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Interactions between autotrophs and heterotrophs are central to carbon (C) exchange across trophic levels in essentially all ecosystems and metabolite exchange is a frequent mechanism for distributing C within spatially structured ecosystems. Yet, despite the importance of C exchange, the timescales at which fixed C is transferred in microbial communities is poorly understood. We employed a stable isotope tracer combined with spatially resolved isotope analysis to quantify photoautotrophic uptake of bicarbonate and track subsequent exchanges across a vertical depth gradient in a stratified microbial mat over a light-driven diel cycle. We observed that C mobility, both across the vertical strata and between taxa, was highest during periods of active photoautotrophy. Parallel experiments with 13C-labeled organic substrates (acetate and glucose) showed comparably less exchange of C within the mat. Metabolite analysis showed rapid incorporation of 13C into molecules that can both comprise a portion of the extracellular polymeric substances in the system and serve to transport C between photoautotrophs and heterotrophs. Stable isotope proteomic analysis revealed rapid C exchange between cyanobacterial and associated heterotrophic community members during the day with decreased exchange at night. We observed strong diel control on the spatial exchange of freshly fixed C within tightly interacting mat communities suggesting a rapid redistribution, both spatially and taxonomically, primarily during daylight periods.

The spore forming pathogen Bacillus anthracis is the etiologic agent of anthrax in humans and animals. It cycles through infected hosts as vegetative cells and is eventually introduced into the environment where it generates an endospore resistant to many harsh conditions. The endospores are subsequently taken up by another host to begin the next cycle. Outbreaks of anthrax occur regularly worldwide in wildlife and livestock, and the potential for human infection exists whenever humans encounter infected animals. It is also possible to encounter intentional releases of anthrax spores, as was the case in October 2001. Consequently, it is important to be able to rapidly establish the provenance of infectious strains of B. anthracis. Here, we compare protein expression in seven low-passage wild isolates and four laboratory strains of B. anthracis grown under identical conditions using LC-MS/MS proteomic analysis. Of the 1,023 total identified proteins, 96 had significant abundance differences between wild and laboratory strains. Of those, 28 proteins directly related to sporulation were upregulated in wild isolates, with expression driven by Spo0A, CodY, and AbrB/ScoC. In addition, we observed evidence of changes in cell division and fatty acid biosynthesis between the two classes of strains, despite being grown under identical experimental conditions. These results suggest wild B. anthracis cells are more highly tuned to sporulate than their laboratory cousins, and this difference should be exploited as a method to differentiate between laboratory and low passage wild strains isolated during an anthrax outbreak. This knowledge should distinguish between intentional releases and exposure to strains in nature, providing a basis for the type of response by public health officials and investigators.

The bacterial pathogen Yersinia pestis, the cause of plague in humans and animals, normally has a sylvatic lifestyle, cycling between fleas and mammals. In contrast, laboratory-grown Y. pestis experiences a more constant environment and conditions that it would not normally encounter. The transition from the natural environment to the laboratory results in a vastly different set of selective pressures, and represents what could be considered domestication. Understanding the kinds of adaptations Y. pestis undergoes as it becomes domesticated will contribute to understanding the basic biology of this important pathogen. In this study, we performed a parallel serial passage experiment (PSPE) to explore the mechanisms by which Y. pestis adapts to laboratory conditions, hypothesizing that cells would undergo significant changes in virulence and nutrient acquisition systems. Two wild strains were serially passaged in 12 independent populations each for ~750 generations, after which each population was analyzed using whole-genome sequencing, LC-MS/MS proteomic analysis, and GC/MS metabolomics. We observed considerable parallel evolution in the endpoint populations, detecting multiple independent mutations in ail, pepA, and zwf, suggesting that specific selective pressures are shaping evolutionary responses. Complementary LC-MS/MS proteomic data provide physiological context to the observed mutations, and reveal regulatory changes not necessarily associated with specific mutations, including changes in amino acid metabolism and cell envelope biogenesis. Proteomic data support hypotheses generated by genomic data in addition to suggesting future mechanistic studies, indicating that future whole-genome sequencing studies be designed to leverage proteomics as a critical complement.

Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water. We then employed infrared spectromicroscopy to detect in real time the flux of H atoms in these same cells. Importantly, both of these techniques indicate that the H and O fluxes are dependent on metabolic processes; cells that are in lag phase or are quiescent exhibit a much smaller flux. In addition, water extracted from the muscle tissue of rats contained a population of H and O atoms that were isotopically distinct from body water, consistent with the results obtained using the cultured Rat-1 fibroblasts. Together these data demonstrate that metabolic processes produce fluxes of H and O atoms into intracellular water, and that these fluxes can be detected and measured in both cultured mammalian cells and in mammalian tissue.

The rapid pace of bacterial evolution enables organisms to adapt to the laboratory environment with repeated passage and thus diverge from naturally-occurring environmental ("wild") strains. Distinguishing wild and laboratory strains is clearly important for biodefense and bioforensics; however, DNA sequence data alone has thus far not provided a clear signature, perhaps due to lack of understanding of how diverse genome changes lead to convergent phenotypes, difficulty in detecting certain types of mutations, or perhaps because some adaptive modifications are epigenetic. Monitoring protein abundance, a molecular measure of phenotype, can overcome some of these difficulties. We have assembled a collection of Yersinia pestis proteomics datasets from our own published and unpublished work, and from a proteomics data archive, and demonstrated that protein abundance data can clearly distinguish laboratory-adapted from wild. We developed a lasso logistic regression classifier that uses binary (presence/absence) or quantitative protein abundance measures to predict whether a sample is laboratory-adapted or wild that proved to be ~98% accurate, as judged by replicated 10-fold cross-validation. Protein features selected by the classifier accord well with our previous study of laboratory adaptation in Y. pestis. The input data was derived from a variety of unrelated experiments and contained significant confounding variables. We show that the classifier is robust with respect to these variables. The methodology is able to discover signatures for laboratory facility and culture medium that are largely independent of the signature of laboratory adaptation. Going beyond our previous laboratory evolution study, this work suggests that proteomic differences between laboratory-adapted and wild Y. pestis are general, potentially pointing to a process that could apply to other species as well. Additionally, we show that proteomics datasets (even archived data collected for different purposes) contain the information necessary to distinguish wild and laboratory samples. This work has clear applications in biomarker detection as well as biodefense.

Adaptation to ecologically complex environments can provide insights into the evolutionary dynamics and functional constraints encountered by organisms during natural selection. Adaptation to a new environment with abundant and varied resources can be difficult to achieve by small incremental changes if many mutations are required to achieve even modest gains in fitness. Since changing complex environments are quite common in nature, we investigated how such an epistatic bottleneck can be avoided to allow rapid adaptation. We show that adaptive mutations arise repeatedly in independently evolved populations in the context of greatly increased genetic and phenotypic diversity. We go on to show that weak selection requiring substantial metabolic reprogramming can be readily achieved by mutations in the global response regulator arcA and the stress response regulator rpoS. We identified 46 unique single-nucleotide variants of arcA and 18 mutations in rpoS, nine of which resulted in stop codons or large deletions, suggesting that subtle modulations of ArcA function and knockouts of rpoS are largely responsible for the metabolic shifts leading to adaptation. These mutations allow a higher order metabolic selection that eliminates epistatic bottlenecks, which could occur when many changes would be required. Proteomic and carbohydrate analysis of adapting E. coli populations revealed an up-regulation of enzymes associated with the TCA cycle and amino acid metabolism, and an increase in the secretion of putrescine. The overall effect of adaptation across populations is to redirect and efficiently utilize uptake and catabolism of abundant amino acids. Concomitantly, there is a pronounced spread of more ecologically limited strains that results from specialization through metabolic erosion. Remarkably, the global regulators arcA and rpoS can provide a "one-step" mechanism of adaptation to a novel environment, which highlights the importance of global resource management as a powerful strategy to adaptation.