David H. Keating

Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid

The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti contains three replicons: pSymA, pSymB, and the chromosome. We report here the complete 1,354,226-nt sequence of pSymA. In addition to a large fraction of the genes known to be specifically involved in symbiosis, pSymA contains genes likely to be involved in nitrogen and carbon metabolism, transport, stress, and resistance responses, and other functions that give S. meliloti an advantage in its specialized niche.

The Intracellular Concentration of Acetyl Phosphate in Escherichia coli Is Sufficient for Direct Phosphorylation of Two-Component Response Regulators

Acetyl phosphate, the intermediate of the AckA-Pta pathway, acts as a global signal in Escherichia coli. Although acetyl phosphate clearly signals through two-component response regulators, it remains unclear whether acetyl phosphate acts as a direct phospho donor or functions through an indirect mechanism. We used two-dimensional thin-layer chromatography to measure the relative concentrations of acetyl phosphate, acetyl coenzyme A, ATP, and GTP over the course of the entire growth curve. We estimated that the intracellular concentration of acetyl phosphate in wild-type cells reaches at least 3 mM, a concentration sufficient to activate two-component response regulators via direct phosphoryl transfer.

Structure-Function Analysis of Nod Factor-Induced Root Hair Calcium Spiking in Rhizobium-Legume Symbiosis

In the Rhizobium-legume symbiosis, compatible bacteria and host plants interact through an exchange of signals: Host compounds promote the expression of bacterial biosynthetic nod(nodulation) genes leading to the production of a lipochito-oligosaccharide signal, the Nod factor (NF). The particular array of nod genes carried by a given species ofRhizobium determines the NF structure synthesized and defines the range of legume hosts by which the bacterium is recognized. Purified NF can induce early host responses even in the absence of live Rhizobium One of the earliest known host responses to NF is an oscillatory behavior of cytoplasmic calcium, or calcium spiking, in root hair cells, initially observed in Medicago spp. and subsequently characterized in four other genera (D.W. Ehrhardt, R. Wais, S.R. Long [1996] Cell 85: 673–681; S.A. Walker, V. Viprey, J.A. Downie [2000] Proc Natl Acad Sci USA 97: 13413–13418; D.W. Ehrhardt, J.A. Downie, J. Harris, R.J. Wais, and S.R. Long, unpublished data). We sought to determine whether live Rhizobium trigger a rapid calcium spiking response and whether this response is NF dependent. We show that, in the Sinorhizobium meliloti-Medicago truncatula interaction, bacteria elicit a calcium spiking response that is indistinguishable from the response to purified NF. We determine that calcium spiking is a nod gene-dependent host response. Studies of calcium spiking in M. truncatula and alfalfa (Medicago sativa) also uncovered the possibility of differences in early NF signal transduction. We further demonstrate the sufficiency of thenod genes for inducing calcium spiking by usingEscherichia coli BL21 (DE3) engineered to express 11S. meliloti nod genes.

Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria

Significance Microbially produced aliphatic alcohols are important biocommodities but exert toxic effects on cells. Understanding the mechanisms by which these alcohols inhibit microbial growth and generate resistant microbes will provide insight into microbial physiology and improve prospects for microbial biotechnology and biofuel production. We find that Escherichia coli ribosomes and RNA polymerase are mechanistically affected by ethanol, identifying the ribosome decoding center as a likely target of ethanol-mediated conformational disruption and showing that ethanol inhibits transcript elongation via direct effects on RNA polymerase. Our findings provide conceptual frameworks for the study of ethanol toxicity in microbes and for the engineering of ethanol tolerance that may be extensible to other microbes and to other short-chain alcohols. The molecular mechanisms of ethanol toxicity and tolerance in bacteria, although important for biotechnology and bioenergy applications, remain incompletely understood. Genetic studies have identified potential cellular targets for ethanol and have revealed multiple mechanisms of tolerance, but it remains difficult to separate the direct and indirect effects of ethanol. We used adaptive evolution to generate spontaneous ethanol-tolerant strains of Escherichia coli, and then characterized mechanisms of toxicity and resistance using genome-scale DNAseq, RNAseq, and ribosome profiling coupled with specific assays of ribosome and RNA polymerase function. Evolved alleles of metJ, rho, and rpsQ recapitulated most of the observed ethanol tolerance, implicating translation and transcription as key processes affected by ethanol. Ethanol induced miscoding errors during protein synthesis, from which the evolved rpsQ allele protected cells by increasing ribosome accuracy. Ribosome profiling and RNAseq analyses established that ethanol negatively affects transcriptional and translational processivity. Ethanol-stressed cells exhibited ribosomal stalling at internal AUG codons, which may be ameliorated by the adaptive inactivation of the MetJ repressor of methionine biosynthesis genes. Ethanol also caused aberrant intragenic transcription termination for mRNAs with low ribosome density, which was reduced in a strain with the adaptive rho mutation. Furthermore, ethanol inhibited transcript elongation by RNA polymerase in vitro. We propose that ethanol-induced inhibition and uncoupling of mRNA and protein synthesis through direct effects on ribosomes and RNA polymerase conformations are major contributors to ethanol toxicity in E. coli, and that adaptive mutations in metJ, rho, and rpsQ help protect these central dogma processes in the presence of ethanol.

Death by a thousand cuts: the challenges and diverse landscape of lignocellulosic hydrolysate inhibitors

Lignocellulosic hydrolysate (LCH) inhibitors are a large class of bioactive molecules that arise from pretreatment, hydrolysis, and fermentation of plant biomass. These diverse compounds reduce lignocellulosic biofuel yields by inhibiting cellular processes and diverting energy into cellular responses. LCH inhibitors present one of the most significant challenges to efficient biofuel production by microbes. Development of new strains that lessen the effects of LCH inhibitors is an economically favorable strategy relative to expensive detoxification methods that also can reduce sugar content in deconstructed biomass. Systems biology analyses and metabolic modeling combined with directed evolution and synthetic biology are successful strategies for biocatalyst development, and methods that leverage state-of-the-art tools are needed to overcome inhibitors more completely. This perspective considers the energetic costs of LCH inhibitors and technologies that can be used to overcome their drain on conversion efficiency. We suggest academic and commercial research groups could benefit by sharing data on LCH inhibitors and implementing “translational biofuel research.”

Engineering and two-stage evolution of a lignocellulosic hydrolysate-tolerant Saccharomyces cerevisiae strain for anaerobic fermentation of xylose from AFEX pretreated corn stover.

The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.

Requirement of the Type II Secretion System for Utilization of Cellulosic Substrates by Cellvibrio japonicus

ABSTRACT Cellulosic biofuels represent a powerful alternative to petroleum but are currently limited by the inefficiencies of the conversion process. While Gram-positive and fungal organisms have been widely explored as sources of cellulases and hemicellulases for biomass degradation, Gram-negative organisms have received less experimental attention. We investigated the ability of Cellvibrio japonicus, a recently sequenced Gram-negative cellulolytic bacterium, to degrade bioenergy-related feedstocks. Using a newly developed biomass medium, we showed that C. japonicus is able to utilize corn stover and switchgrass as sole sources of carbon and energy for growth. We also developed tools for directed gene disruptions in C. japonicus and used this system to construct a mutant in the gspD gene, which is predicted to encode a component of the type II secretion system. The gspD::pJGG1 mutant displayed a greater-than-2-fold decrease in endoglucanase secretion compared to wild- type C. japonicus. In addition, the mutant strain showed a pronounced growth defect in medium with biomass as a carbon source, yielding 100-fold fewer viable cells than the wild type. To test the potential of C. japonicus to undergo metabolic engineering, we constructed a strain able to produce small amounts of ethanol from biomass. Collectively, these data suggest that C. japonicus is a useful platform for biomass conversion and biofuel production.

Complex Physiology and Compound Stress Responses during Fermentation of Alkali-Pretreated Corn Stover Hydrolysate by an Escherichia coli Ethanologen

ABSTRACT The physiology of ethanologenic Escherichia coli grown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into how E. coli responds to such hydrolysates, we studied an E. coli K-12 ethanologen fermenting a hydrolysate prepared from corn stover pretreated by ammonia fiber expansion. Despite the high sugar content (∼6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate, E. coli ceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses during an exponential growth phase, a transition phase, and the glycolytically active stationary phase. During the exponential and transition phases, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate. However, after the majority of amino acids were depleted, the cells entered stationary phase, and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin, and ethanol stress collectively limits growth, sugar utilization rates, and ethanol yields in alkali-pretreated lignocellulosic hydrolysates.

Sinorhizobium meliloti sulfotransferase that modifies lipopolysaccharide

Sinorhizobium meliloti is a gram-negative soil bacterium found either in free-living form or as a nitrogen-fixing endosymbiont of a plant structure called the nodule. Symbiosis between S. meliloti and its plant host alfalfa is dependent on bacterial transcription of nod genes, which encode the enzymes responsible for synthesis of Nod factor. S. meliloti Nod factor is a lipochitooligosaccharide that undergoes a sulfate modification essential for its biological activity. Sulfate also modifies the carbohydrate substituents of the bacterial cell surface, including lipopolysaccharide (LPS) and capsular polysaccharide (K-antigen) (R. A. Cedergren, J. Lee, K. L. Ross, and R. I. Hollingsworth, Biochemistry 34:4467-4477, 1995). We utilized the genomic sequence of S. meliloti to identify an open reading frame, SMc04267 (which we now propose to name lpsS), which encodes an LPS sulfotransferase activity. We expressed LpsS in Escherichia coli and demonstrated that the purified protein functions as an LPS sulfotransferase. Mutants lacking LpsS displayed an 89% reduction in LPS sulfotransferase activity in vitro. However, lpsS mutants retain approximately wild-type levels of sulfated LPS when assayed in vivo, indicating the presence of an additional LPS sulfotransferase activity(ies) in S. meliloti that can compensate for the loss of LpsS. The lpsS mutant did show reduced LPS sulfation, compared to that of the wild type, under conditions that promote nod gene expression, and it elicited a greater number of nodules than did the wild type during symbiosis with alfalfa. These results suggest that sulfation of cell surface polysaccharides and Nod factor may compete for a limiting pool of intracellular sulfate and that LpsS is required for optimal LPS sulfation under these conditions.

Systems biology defines the biological significance of redox-active proteins during cellulose degradation in an aerobic bacterium

Microbial depolymerization of plant cell walls contributes to global carbon balance and is a critical component of renewable energy. The genomes of lignocellulose degrading microorganisms encode diverse classes of carbohydrate modifying enzymes, although currently there is a paucity of knowledge on the role of these proteins in vivo. We report the comprehensive analysis of the cellulose degradation system in the saprophytic bacterium Cellvibrio japonicus. Gene expression profiling of C. japonicus demonstrated that three of the 12 predicted β‐1,4 endoglucanases (cel5A, cel5B, and cel45A) and the sole predicted cellobiohydrolase (cel6A) showed elevated expression during growth on cellulose. Targeted gene disruptions of all 13 predicted cellulase genes showed that only cel5B and cel6A were required for optimal growth on cellulose. Our analysis also identified three additional genes required for cellulose degradation: lpmo10B encodes a lytic polysaccharide monooxygenase (LPMO), while cbp2D and cbp2E encode proteins containing carbohydrate binding modules and predicted cytochrome domains for electron transfer. CjLPMO10B oxidized cellulose and Cbp2D demonstrated spectral properties consistent with redox function. Collectively, this report provides insight into the biological role of LPMOs and redox proteins in cellulose utilization and suggests that C. japonicus utilizes a combination of hydrolytic and oxidative cleavage mechanisms to degrade cellulose.