Sea Urchin Polyketide Synthase SpPks1 Produces the Naphthalene Precursor to Echinoderm Pigments.

Nearly every animal species on Earth contains a unique polyketide synthase (PKS) encoded in its genome, yet no animal-clade PKS has been biochemically characterized, and even the chemical products of these ubiquitous enzymes are known in only a few cases. The earliest animal genome-encoded PKS gene to be identified was SpPks1 from sea urchins. Previous genetic knockdown experiments implicated SpPks1 in synthesis of the sea urchin pigment echinochrome. Here, we express and purify SpPks1, performing biochemical experiments to demonstrate that the sea urchin protein is responsible for the synthesis of 2-acetyl-1,3,6,8-tetrahydroxynaphthalene (ATHN). Since ATHN is a plausible precursor of echinochromes, this result defines a biosynthetic pathway to the ubiquitous echinoderm pigments and rewrites the previous hypothesis for echinochrome biosynthesis. Truncation experiments showed that, unlike other type I iterative PKSs so far characterized, SpPks1 produces the naphthalene core using solely ketoacylsynthase (KS), acyltransferase, and acyl carrier protein domains, delineating a unique class of animal nonreducing aromatic PKSs (aPKSs). A series of amino acids in the KS domain define the family and are likely crucial in cyclization activity. Phylogenetic analyses indicate that SpPks1 and its homologs are widespread in echinoderms and their closest relatives, the acorn worms, reinforcing their fundamental importance to echinoderm biology. While the animal microbiome is known to produce aromatic polyketides, this work provides biochemical evidence that animals themselves also harbor ancient, convergent, dedicated pathways to carbocyclic aromatic polyketides. More fundamentally, biochemical analysis of SpPks1 begins to define the vast and unexplored biosynthetic space of the ubiquitous animal PKS family.

Diploid genomic architecture of Nitzschia inconspicua, an elite biomass production diatom.

A near-complete diploid nuclear genome and accompanying circular mitochondrial and chloroplast genomes have been assembled from the elite commercial diatom species Nitzschia inconspicua. The 50 Mbp haploid size of the nuclear genome is nearly double that of model diatom Phaeodactylum tricornutum, but 30% smaller than closer relative Fragilariopsis cylindrus. Diploid assembly, which was facilitated by low levels of allelic heterozygosity (2.7%), included 14 candidate chromosome pairs composed of long, syntenic contigs, covering 93% of the total assembly. Telomeric ends were capped with an unusual 12-mer, G-rich, degenerate repeat sequence. Predicted proteins were highly enriched in strain-specific marker domains associated with cell-surface adhesion, biofilm formation, and raphe system gliding motility. Expanded species-specific families of carbonic anhydrases suggest potential enhancement of carbon concentration efficiency, and duplicated glycolysis and fatty acid synthesis pathways across cytosolic and organellar compartments may enhance peak metabolic output, contributing to competitive success over other organisms in mixed cultures. The N. inconspicua genome delivers a robust new reference for future functional and transcriptomic studies to illuminate the physiology of benthic pennate diatoms and harness their unique adaptations to support commercial algae biomass and bioproduct production.

Real-Time Mass Spectrometry Measurements of Respiration Rates in Biological Systems.

Many organisms process carbon and other nutrients to generate energy through aerobic respiration where organic carbon compounds are broken down and oxygen is consumed, producing carbon dioxide and water. Respiration is indicative of active metabolism, and respiration rates are proportional to the amount of living biomass in an ecosystem. Although there are many methods for measuring respiration rates in the laboratory, current systems, such as infrared gas analyzers, are limited in their ability to independently resolve isotopomer fluxes across a range of relevant gases including both CO2 and O2 in real-time. Therefore, monitoring of biological respiration in real time under controlled laboratory conditions would enable better understanding of cellular physiology. To address this challenge, we developed a real time mass spectrometry (RTMS) manifold that simultaneously measures production and consumption of multiple gases and their isotopologues in seconds with the speed and sensitivity necessary to characterize rapidly changing respiration events as they occur. This universal manifold can be fitted to a variety of instruments and affords the same analytical precision and accuracy of the instrument while allowing for the real time measurements. Here, we paired the manifold to a single quad MS with an electron impact (EI) source operated in scan mode to detect extracted target gases by their respective masses (e.g., 12CO2 at mass 44, 13CO2 at 45). We demonstrated applicability of the RTMS instrument to different biological ecosystems (bacterial cultures, plants, and soil), and in all cases, we were able to detect simultaneous and rapid measurements of multiple gases in real time, providing novel insights into complex respiratory metabolism and the influence of biological and environmental factors.

Species-specific transcriptomic network inference of interspecies interactions.

The advent of high-throughput 'omics approaches coupled with computational analyses to reconstruct individual genomes from metagenomes provides a basis for species-resolved functional studies. Here, a mutual information approach was applied to build a gene association network of a commensal consortium, in which a unicellular cyanobacterium Thermosynechococcus elongatus BP1 supported the heterotrophic growth of Meiothermus ruber strain A. Specifically, we used the context likelihood of relatedness (CLR) algorithm to generate a gene association network from 25 transcriptomic datasets representing distinct growth conditions. The resulting interspecies network revealed a number of linkages between genes in each species. While many of the linkages were supported by the existing knowledge of phototroph-heterotroph interactions and the metabolism of these two species several new interactions were inferred as well. These include linkages between amino acid synthesis and uptake genes, as well as carbohydrate and vitamin metabolism, terpenoid metabolism and cell adhesion genes. Further topological examination and functional analysis of specific gene associations suggested that the interactions are likely to center around the exchange of energetically costly metabolites between T. elongatus and M. ruber. Both the approach and conclusions derived from this work are widely applicable to microbial communities for identification of the interactions between species and characterization of community functioning as a whole.

Indirect Interspecies Regulation: Transcriptional and Physiological Responses of a Cyanobacterium to Heterotrophic Partnership.

The mechanisms by which microbes interact in communities remain poorly understood. Here, we interrogated specific interactions between photoautotrophic and heterotrophic members of a model consortium to infer mechanisms that mediate metabolic coupling and acclimation to partnership. This binary consortium was composed of a cyanobacterium, Thermosynechococcus elongatus BP-1, which supported growth of an obligate aerobic heterotroph, Meiothermus ruber strain A, by providing organic carbon, O2, and reduced nitrogen. Species-resolved transcriptomic analyses were used in combination with growth and photosynthesis kinetics to infer interactions and the environmental context under which they occur. We found that the efficiency of biomass production and resistance to stress induced by high levels of dissolved O2 increased, beyond axenic performance, as a result of heterotrophic partnership. Coordinated transcriptional responses transcending both species were observed and used to infer specific interactions resulting from the synthesis and exchange of resources. The cyanobacterium responded to heterotrophic partnership by altering expression of core genes involved with photosynthesis, carbon uptake/fixation, vitamin synthesis, and scavenging of reactive oxygen species (ROS). IMPORTANCE This study elucidates how a cyanobacterial primary producer acclimates to heterotrophic partnership by modulating the expression levels of key metabolic genes. Heterotrophic bacteria can indirectly regulate the physiology of the photoautotrophic primary producers, resulting in physiological changes identified here, such as increased intracellular ROS. Some of the interactions inferred from this model system represent putative principles of metabolic coupling in phototrophic-heterotrophic partnerships.

A flexible microbial co-culture platform for simultaneous utilization of methane and carbon dioxide from gas feedstocks.

A new co-cultivation technology is presented that converts greenhouse gasses, CH4 and CO2, into microbial biomass. The methanotrophic bacterium, Methylomicrobium alcaliphilum 20z, was coupled to a cyanobacterium, Synechococcus PCC 7002 via oxygenic photosynthesis. The system exhibited robust growth on diverse gas mixtures ranging from biogas to those representative of a natural gas feedstock. A continuous processes was developed on a synthetic natural gas feed that achieved steady-state by imposing coupled light and O2 limitations on the cyanobacterium and methanotroph, respectively. Continuous co-cultivation resulted in an O2 depleted reactor and does not require CH4/O2 mixtures to be fed into the system, thereby enhancing process safety considerations over traditional methanotroph mono-culture platforms. This co-culture technology is scalable with respect to its ability to utilize different gas streams and its biological components constructed from model bacteria that can be metabolically customized to produce a range of biofuels and bioproducts.

Dinitrogenase-Driven Photobiological Hydrogen Production Combats Oxidative Stress in Cyanothece sp. Strain ATCC 51142.

Photobiologically synthesized hydrogen (H2) gas is carbon neutral to produce and clean to combust, making it an ideal biofuel. Cyanothece sp. strain ATCC 51142 is a cyanobacterium capable of performing simultaneous oxygenic photosynthesis and H2 production, a highly perplexing phenomenon because H2 evolving enzymes are O2 sensitive. We employed a system-level in vivo chemoproteomic profiling approach to explore the cellular dynamics of protein thiol redox and how thiol redox mediates the function of the dinitrogenase NifHDK, an enzyme complex capable of aerobic hydrogenase activity. We found that NifHDK responds to intracellular redox conditions and may act as an emergency electron valve to prevent harmful reactive oxygen species formation in concert with other cell strategies for maintaining redox homeostasis. These results provide new insight into cellular redox dynamics useful for advancing photolytic bioenergy technology and reveal a new understanding for the biological function of NifHDK. IMPORTANCE: Here, we demonstrate that high levels of hydrogen synthesis can be induced as a protection mechanism against oxidative stress via the dinitrogenase enzyme complex in Cyanothece sp. strain ATCC 51142. This is a previously unknown feature of cyanobacterial dinitrogenase, and we anticipate that it may represent a strategy to exploit cyanobacteria for efficient and scalable hydrogen production. We utilized a chemoproteomic approach to capture the in situ dynamics of reductant partitioning within the cell, revealing proteins and reactive thiols that may be involved in redox sensing and signaling. Additionally, this method is widely applicable across biological systems to achieve a greater understanding of how cells navigate their environment and how redox chemistry can be utilized to alter metabolism and achieve homeostasis.

Network analysis of transcriptomics expands regulatory landscapes in Synechococcus sp. PCC 7002.

Cyanobacterial regulation of gene expression must contend with a genome organization that lacks apparent functional context, as the majority of cellular processes and metabolic pathways are encoded by genes found at disparate locations across the genome and relatively few transcription factors exist. In this study, global transcript abundance data from the model cyanobacterium Synechococcus sp. PCC 7002 grown under 42 different conditions was analyzed using Context-Likelihood of Relatedness (CLR). The resulting network, organized into 11 modules, provided insight into transcriptional network topology as well as grouping genes by function and linking their response to specific environmental variables. When used in conjunction with genome sequences, the network allowed identification and expansion of novel potential targets of both DNA binding proteins and sRNA regulators. These results offer a new perspective into the multi-level regulation that governs cellular adaptations of the fast-growing physiologically robust cyanobacterium Synechococcus sp. PCC 7002 to changing environmental variables. It also provides a methodological high-throughput approach to studying multi-scale regulatory mechanisms that operate in cyanobacteria. Finally, it provides valuable context for integrating systems-level data to enhance gene grouping based on annotated function, especially in organisms where traditional context analyses cannot be implemented due to lack of operon-based functional organization.

Unlocking the Constraints of Cyanobacterial Productivity: Acclimations Enabling Ultrafast Growth.

UNLABELLED: Harnessing the metabolic potential of photosynthetic microbes for next-generation biotechnology objectives requires detailed scientific understanding of the physiological constraints and regulatory controls affecting carbon partitioning between biomass, metabolite storage pools, and bioproduct synthesis. We dissected the cellular mechanisms underlying the remarkable physiological robustness of the euryhaline unicellular cyanobacterium Synechococcus sp. strain PCC 7002 (Synechococcus 7002) and identify key mechanisms that allow cyanobacteria to achieve unprecedented photoautotrophic productivities (~2.5-h doubling time). Ultrafast growth of Synechococcus 7002 was supported by high rates of photosynthetic electron transfer and linked to significantly elevated transcription of precursor biosynthesis and protein translation machinery. Notably, no growth or photosynthesis inhibition signatures were observed under any of the tested experimental conditions. Finally, the ultrafast growth in Synechococcus 7002 was also linked to a 300% expansion of average cell volume. We hypothesize that this cellular adaptation is required at high irradiances to support higher cell division rates and reduce deleterious effects, corresponding to high light, through increased carbon and reductant sequestration. IMPORTANCE: Efficient coupling between photosynthesis and productivity is central to the development of biotechnology based on solar energy. Therefore, understanding the factors constraining maximum rates of carbon processing is necessary to identify regulatory mechanisms and devise strategies to overcome productivity constraints. Here, we interrogate the molecular mechanisms that operate at a systems level to allow cyanobacteria to achieve ultrafast growth. This was done by considering growth and photosynthetic kinetics with global transcription patterns. We have delineated putative biological principles that allow unicellular cyanobacteria to achieve ultrahigh growth rates through photophysiological acclimation and effective management of cellular resource under different growth regimes.

Multi-Omic Dynamics Associate Oxygenic Photosynthesis with Nitrogenase-Mediated H2 Production in Cyanothece sp. ATCC 51142.

To date, the proposed mechanisms of nitrogenase-driven photosynthetic H2 production by the diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142 have assumed that reductant and ATP requirements are derived solely from glycogen oxidation and cyclic-electron flow around photosystem I. Through genome-scale transcript and protein profiling, this study presents and tests a new hypothesis on the metabolic relationship between oxygenic photosynthesis and nitrogenase-mediated H2 production in Cyanothece 51142. Our results show that net-positive rates of oxygenic photosynthesis and increased expression of photosystem II reaction centers correspond and are synchronized with nitrogenase expression and H2 production. These findings provide a new and more complete view on the metabolic processes contributing to the energy budget of photosynthetic H2 production and highlight the role of concurrent photocatalytic H2O oxidation as a participating process.

Lauric Acid Production in a Glycogen-Less Strain of Synechococcus sp. PCC 7002.

The cyanobacterium Synechococcus sp. Pasteur culture collection 7002 was genetically engineered to synthesize biofuel-compatible medium-chain fatty acids (FAs) during photoautotrophic growth. Expression of a heterologous lauroyl-acyl carrier protein (C12:0-ACP) thioesterase with concurrent deletion of the endogenous putative acyl-ACP synthetase led to secretion of transesterifiable C12:0 FA in CO2-supplemented batch cultures. When grown at steady state over a range of light intensities in a light-emitting diode turbidostat photobioreactor, the C12-secreting mutant exhibited a modest reduction in growth rate and increased O2 evolution relative to the wild-type (WT). Inhibition of (i) glycogen synthesis by deletion of the glgC-encoded ADP-glucose pyrophosphorylase (AGPase) and (ii) protein synthesis by nitrogen deprivation were investigated as potential mechanisms for metabolite redistribution to increase FA synthesis. Deletion of AGPase led to a 10-fold decrease in reducing carbohydrates and secretion of organic acids during nitrogen deprivation consistent with an energy spilling phenotype. When the carbohydrate-deficient background (ΔglgC) was modified for C12 secretion, no increase in C12 was achieved during nutrient replete growth, and no C12 was recovered from any strain upon nitrogen deprivation under the conditions used. At steady state, the growth rate of the ΔglgC strain saturated at a lower light intensity than the WT, but O2 evolution was not compromised and became increasingly decoupled from growth rate with rising irradiance. Photophysiological properties of the ΔglgC strain suggest energy dissipation from photosystem II and reconfiguration of electron flow at the level of the plastoquinone pool.

Integrated in silico Analyses of Regulatory and Metabolic Networks of Synechococcus sp. PCC 7002 Reveal Relationships between Gene Centrality and Essentiality.

Cyanobacteria dynamically relay environmental inputs to intracellular adaptations through a coordinated adjustment of photosynthetic efficiency and carbon processing rates. The output of such adaptations is reflected through changes in transcriptional patterns and metabolic flux distributions that ultimately define growth strategy. To address interrelationships between metabolism and regulation, we performed integrative analyses of metabolic and gene co-expression networks in a model cyanobacterium, Synechococcus sp. PCC 7002. Centrality analyses using the gene co-expression network identified a set of key genes, which were defined here as "topologically important." Parallel in silico gene knock-out simulations, using the genome-scale metabolic network, classified what we termed as "functionally important" genes, deletion of which affected growth or metabolism. A strong positive correlation was observed between topologically and functionally important genes. Functionally important genes exhibited variable levels of topological centrality; however, the majority of topologically central genes were found to be functionally essential for growth. Subsequent functional enrichment analysis revealed that both functionally and topologically important genes in Synechococcus sp. PCC 7002 are predominantly associated with translation and energy metabolism, two cellular processes critical for growth. This research demonstrates how synergistic network-level analyses can be used for reconciliation of metabolic and gene expression data to uncover fundamental biological principles.

Starter unit flexibility for engineered product synthesis by the nonreducing polyketide synthase PksA.

Nonreducing polyketide synthases (NR-PKSs) are unique among PKSs in their domain structure, notably including a starter unit:acyl-carrier protein (ACP) transacylase (SAT) domain that selects an acyl group as the primer for biosynthesis, most commonly acetyl-CoA from central metabolism. This clan of mega-enzymes resembles fatty acid synthases (FASs) by sharing both their central chain elongation steps and their capacity for iterative catalysis. In this mode of synthesis, catalytic domains involved in chain extension exhibit substrate plasticity to accommodate growing chains as small as two carbons to 20 or more. PksA is the NR-PKS central to the biosynthesis of the mycotoxin aflatoxin B1 whose SAT domain accepts an unusual hexanoyl starter from a dedicated yeast-like FAS. Explored in this paper is the ability of PksA to utilize a selection of potential starter units as substrates to initiate and sustain extension and cyclization to on-target, programmed polyketide synthesis. Most of these starter units were successfully accepted and properly processed by PksA to achieve biosynthesis of the predicted naphthopyrone product. Analysis of the on-target and derailment products revealed trends of tolerance by individual PksA domains to alternative starter units. In addition, natural and un-natural variants of the active site cysteine were examined and found to be capable of biosynthesis, suggesting possible direct loading of starter units onto the ß-ketoacyl synthase (KS) domain. In light of the data assembled here, the predictable synthesis of unnatural products by NR-PKSs is more fully defined.

Effect of mono- and dichromatic light quality on growth rates and photosynthetic performance of Synechococcus sp. PCC 7002.

Synechococcus sp. PCC 7002 was grown to steady state in optically thin turbidostat cultures under conditions for which light quantity and quality was systematically varied by modulating the output of narrow-band LEDs. Cells were provided photons absorbed primarily by chlorophyll (680 nm) or phycocyanin (630 nm) as the organism was subjected to four distinct mono- and dichromatic regimes. During cultivation with dichromatic light, growth rates were generally proportional to the total incident irradiance at values <275 µmol photons m(-2) · s(-1) and were not affected by the ratio of 630:680 nm wavelengths. Notably, under monochromatic light conditions, cultures exhibited similar growth rates only when they were irradiated with 630 nm light; cultures irradiated with only 680 nm light grew at rates that were 60-70% of those under other light quality regimes at equivalent irradiances. The functionality of photosystem II and associated processes such as maximum rate of photosynthetic electron transport, rate of cyclic electron flow, and rate of dark respiration generally increased as a function of growth rate. Nonetheless, some of the photophysiological parameters measured here displayed distinct patterns with respect to growth rate of cultures adapted to a single wavelength including phycobiliprotein content, which increased under severely light-limited growth conditions. Additionally, the ratio of photosystem II to photosystem I increased ~40% over the range of growth rates, although cells grown with 680 nm light only had the highest ratios. These results suggest the presence of effective mechanisms which allow acclimation of Synechococcus sp. PCC 7002 acclimation to different irradiance conditions.

Characterization of protein redox dynamics induced during light-to-dark transitions and nutrient limitation in cyanobacteria.

Protein redox chemistry constitutes a major void in knowledge pertaining to photoautotrophic system regulation and signaling processes. We have employed a chemical biology approach to analyze redox sensitive proteins in live Synechococcus sp. PCC 7002 cells in both light and dark periods, and to understand how cellular redox balance is disrupted during nutrient perturbation. The present work identified 300 putative redox-sensitive proteins that are involved in the generation of reductant, macromolecule synthesis, and carbon flux through central metabolic pathways, and may be involved in cell signaling and response mechanisms. Furthermore, our research suggests that dynamic redox changes in response to specific nutrient limitations, including carbon and nitrogen limitations, contribute to the regulatory changes driven by a shift from light to dark. Taken together, these results contribute to a high-level understanding of post-translational mechanisms regulating flux distributions and suggest potential metabolic engineering targets for redirecting carbon toward biofuel precursors.

Inference of interactions in cyanobacterial-heterotrophic co-cultures via transcriptome sequencing.

We used deep sequencing technology to identify transcriptional adaptation of the euryhaline unicellular cyanobacterium Synechococcus sp. PCC 7002 and the marine facultative aerobe Shewanella putrefaciens W3-18-1 to growth in a co-culture and infer the effect of carbon flux distributions on photoautotroph-heterotroph interactions. The overall transcriptome response of both organisms to co-cultivation was shaped by their respective physiologies and growth constraints. Carbon limitation resulted in the expansion of metabolic capacities, which was manifested through the transcriptional upregulation of transport and catabolic pathways. Although growth coupling occurred via lactate oxidation or secretion of photosynthetically fixed carbon, there was evidence of specific metabolic interactions between the two organisms. These hypothesized interactions were inferred from the excretion of specific amino acids (for example, alanine and methionine) by the cyanobacterium, which correlated with the downregulation of the corresponding biosynthetic machinery in Shewanella W3-18-1. In addition, the broad and consistent decrease of mRNA levels for many Fe-regulated Synechococcus 7002 genes during co-cultivation may indicate increased Fe availability as well as more facile and energy-efficient mechanisms for Fe acquisition by the cyanobacterium. Furthermore, evidence pointed at potentially novel interactions between oxygenic photoautotrophs and heterotrophs related to the oxidative stress response as transcriptional patterns suggested that Synechococcus 7002 rather than Shewanella W3-18-1 provided scavenging functions for reactive oxygen species under co-culture conditions. This study provides an initial insight into the complexity of photoautotrophic-heterotrophic interactions and brings new perspectives of their role in the robustness and stability of the association.

In situ molecular imaging of a hydrated biofilm in a microfluidic reactor by ToF-SIMS.

A novel microfluidic reactor for biofilm growth and in situ characterization using time-of-flight secondary ion mass spectrometry (ToF-SIMS) was constructed to enable two-dimensional chemical imaging of hydrated biofilms. We demonstrate the detection of characteristic fatty acid fragments from microfluidic reactor-grown biofilms and illustrate advantages of hydrated-state ToF-SIMS imaging.

Live cell chemical profiling of temporal redox dynamics in a photoautotrophic cyanobacterium.

Protein reduction-oxidation (redox) modification is an important mechanism that allows microorganisms to sense environmental changes and initiate cellular responses. We have developed a quantitative chemical probe approach for live cell labeling and imaging of proteins that are sensitive to redox modifications. We utilize this in vivo strategy to identify 176 proteins undergoing ∼5-10-fold dynamic redox change in response to nutrient limitation and subsequent replenishment in the photoautotrophic cyanobacterium Synechococcus sp. PCC 7002. We detect redox changes in as little as 30 s after nutrient perturbation and oscillations in reduction and oxidation for 60 min following the perturbation. Many of the proteins undergoing dynamic redox transformations participate in the major components for the production (photosystems and electron transport chains) or consumption (Calvin-Benson cycle and protein synthesis) of reductant and/or energy in photosynthetic organisms. Thus, our in vivo approach reveals new redox-susceptible proteins and validates those previously identified in vitro.

Contributions of the [NiFe]- and [FeFe]-hydrogenase to H2 production in Shewanella oneidensis MR-1 as revealed by isotope ratio analysis of evolved H(2).

Shewanella oneidensis MR-1 encodes both a [NiFe]- and an [FeFe]-hydrogenase. While the output of these proteins has been characterized in mutant strains expressing only one of the enzymes, the contribution of each to H2 synthesis in the wild-type organism is not clear. Here, we use stable isotope analysis of H2 in the culture headspace, along with transcription data and measurements of the concentrations of gases in the headspace, to characterize H2 production in the wild-type strain. After most of the O2 in the headspace had been consumed, H2 was produced and then consumed by the bidirectional [NiFe]-hydrogenase. Once the cultures were completely anaerobic, a new burst of H2 synthesis catalyzed by both enzymes took place. Our data are consistent with the hypothesis that at this point in the culture cycle, a pool of electrons is shunted toward both hydrogenases in the wild-type organisms, but that in the absence of one of the hydrogenases, the flux is redirected to the available enzyme. To our knowledge, this is the first use of natural-abundance stable isotope analysis of a metabolic product to elucidate substrate flux through two alternative enzymes in the same cellular system.

Changes in translational efficiency is a dominant regulatory mechanism in the environmental response of bacteria.

To understand how cell physiological state affects mRNA translation, we used Shewanella oneidensis MR-1 grown under steady state conditions at either 20% or 8.5% O2. Using a combination of quantitative proteomics and RNA-Seq, we generated high-confidence data on >1000 mRNA and protein pairs. By using a steady state model, we found that differences in protein-mRNA ratios were primarily due to differences in the translational efficiency of specific genes. When oxygen levels were lowered, 28% of the proteins showed at least a 2-fold change in expression. Transcription levels were sp. significantly altered for 26% of the protein changes; translational efficiency was significantly altered for 46% and a combination of both was responsible for the remaining 28%. Changes in translational efficiency were significantly correlated with the codon usage pattern of the genes and measurable tRNA pools changed in response to altered O2 levels. Our results suggest that changes in the translational efficiency of proteins, in part due to altered tRNA pools, is a major determinant of regulated alterations in protein expression levels in bacteria.