Integration of Phenotypes in Microbiome Networks for Designing Synthetic Communities: a Study of Mycobiomes in the Grafted Tomato System.

Understanding factors influencing microbial interactions, and designing methods to identify key taxa that are candidates for synthetic communities, or SynComs, are complex challenges for achieving microbiome-based agriculture. Here, we study how grafting and the choice of rootstock influences root-associated fungal communities in a grafted tomato system. We studied three tomato rootstocks (BHN589, RST-04-106, and Maxifort) grafted to a BHN589 scion and profiled the fungal communities in the endosphere and rhizosphere by sequencing the internal transcribed spacer (ITS2). The data provided evidence for a rootstock effect (explaining ~2% of the total captured variation, P < 0.01) on the fungal community. Moreover, the most productive rootstock, Maxifort, supported greater fungal species richness than the other rootstocks or controls. We then constructed a phenotype-operational taxonomic unit (OTU) network analysis (PhONA) using an integrated machine learning and network analysis approach based on fungal OTUs and associated tomato yield as the phenotype. PhONA provides a graphical framework to select a testable and manageable number of OTUs to support microbiome-enhanced agriculture. We identified differentially abundant OTUs specific to each rootstock in both endosphere and rhizosphere compartments. Subsequent analyses using PhONA identified OTUs that were directly associated with tomato fruit yield and others that were indirectly linked to yield through their links to these OTUs. Fungal OTUs that are directly or indirectly linked with tomato yield may represent candidates for synthetic communities to be explored in agricultural systems. IMPORTANCE The realized benefits of microbiome analyses for plant health and disease management are often limited by the lack of methods to select manageable and testable synthetic microbiomes. We evaluated the composition and diversity of root-associated fungal communities from grafted tomatoes. We then constructed a phenotype-OTU network analysis (PhONA) using these linear and network models. By incorporating yield data in the network, PhONA identified OTUs that were directly predictive of tomato yield and others that were indirectly linked to yield through their links to these OTUs. Follow-up functional studies of taxa associated with effective rootstocks, identified using approaches such as PhONA, could support the design of synthetic fungal communities for microbiome-based crop production and disease management. The PhONA framework is flexible for incorporation of other phenotypic data, and the underlying models can readily be generalized to accommodate other microbiome or 'omics data.

Spatial Structure Formation by RsmE-Regulated Extracellular Secretions in Pseudomonas fluorescens Pf0-1.

Cells in microbial communities on surfaces live and divide in close proximity, which greatly enhances the potential for social interactions. Spatiogenetic structures are manifested through competitive and cooperative interactions among the same and different genotypes within a shared space, and extracellular secretions appear to function dynamically at the forefront. A previous experimental evolution study utilizing Pseudomonas fluorescens Pf0-1 colonies demonstrated that diverse mutations in the rsmE gene were repeatedly and exclusively selected through the formation of a dominant spatial structure. RsmE's primary molecular function is translation repression, and its homologs regulate various social and virulence phenotypes. Pseudomonas spp. possess multiple paralogs of Rsm proteins, and RsmA, RsmE, and RsmI are the most prevalent. Here, we demonstrate that the production of a mucoid polymer and a biosurfactant are exclusively regulated through RsmE, contradicting the generalized notion of functional redundancy among the Rsm paralogs. Furthermore, we identified the biosurfactant as the cyclic lipopeptide gacamide A. Competition and microscopy analyses showed that the mucoid polymer is solely responsible for creating a space of low cellular density, which is shared exclusively by the same genotype. Gacamide A and other RsmE-regulated products appear to establish a physical boundary that prevents the encroachment of the competing genotype into the newly created space. Although cyclic lipopeptides and other biosurfactants are best known for their antimicrobial properties and reducing surface tension to promote the spreading of cells on various surfaces, they also appear to help define spatial structure formation within a dense community. IMPORTANCE In densely populated colonies of the bacterium Pseudomonas fluorescens Pf0-1, diverse mutations in the rsmE gene are naturally selected by solving the problem of overcrowding. Here, we show that RsmE-regulated secretions function together to create and protect space of low cell density. A biosurfactant generally promotes the spreading of bacterial cells on abiotic surfaces; however, it appears to function atypically within a crowded population by physically defining genotypic boundaries. Another significant finding is that these secretions are not regulated by RsmE's paralogs that share high sequence similarity. The experimental pipeline described in this study is highly tractable and should facilitate future studies to explore additional RsmE-regulated products and address why RsmE is functionally unique from its paralogs.

Identification of Two Variants of Acinetobacter baumannii Strain ATCC 17978 with Distinct Genotypes and Phenotypes.

Acinetobacter baumannii is a nosocomial pathogen that exhibits substantial genomic plasticity. Here, the identification of two variants of A. baumannii ATCC 17978 that differ based on the presence of a 44-kb accessory locus, named AbaAL44 (A. baumannii accessory locus 44 kb), is described. Analyses of existing deposited data suggest that both variants are found in published studies of A. baumannii ATCC 17978 and that American Type Culture Collection (ATCC)-derived laboratory stocks comprise a mix of these two variants. Yet, each variant exhibits distinct interactions with the host in vitro and in vivo. Infection with the variant that harbors AbaAL44 (A. baumannii 17978 UN) results in decreased bacterial burdens and increased neutrophilic lung inflammation in a mouse model of pneumonia, and affects the production of interleukin 1 beta (IL-1ß) and IL-10 by infected macrophages. AbaAL44 harbors putative pathogenesis genes, including those predicted to encode a type I pilus cluster, a catalase, and a cardiolipin synthase. The accessory catalase increases A. baumannii resistance to oxidative stress and neutrophil-mediated killing in vitro. The accessory cardiolipin synthase plays a dichotomous role by promoting bacterial uptake and increasing IL-1ß production by macrophages, but also by enhancing bacterial resistance to cell envelope stress. Collectively, these findings highlight the phenotypic consequences of the genomic dynamism of A. baumannii through the evolution of two variants of a common type strain with distinct infection-related attributes.

Genomic and Phenotypic Diversity among Ten Laboratory Isolates of Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an opportunistic pathogen found ubiquitously in the environment and commonly associated with airway infection in patients with cystic fibrosis. P. aeruginosa strain PAO1 is one of the most commonly used laboratory-adapted research strains and is a standard laboratory-adapted strain in multiple laboratories and strain banks worldwide. Due to potential isolate-to-isolate variability, we investigated the genomic and phenotypic diversity among 10 PAO1 strains (henceforth called sublines) obtained from multiple research laboratories and commercial sources. Genomic analysis predicted a total of 5,682 genes, with 5,434 (95.63%) being identical across all 10 strains. Phenotypic analyses revealed comparable growth phenotypes in rich media and biofilm formation profiles. Limited differences were observed in antibiotic susceptibility profiles and immunostimulatory potential, measured using heat-killed whole-cell preparations in four immortalized cell lines followed by quantification of interleukin-6 (IL-6) and IL-1ß secretion. However, variability was observed in the profiles of secreted molecular products, most notably, in rhamnolipid, pyoverdine, pyocyanin, Pseudomonas quinolone signal (PQS), extracellular DNA, exopolysaccharide, and outer membrane vesicle production. Many of the observed phenotypic differences did not correlate with subline-specific genetic changes, suggesting alterations in transcriptional and translational regulation. Taken together, these results suggest that individually maintained sublines of PAO1, even when acquired from the same parent subline, are continuously undergoing microevolution during culture and storage that results in alterations in phenotype, potentially affecting the outcomes of in vitro phenotypic analyses and in vivo pathogenesis studies.IMPORTANCE Laboratory-adapted strains of bacteria are used throughout the world for microbiology research. These prototype strains help keep research data consistent and comparable between laboratories. However, we have observed phenotypic variability when using different strains of Pseudomonas aeruginosa PAO1, one of the major laboratory-adopted research strains. Here, we describe the genomic and phenotypic differences among 10 PAO1 strains acquired from independent sources over 15 years to understand how individual maintenance affects strain characteristics. We observed limited genomic changes but variable phenotypic changes, which may have consequences for cross-comparison of data generated using different PAO1 strains. Our research highlights the importance of limiting practices that may promote the microevolution of model strains and calls for researchers to specify the strain origin to ensure reproducibility.

A shift between mineral and nonmineral sources of iron and sulfur causes proteome-wide changes in Methanosarcina barkeri.

Iron (Fe) and sulfur (S) are required elements for life, and changes in their availability can limit the ecological distribution and function of microorganisms. In anoxic environments, soluble Fe typically exists as ferrous iron [Fe(II)] and S as sulfide (HS-). These species exhibit a strong affinity that ultimately drives the formation of sedimentary pyrite (FeS2). Recently, paradigm-shifting studies indicate that Fe and S in FeS2 can be made bioavailable by methanogens through a reductive dissolution process. However, the impact of the utilization of FeS2, as opposed to canonical Fe and S sources, on the phenotype of cells is not fully understood. Here, shotgun proteomics was utilized to measure changes in the phenotype of Methanosarcina barkeri MS grown with FeS2, Fe(II)/HS-, or Fe(II)/cysteine. Shotgun proteomics tracked 1,019 proteins overall, with 307 observed to change between growth conditions. Functional characterization and pathway analyses revealed these changes to be systemic and largely tangential to Fe/S metabolism. As a final step, the proteomics data were viewed with respect to previously collected transcriptomics data to deepen the analysis. Presented here is evidence that M. barkeri adopts distinct phenotypes to exploit specific sources of Fe and S in its environment. This is supported by observed protein abundance changes across broad categories of cellular biology. DNA adjacent metabolism, central carbon metabolism methanogenesis, metal trafficking, quorum sensing, and porphyrin biosynthesis pathways are all features in the phenotypic differentiation. Differences in trace metal availability attributed to complexation with HS-, either as a component of the growth medium [Fe(II)/HS-] or generated through reduction of FeS2, were likely a major factor underpinning these phenotypic differences.IMPORTANCEThe methanogenic archaeon Methanosarcina barkeri holds great potential for industrial bio-mining and energy generation technologies. Much of the biochemistry of this microbe is poorly understood, and its characterization will provide a glimpse into biological processes that evolved close to life's origin. The discovery of its ability to extract iron and sulfur from bulk, solid-phase minerals shifted a longstanding paradigm that these elements were inaccessible to biological systems. The full elucidation of this process has the potential to help scientists and engineers extract valuable metals from low-grade ore and mine waste generating energy in the form of methane while doing so.

Detection of Atypical Salmonella Infantis Phenotypes in Broiler Environmental Samples.

In numerous countries, strict and targeted measures concerning Salmonella monitoring and control are implemented and high quality of surveillance is ensured by obligatory investigation of samples from the primary production level of animals according to EN/ISO standards. Here, 2 phenotypic characteristics of Salmonella exhibited on compulsory media are crucial, namely, motility demonstrated on modified semisolid Rappaport Vassiliadis agar (MSRV), and production of hydrogen sulfide (H2S) on xylose lysine deoxycholate agar (XLD). In the present study, we describe the detection of Salmonella Infantis variants found in broiler environmental samples with major alterations in their growth characteristics on MSRV, XLD, and brilliant green-phenol red-agar (BPLS). The variants proved to be non-motile on MSRV and displayed non-confirming colony appearances on the previously mentioned selective agars. The growth spectrum comprised pinhead sized yellow colonies with small black centers, but also pinpoint sized colorless colonies, both colony types of regular shape. Our work contributes to highlight the finding of S. Infantis variants which possess more than one phenotypic deviation from the "typical" growth characteristics and by this limit the detection power of the actual obligatory used media. IMPORTANCE Salmonellosis caused by non-typhoidal Salmonella serovars is the second most frequently reported zoonotic disease in humans in the EU. The transmission of these agents is mainly via contaminated food of animal origin. In this context, poultry products are the main source of infection. Therefore, continuous and standardized surveillance of the prevalence of such Salmonella serovars at the primary production level is essential. Our findings show the phenotypic heterogeneity of the serovar Infantis and provide growth characteristics of atypical variants. Such variants pass unnoticed official screening methods, resulting in incorrect identification and being underrepresented in epidemiological surveillance programs.

Variable Phenotypes of Epilepsy, Intellectual Disability, and Schizophrenia Caused by 12p13.33-p13.32 Terminal Microdeletion in a Korean Family: A Case Report and Literature Review.

A simultaneous analysis of nucleotide changes and copy number variations (CNVs) based on exome sequencing data was demonstrated as a potential new first-tier diagnosis strategy for rare neuropsychiatric disorders. In this report, using depth-of-coverage analysis from exome sequencing data, we described variable phenotypes of epilepsy, intellectual disability (ID), and schizophrenia caused by 12p13.33-p13.32 terminal microdeletion in a Korean family. We hypothesized that CACNA1C and KDM5A genes of the six candidate genes located in this region were the best candidates for explaining epilepsy, ID, and schizophrenia and may be responsible for clinical features reported in cases with monosomy of the 12p13.33 subtelomeric region. On the background of microdeletion syndrome, which was described in clinical cases with mild, moderate, and severe neurodevelopmental manifestations as well as impairments, the clinician may determine whether the patient will end up with a more severe or milder end-phenotype, which in turn determines disease prognosis. In our case, the 12p13.33-p13.32 terminal microdeletion may explain the variable expressivity in the same family. However, further comprehensive studies with larger cohorts focusing on careful phenotyping across the lifespan are required to clearly elucidate the possible contribution of genetic modifiers and the environmental influence on the expressivity of 12p13.33 microdeletion and associated characteristics.

Species Designations Belie Phenotypic and Genotypic Heterogeneity in Oral Streptococci.

Health-associated oral Streptococcus species are promising probiotic candidates to protect against dental caries. Ammonia production through the arginine deiminase system (ADS), which can increase the pH of oral biofilms, and direct antagonism of caries-associated bacterial species are desirable properties for oral probiotic strains. ADS and antagonistic activities can vary dramatically among individuals, but the genetic basis for these differences is unknown. We sequenced whole genomes of a diverse set of clinical oral Streptococcus isolates and examined the genetic basis of variability in ADS and antagonistic activities. A total of 113 isolates were included and represented 10 species: Streptococcus australis, A12-like, S. cristatus, S. gordonii, S. intermedius, S. mitis, S. oralis including S. oralis subsp. dentisani, S. parasanguinis, S. salivarius, and S. sanguinis. Mean ADS activity and antagonism on Streptococcus mutans UA159 were measured for each isolate, and each isolate was whole genome shotgun sequenced on an Illumina MiSeq. Phylogenies were built of genes known to be involved in ADS activity and antagonism. Several approaches to correlate the pan-genome with phenotypes were performed. Phylogenies of genes previously identified in ADS activity and antagonism grouped isolates by species, but not by phenotype. A genome-wide association study (GWAS) identified additional genes potentially involved in ADS activity or antagonism across all the isolates we sequenced as well as within several species. Phenotypic heterogeneity in oral streptococci is not necessarily reflected by genotype and is not species specific. Probiotic strains must be carefully selected based on characterization of each strain and not based on inclusion within a certain species. IMPORTANCE Representative type strains are commonly used to characterize bacterial species, yet species are phenotypically and genotypically heterogeneous. Conclusions about strain physiology and activity based on a single strain therefore may be inappropriate and misleading. When selecting strains for probiotic use, the assumption that all strains within a species share the same desired probiotic characteristics may result in selection of a strain that lacks the desired traits, and therefore makes a minimally effective or ineffective probiotic. Health-associated oral streptococci are promising candidates for anticaries probiotics, but strains need to be carefully selected based on observed phenotypes. We characterized the genotypes and anticaries phenotypes of strains from 10 species of oral streptococci and demonstrate poor correlation between genotype and phenotype across all species.