Machine learning analysis of RB-TnSeq fitness data predicts functional gene modules in Pseudomonas putida KT2440.

There is growing interest in engineering Pseudomonas putida KT2440 as a microbial chassis for the conversion of renewable and waste-based feedstocks, and metabolic engineering of P. putida relies on the understanding of the functional relationships between genes. In this work, independent component analysis (ICA) was applied to a compendium of existing fitness data from randomly barcoded transposon insertion sequencing (RB-TnSeq) of P. putida KT2440 grown in 179 unique experimental conditions. ICA identified 84 independent groups of genes, which we call fModules ("functional modules"), where gene members displayed shared functional influence in a specific cellular process. This machine learning-based approach both successfully recapitulated previously characterized functional relationships and established hitherto unknown associations between genes. Selected gene members from fModules for hydroxycinnamate metabolism and stress resistance, acetyl coenzyme A assimilation, and nitrogen metabolism were validated with engineered mutants of P. putida. Additionally, functional gene clusters from ICA of RB-TnSeq data sets were compared with regulatory gene clusters from prior ICA of RNAseq data sets to draw connections between gene regulation and function. Because ICA profiles the functional role of several distinct gene networks simultaneously, it can reduce the time required to annotate gene function relative to manual curation of RB-TnSeq data sets. IMPORTANCE: This study demonstrates a rapid, automated approach for elucidating functional modules within complex genetic networks. While Pseudomonas putida randomly barcoded transposon insertion sequencing data were used as a proof of concept, this approach is applicable to any organism with existing functional genomics data sets and may serve as a useful tool for many valuable applications, such as guiding metabolic engineering efforts in other microbes or understanding functional relationships between virulence-associated genes in pathogenic microbes. Furthermore, this work demonstrates that comparison of data obtained from independent component analysis of transcriptomics and gene fitness datasets can elucidate regulatory-functional relationships between genes, which may have utility in a variety of applications, such as metabolic modeling, strain engineering, or identification of antimicrobial drug targets.

RB-TnSeq identifies genetic targets for improved tolerance of Pseudomonas putida towards compounds relevant to lignin conversion.

Lignin-derived mixtures intended for bioconversion commonly contain high concentrations of aromatic acids, aliphatic acids, and salts. The inherent toxicity of these chemicals places a significant bottleneck upon the effective use of microbial systems for the valorization of these mixtures. Pseudomonas putida KT2440 can tolerate stressful quantities of several lignin-related compounds, making this bacterium a promising host for converting these chemicals to valuable bioproducts. Nonetheless, further increasing P. putida tolerance to chemicals in lignin-rich substrates has the potential to improve bioprocess performance. Accordingly, we employed random barcoded transposon insertion sequencing (RB-TnSeq) to reveal genetic determinants in P. putida KT2440 that influence stress outcomes during exposure to representative constituents found in lignin-rich process streams. The fitness information obtained from the RB-TnSeq experiments informed engineering of strains via deletion or constitutive expression of several genes. Namely, ΔgacAS, ΔfleQ, ΔlapAB, ΔttgR::Ptac:ttgABC, Ptac:PP_1150:PP_1152, ΔrelA, and ΔPP_1430 mutants showed growth improvement in the presence of single compounds, and some also exhibited greater tolerance when grown using a complex chemical mixture representative of a lignin-rich chemical stream. Overall, this work demonstrates the successful implementation of a genome-scale screening tool for the identification of genes influencing stress tolerance against notable compounds within lignin-enriched chemical streams, and the genetic targets identified herein offer promising engineering targets for improving feedstock tolerance in lignin valorization strains of P. putida KT2440.

Experimental and Analytical Approaches for Improving the Resolution of Randomly Barcoded Transposon Insertion Sequencing (RB-TnSeq) Studies.

Randomly barcoded transposon insertion sequencing (RB-TnSeq) is an efficient, multiplexed method to determine microbial gene function during growth under a selection condition of interest. This technique applies to growth, tolerance, and persistence studies in a variety of hosts, but the wealth of data generated can complicate the identification of the most critical gene targets. Experimental and analytical methods for improving the resolution of RB-TnSeq are proposed, using Pseudomonas putida KT2440 as an example organism. Several key parameters, such as baseline media selection, substantially influence the determination of gene fitness. We also present options to increase statistical confidence in gene fitness, including increasing the number of biological replicates and passaging the baseline culture in parallel with selection conditions. These considerations provide practitioners with several options to identify genes of importance in TnSeq data sets, thereby streamlining metabolic characterization.

2-Aminoacrylate stress damages diverse PLP-dependent enzymes in vivo.

Pyridoxal 5'-phosphate (PLP) is an essential cofactor for a class of enzymes that catalyze diverse reactions in central metabolism. The catalytic mechanism of some PLP-dependent enzymes involves the generation of reactive enamine intermediates like 2-aminoacrylate (2AA). 2AA can covalently modify PLP in the active site of some PLP-dependent enzymes and subsequently inactivate the enzyme through the formation of a PLP-pyruvate adduct. In the absence of the enamine/imine deaminase RidA, Salmonella enterica experiences 2AA-mediated metabolic stress. Surprisingly, PLP-dependent enzymes that generate endogenous 2AA appear to be immune to its attack, while other PLP-dependent enzymes accumulate damage in the presence of 2AA stress; however, structural determinants of 2AA sensitivity are unclear. In this study, we refined a molecular method to query proteins from diverse systems for their sensitivity to 2AA in vivo. This method was then used to examine active site residues of Alr, a 2AA-sensitive PLP-dependent enzyme, that affect its sensitivity to 2AA in vivo. Unexpectedly, our data also showed that a low level of 2AA stress can persist even in the presence of a functional RidA. In summary, this study expands our understanding of 2AA metabolism and takes an initial step toward characterizing the structural determinants influencing enzyme susceptibility to damage by free 2AA.

Challenges and opportunities in biological funneling of heterogeneous and toxic substrates beyond lignin.

Significant developments in the understanding and manipulation of microbial metabolism have enabled the use of engineered biological systems toward a more sustainable energy and materials economy. While developments in metabolic engineering have primarily focused on the conversion of carbohydrates, substantial opportunities exist for using these same principles to extract value from more heterogeneous and toxic waste streams, such as those derived from lignin, biomass pyrolysis, or industrial waste. Funneling heterogeneous substrates from these streams toward valuable products, termed biological funneling, presents new challenges in balancing multiple catabolic pathways competing for shared cellular resources and engineering against perturbation from toxic substrates. Solutions to many of these challenges have been explored within the field of lignin valorization. This perspective aims to extend beyond lignin to highlight the challenges and discuss opportunities for use of biological systems to upgrade previously inaccessible waste streams.

Two novel fish paralogs provide insights into the Rid family of imine deaminases active in pre-empting enamine/imine metabolic damage.

Reactive Intermediate Deaminase (Rid) protein superfamily includes eight families among which the RidA is conserved in all domains of life. RidA proteins accelerate the deamination of the reactive 2-aminoacrylate (2AA), an enamine produced by some pyridoxal phosphate (PLP)-dependent enzymes. 2AA accumulation inhibits target enzymes with a detrimental impact on fitness. As a consequence of whole genome duplication, teleost fish have two ridA paralogs, while other extant vertebrates contain a single-copy gene. We investigated the biochemical properties of the products of two paralogs, identified in Salmo salar. SsRidA-1 and SsRidA-2 complemented the growth defect of a Salmonella enterica ridA mutant, an in vivo model of 2AA stress. In vitro, both proteins hydrolyzed 2-imino acids (IA) to keto-acids and ammonia. SsRidA-1 was active on IA derived from nonpolar amino acids and poorly active or inactive on IA derived from other amino acids tested. In contrast, SsRidA-2 had a generally low catalytic efficiency, but showed a relatively higher activity with IA derived from L-Glu and aromatic amino acids. The crystal structures of SsRidA-1 and SsRidA-2 provided hints of the remarkably different conformational stability and substrate specificity. Overall, SsRidA-1 is similar to the mammalian orthologs whereas SsRidA-2 displays unique properties likely generated by functional specialization of a duplicated ancestral gene.

Proton Nuclear Magnetic Resonance Metabolomics Corroborates Serine Hydroxymethyltransferase as the Primary Target of 2-Aminoacrylate in a ridA Mutant of Salmonella enterica.

The reactive intermediate deaminase RidA (EC 3.5.99.10) is conserved across all domains of life and deaminates reactive enamine species. When Salmonella enterica ridA mutants are grown in minimal medium, 2-aminoacrylate (2AA) accumulates, damages several pyridoxal 5'-phosphate (PLP)-dependent enzymes, and elicits an observable growth defect. Genetic studies suggested that damage to serine hydroxymethyltransferase (GlyA), and the resultant depletion of 5,10-methelenetetrahydrofolate (5,10-mTHF), was responsible for the observed growth defect. However, the downstream metabolic consequence from GlyA damage by 2AA remains relatively unexplored. This study sought to use untargeted proton nuclear magnetic resonance (1H NMR) metabolomics to determine whether the metabolic state of an S. enterica ridA mutant was accurately reflected by characterizing growth phenotypes. The data supported the conclusion that metabolic changes in a ridA mutant were due to the IlvA-dependent generation of 2AA, and that the majority of these changes were a consequence of damage to GlyA. While many of the metabolic differences for a ridA mutant could be explained, changes in some metabolites were not easily modeled, suggesting that additional levels of metabolic complexity remain to be unraveled.IMPORTANCE The accumulation of the reactive enamine intermediate 2-aminoacrylate (2AA) elicits global metabolic stress in many prokaryotes and eukaryotes by simultaneously damaging multiple pyridoxal 5'-phosphate (PLP)-dependent enzymes. This work employed 1H NMR to expand our understanding of the consequence(s) of 2AA stress on metabolite pools and effectively identify the metabolic changes stemming from one damaged target: GlyA. This study shows that nutrient supplementation during 1H NMR metabolomics experiments can disentangle complex metabolic outcomes stemming from a general metabolic stress. Metabolomics shows great potential to complement classical reductionist approaches to cost-effectively accelerate the rate of progress in expanding our global understanding of metabolic network structure and physiology. To that end, this work demonstrates the utility in implementing nutrient supplementation and genetic perturbation into metabolomics workflows as a means to connect metabolic outputs to physiological phenomena and establish causal relationships.

Integrated Metabolomics and Transcriptomics Suggest the Global Metabolic Response to 2-Aminoacrylate Stress in Salmonella enterica.

In Salmonella enterica, 2-aminoacrylate (2AA) is a reactive enamine intermediate generated during a number of biochemical reactions. When the 2-iminobutanoate/2-iminopropanoate deaminase (RidA; EC: 3.5.99.10) is eliminated, 2AA accumulates and inhibits the activity of multiple pyridoxal 5'-phosphate(PLP)-dependent enzymes. In this study, untargeted proton nuclear magnetic resonance (1H NMR) metabolomics and transcriptomics data were used to uncover the global metabolic response of S. enterica to the accumulation of 2AA. The data showed that elimination of RidA perturbed folate and branched chain amino acid metabolism. Many of the resulting perturbations were consistent with the known effect of 2AA stress, while other results suggested additional potential enzyme targets of 2AA-dependent damage. The majority of transcriptional and metabolic changes appeared to be the consequence of downstream effects on the metabolic network, since they were not directly attributable to a PLP-dependent enzyme. In total, the results highlighted the complexity of changes stemming from multiple perturbations of the metabolic network, and suggested hypotheses that will be valuable in future studies of the RidA paradigm of endogenous 2AA stress.

Reactive Enamines and Imines In Vivo: Lessons from the RidA Paradigm.

Metabolic networks are webs of integrated reactions organized to maximize growth and replication while minimizing the detrimental impact that reactive metabolites can have on fitness. Enamines and imines, such as 2-aminoacrylate (2AA), are reactive metabolites produced as short-lived intermediates in a number of enzymatic processes. Left unchecked, the inherent reactivity of enamines and imines may perturb the metabolic network. Genetic and biochemical studies have outlined a role for the broadly conserved reactive intermediate deaminase (Rid) (YjgF/YER057c/UK114) protein family, in particular RidA, in catalyzing the hydrolysis of enamines and imines to their ketone product. Herein, we discuss new findings regarding the biological significance of enamine and imine production and outline the importance of RidA in controlling the accumulation of reactive metabolites.

Analyses of variants of the Ser/Thr dehydratase IlvA provide insight into 2-aminoacrylate metabolism in Salmonella enterica.

RidA is a conserved and broadly distributed protein that has enamine deaminase activity. In a variety of organisms tested thus far, lack of RidA results in the accumulation of the reactive metabolite 2-aminoacrylate (2AA), an obligate intermediate in the catalytic mechanism of several pyridoxal 5'-phosphate (PLP)-dependent enzymes. This study reports the characterization of variants of the biosynthetic serine/threonine dehydratase (EC 4.3.1.19; IlvA), which is a significant generator of 2AA in the bacteria Salmonella enterica, Escherichia coli, and Pseudomonas aeruginosa and the yeast Saccharomyces cerevisiae Two previously identified mutations, ilvA3210 and ilvA3211, suppressed the phenotypic growth consequences of 2AA accumulation in S. enterica Characterization of the respective protein variants suggested that they affect 2AA metabolism in vivo by two different catalytic mechanisms, both leading to an overall reduction in serine dehydratase activity. To emphasize the physiological relevance of the in vitro enzyme characterization, we sought to explain in vivo phenotypes using these data. A simple mathematical model describing the impact these catalytic deficiencies had on 2AA production was generally supported by our data. However, caveats arose when kinetic parameters, determined in vitro, were used to predict formation of the isoleucine precursor 2-ketobutyrate and model in vivo (growth) behaviors. Altogether, our data support the need for a holistic approach, including in vivo and in vitro analyses, to generate data used in understanding and modeling metabolism.

Perturbation of the metabolic network in Salmonella enterica reveals cross-talk between coenzyme A and thiamine pathways.

Microorganisms respond to a variety of metabolic perturbations by repurposing or recruiting pathways to reroute metabolic flux and overcome the perturbation. Elimination of the 2-dehydropantoate 2-reductase, PanE, both reduces total coenzyme A (CoA) levels and causes a conditional HMP-P auxotrophy in Salmonella enterica. CoA or acetyl-CoA has no demonstrable effect on the HMP-P synthase, ThiC, in vitro. Suppressors aimed at probing the connection between the biosynthesis of thiamine and CoA contained mutations in the gene encoding the ilvC transcriptional regulator, ilvY. These mutations may help inform the structure and mechanism of action for the effector-binding domain, as they represent the first sequenced substitutions in the effector-binding domain of IlvY that cause constitutive expression of ilvC. Since IlvC moonlights as a 2-dehydropantoate 2-reductase, the resultant increase in ilvC transcription increased synthesis of CoA. This study failed to identify mutations overcoming the need for CoA for thiamine synthesis in S. enterica panE mutants, suggesting that a more integrated approach may be necessary to uncover the mechanism connecting CoA and ThiC activity in vivo.

Endogenously generated 2-aminoacrylate inhibits motility in Salmonella enterica.

Members of the broadly distributed Rid/YER057c/UK114 protein family have imine/enamine deaminase activity, notably on 2-aminoacrylate (2AA). Strains of Salmonella enterica, and other organisms lacking RidA, have diverse growth phenotypes, attributed to the accumulation of 2AA. In S. enterica, 2AA inactivates a number of pyridoxal 5'-phosephate(PLP)-dependent enzymes, some of which have been linked to the growth phenotypes of a ridA mutant. This study used transcriptional differences between S. enterica wild-type and ridA strains to explore the breadth of the cellular consequences that resulted from accumulation of 2AA. Accumulation of endogenously generated 2AA in a ridA mutant resulted in lower expression of genes encoding many flagellar assembly components, which led to a motility defect. qRT-PCR results were consistent with the motility phenotype of a ridA mutant resulting from a defect in FlhD4C2 activity. In total, the results of comparative transcriptomics correctly predicted a 2AA-dependent motility defect and identified additional areas of metabolism impacted by the metabolic stress of 2AA in Salmonella enterica. Further, the data emphasized the value of integrating global approaches with biochemical genetic approaches to understand the complex system of microbial metabolism.

The Response to 2-Aminoacrylate Differs in Escherichia coli and Salmonella enterica, despite Shared Metabolic Components.

The metabolic network of an organism includes the sum total of the biochemical reactions present. In microbes, this network has an impeccable ability to sense and respond to perturbations caused by internal or external stimuli. The metabolic potential (i.e., network structure) of an organism is often drawn from the genome sequence, based on the presence of enzymes deemed to indicate specific pathways. Escherichia coli and Salmonella enterica are members of the Enterobacteriaceae family of Gram-negative bacteria that share the majority of their metabolic components and regulatory machinery as the "core genome." In S. enterica, the ability of the enamine intermediate 2-aminoacrylate (2AA) to inactivate a number of pyridoxal 5'-phosphate (PLP)-dependent enzymes has been established in vivo In this study, 2AA metabolism and the consequences of its accumulation were investigated in E. coli The data showed that despite the conservation of all relevant enzymes, S. enterica and E. coli differed in both the generation and detrimental consequences of 2AA. In total, these findings suggest that the structure of the metabolic network surrounding the generation and response to endogenous 2AA stress differs between S. enterica and E. coliIMPORTANCE This work compared the metabolic networks surrounding the endogenous stressor 2-aminoacrylate in two closely related members of the Enterobacteriaceae The data showed that despite the conservation of all relevant enzymes in this metabolic node, the two closely related organisms diverged in their metabolic network structures. This work highlights how a set of conserved components can generate distinct network architectures and how this can impact the physiology of an organism. This work defines a model to expand our understanding of the 2-aminoacrylate stress response and the differences in metabolic structures and cellular milieus between S. enterica and E. coli.

Prevalence of toxin-producing Clostridium botulinum associated with the macroalga Cladophora in three Great Lakes: growth and management.

The reemergence of avian botulism caused by Clostridium botulinum type E has been observed across the Great Lakes in recent years. Evidence suggests an association between the nuisance algae, Cladophora spp., and C. botulinum in nearshore areas of the Great Lakes. However, the nature of the association between Cladophora and C. botulinum is not fully understood due, in part, to the complex food web interactions in this disease etiology. In this study, we extensively evaluated their association by quantitatively examining population size and serotypes of C. botulinum in algal mats collected from wide geographic areas in lakes Michigan, Ontario, and Erie in 2011-2012 and comparing them with frequencies in other matrices such as sand and water. A high prevalence (96%) of C. botulinum type E was observed in Cladophora mats collected from shorelines of the Great Lakes in 2012. Among the algae samples containing detectable C. botulinum, the population size of C. Botulinum type E was 10(0)-10(4) MPN/g dried algae, which was much greater (up to 10(3) fold) than that found in sand or the water column, indicating that Cladophora mats are sources of this pathogen. Mouse toxinantitoxin bioassays confirmed that the putative C. botulinum belonged to the type E serotype. Steam treatment was effective in reducing or eliminating C. botulinum type E viable cells in Cladophora mats, thereby breaking the potential transmission route of toxin up to the food chain. Consequently, our data suggest that steam treatment incorporated with a beach cleaning machine may be an effective treatment of Cladophora-borne C. botulinum and may reduce bird mortality and human health risks.

Decay of genetic markers for fecal bacterial indicators and pathogens in sand from Lake Superior.

Beach sands impact water quality and pathogen loads, however, the comparative decay of the fecal indicator bacteria (FIB) Enterococcus spp. and Escherichia coli, and pathogens in freshwater sand have not been examined. In this study, freshwater sand microcosms were inoculated with sewage and pure cultures of bacterial pathogens to compare relative decay rates. The abundance of culturable Enterococcus spp. and E. coli, genetic markers for Enterococcus spp. (Entero1), total Bacteroides (AllBac), and human-specific Bacteroides (HF183), and genetic markers for the pathogens Campylobacter jejuni, methicillin-resistant Staphylococcus aureus (MRSA), Salmonella enterica subsp. enterica serovar Typhimurium, and Shigella flexneri were monitored over the course of two weeks using conventional culture methods and quantitative PCR (qPCR). The effect of moisture on the persistence of culturable FIB and all genetic markers was also determined. In addition, propidium monoazide (PMA) treatment was used to examine differences in the persistence of total genetic markers and those from live cells. Decay rates were statistically compared using Tukey's test. Moisture had a significant (p ≤ 0.05) effect on the decay rates of culturable indicator bacteria, total AllBac markers, and genetic markers for FIB, Salmonella, and MRSA from live cells. At 14% sand moisture, the decay rate of total markers was slower than that of live cells for all qPCR assays, but at 28% moisture, there was no difference in the decay rates of total and live markers for any assay. AllBac and MRSA markers increased in sand at 28% moisture, probably indicating cellular growth. Overall, culturable FIB and HF183 had decay rates that were most comparable to the bacterial pathogen markers examined in this study, whereas Entero1 and AllBac rarely exhibited decay rates similar to the bacterial pathogens in this study. The choice of FIB for assessment of fecal contamination in freshwater sand should take into account the pathogen of concern and sand moisture conditions.