in vitro. Although separately these bacteria did not affect the plant significantly, together they induced systemic resistance against downy mildew and promoted growth of the plant. Moreover, we show that the soil-mediated legacy of a primary population of downy mildew infected plants confers enhanced protection against this pathogen in a second population of plants growing in the same soil. Together our results indicate that plants can adjust their root microbiome upon pathogen infection and specifically recruit a group of disease resistance-inducing and growth-promoting beneficial microbes, therewith potentially maximizing the chance of survival of their offspring that will grow in the same soil.

A meta-proteomics approach to study the interspecies interactions affecting microbial biofilm development in a model community.

Microbial biofilms are omnipresent in nature and relevant to a broad spectrum of industries ranging from bioremediation and food production to biomedical applications. To date little is understood about how multi-species biofilm communities develop and function on a molecular level, due to the complexity of these biological systems. Here we apply a meta-proteomics approach to investigate the mechanisms influencing biofilm formation in a model consortium of four bacterial soil isolates; Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus. Protein abundances in community and single species biofilms were compared to describe occurring inter-species interactions and the resulting changes in active metabolic pathways. To obtain full taxonomic resolution between closely related species and empower correct protein quantification, we developed a novel pipeline for generating reduced reference proteomes for spectral database searches. Meta-proteomics profiling indicated that community development is dependent on cooperative interactions between community members facilitating cross-feeding on specific amino acids. Opposite regulation patterns of fermentation and nitrogen pathways in Paenibacillus amylolyticus and Xanthomonas retroflexus may, however, indicate that competition for limited resources also affects community development. Overall our results demonstrate the multitude of pathways involved in biofilm formation in mixed communities.

From Sample to Multi-Omics Conclusions in under 48 Hours.

Multi-omics methods have greatly advanced our understanding of the biological organism and its microbial associates. However, they are not routinely used in clinical or industrial applications, due to the length of time required to generate and analyze omics data. Here, we applied a novel integrated omics pipeline for the analysis of human and environmental samples in under 48 h. Human subjects that ferment their own foods provided swab samples from skin, feces, oral cavity, fermented foods, and household surfaces to assess the impact of home food fermentation on their microbial and chemical ecology. These samples were analyzed with 16S rRNA gene sequencing, inferred gene function profiles, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics through the Qiita, PICRUSt, and GNPS pipelines, respectively. The human sample microbiomes clustered with the corresponding sample types in the American Gut Project (http://www.americangut.org), and the fermented food samples produced a separate cluster. The microbial communities of the household surfaces were primarily sourced from the fermented foods, and their consumption was associated with increased gut microbial diversity. Untargeted metabolomics revealed that human skin and fermented food samples had separate chemical ecologies and that stool was more similar to fermented foods than to other sample types. Metabolites from the fermented foods, including plant products such as procyanidin and pheophytin, were present in the skin and stool samples of the individuals consuming the foods. Some food metabolites were modified during digestion, and others were detected in stool intact. This study represents a first-of-its-kind analysis of multi-omics data that achieved time intervals matching those of classic microbiological culturing. IMPORTANCE Polymicrobial infections are difficult to diagnose due to the challenge in comprehensively cultivating the microbes present. Omics methods, such as 16S rRNA sequencing, metagenomics, and metabolomics, can provide a more complete picture of a microbial community and its metabolite production, without the biases and selectivity of microbial culture. However, these advanced methods have not been applied to clinical or industrial microbiology or other areas where complex microbial dysbioses require immediate intervention. The reason for this is the length of time required to generate and analyze omics data. Here, we describe the development and application of a pipeline for multi-omics data analysis in time frames matching those of the culture-based approaches often used for these applications. This study applied multi-omics methods effectively in clinically relevant time frames and sets a precedent toward their implementation in clinical medicine and industrial microbiology.

Genome Sequence of Psychrobacter cibarius Strain W1.

Here, we report the draft genome sequence of Psychrobacter cibarius strain W1, which was isolated at a slaughterhouse in Denmark. The 3.63-Mb genome sequence was assembled into 241 contigs.

Genome Sequence of Arthrobacter antarcticus Strain W2, Isolated from a Slaughterhouse.

We report the draft genome sequence ofArthrobacter antarcticusstrain W2, which was isolated from a wall of a small slaughterhouse in Denmark. The 4.43-Mb genome sequence was assembled into 170 contigs.

Genome Sequence of Kocuria palustris Strain W4.

We report the 3.09 Mb draft genome sequence ofKocuria palustrisW4, isolated from a slaughterhouse in Denmark.

Genome Sequence of Kocuria varians G6 Isolated from a Slaughterhouse in Denmark.

We report here the first draft genome sequence ofKocuria variansG6, which was isolated from a meat chopper at a small slaughterhouse in Denmark. The 2.90-Mb genome sequence consists of 95 contigs and contains 2,518 predicted protein-coding genes.