Butanol production by Clostridium acetobutylicum is accompanied by coproduction of acetone and ethanol, which reduces the yield of butanol and increases the production cost. Here, we report development of several clostridial aldehyde/alcohol dehydrogenase (AAD) variants showing increased butanol selectivity by a series of design and analysis procedures, including random mutagenesis, substrate specificity feature analysis, and structure-based butanol selectivity design. The butanol/ethanol ratios (B/E ratios) were dramatically increased to 17.47 and 15.91 g butanol/g ethanol for AADF716L and AADN655H, respectively, which are 5.8-fold and 5.3-fold higher than the ratios obtained with the wild-type AAD. The much-increased B/E ratio obtained was due to the dramatic reduction in ethanol production (0.59 ± 0.01 g/liter) that resulted from engineering the substrate binding chamber and the active site of AAD. This protein design strategy can be applied generally for engineering enzymes to alter substrate selectivity.IMPORTANCE Renewable biofuel represents one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, clostridial strain improvement has been slower than improvement of other microorganisms. Furthermore, fermentation coproducing various by-products requires costly downstream processing for butanol purification. Here, we report the results of enzyme engineering of aldehyde/alcohol dehydrogenase (AAD) to increase butanol selectivity. A metabolically engineered Clostridium acetobutylicum strain expressing the engineered aldehyde/alcohol dehydrogenase gene was capable of producing butanol at a high level of selectivity.

Malonyl-CoA reductase (MCR) is a NADPH-dependent bi-functional enzyme that performs alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities in the N- and C-terminal fragments, respectively. It catalyzes the two-step reduction of malonyl-CoA to 3-hydroxypropionate (3-HP), a key reaction in the autotrophic CO2 fixation cycles of Chloroflexaceae green non-sulfur bacteria and the archaea Crenarchaeota. However, the structural basis underlying substrate selection, coordination, and the subsequent catalytic reactions of full-length MCR is largely unknown. For the first time, we here determined the structure of full-length MCR from the photosynthetic green non-sulfur bacterium Roseiflexus castenholzii (RfxMCR) at 3.35 Å resolution. Furthermore, we determined the crystal structures of the N- and C-terminal fragments bound with reaction intermediates NADP+ and malonate semialdehyde (MSA) at 2.0 Å and 2.3 Å, respectively, and elucidated the catalytic mechanisms using a combination of molecular dynamics simulations and enzymatic analyses. Full-length RfxMCR was a homodimer of two cross-interlocked subunits, each containing four tandemly arranged short-chain dehydrogenase/reductase (SDR) domains. Only the catalytic domains SDR1 and SDR3 incorporated additional secondary structures that changed with NADP+-MSA binding. The substrate, malonyl-CoA, was immobilized in the substrate-binding pocket of SDR3 through coordination with Arg1164 and Arg799 of SDR4 and the extra domain, respectively. Malonyl-CoA was successively reduced through protonation by the Tyr743-Arg746 pair in SDR3 and the catalytic triad (Thr165-Tyr178-Lys182) in SDR1 after nucleophilic attack from NADPH hydrides. IMPORTANCE The bi-functional MCR catalyzes NADPH-dependent reduction of malonyl-CoA to 3-HP, an important metabolic intermediate and platform chemical, from biomass. The individual MCR-N and MCR-C fragments, which contain the alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities, respectively, have previously been structurally investigated and reconstructed into a malonyl-CoA pathway for the biosynthetic production of 3-HP. However, no structural information for full-length MCR has been available to illustrate the catalytic mechanism of this enzyme, which greatly limits our capacity to increase the 3-HP yield of recombinant strains. Here, we report the cryo-electron microscopy structure of full-length MCR for the first time and elucidate the mechanisms underlying substrate selection, coordination, and catalysis in the bi-functional MCR. These findings provide a structural and mechanistic basis for enzyme engineering and biosynthetic applications of the 3-HP carbon fixation pathways.

The opportunistic human pathogen Pseudomonas aeruginosa is known for exhibiting diverse forms of collective behaviors, like swarming motility and biofilm formation. Swarming in P. aeruginosa is a collective movement of the bacterial population over a semisolid surface, but specific swarming signals are not clear. We hypothesize that specific environmental signals induce swarming in P. aeruginosa. We show that under nutrient-limiting conditions, a low concentration of ethanol provides a strong ecological motivation for swarming in P. aeruginosa strain PA14. Ethanol serves as a signal and not a source of carbon under these conditions. Moreover, ethanol-driven swarming relies on the ability of the bacteria to metabolize ethanol to acetaldehyde using a periplasmic quinoprotein alcohol dehydrogenase, ExaA. We found that ErdR, an orphan response regulator linked to ethanol oxidation, is necessary for the transcriptional regulation of a cluster of 17 genes, including exaA, during swarm lag. Further, we show that P. aeruginosa displays characteristic foraging motility on a lawn of Cryptococcus neoformans, a yeast species, in a manner dependent on the ethanol dehydrogenase ErdR and on rhamnolipids. Finally, we show that ethanol, as a volatile, could induce swarming in P. aeruginosa at a distance, suggesting long-range spatial effects of ethanol as a signaling molecule. IMPORTANCE P. aeruginosa, a Gram-negative opportunistic pathogen, can adapt to diverse ecological niches and exhibits several forms of social behavior. Swarming (flagellum-driven collective motility) is a collective behavior of P. aeruginosa exclusively over semisolid surfaces. However, the ecological motivations for swarming are not known. Here, we demonstrate the importance of a specific environmental cue, ethanol, produced by many microbes, in inducing swarming in the P. aeruginosa population during starvation. We show that ethanol is a signal for swarming in P. aeruginosa. Our study provides a framework to understand swarming as a chemotactic response of bacterium to a food source via a foraging signal, ethanol.

A recent surprising discovery of the activity of rare earth metals (lanthanides) as enzyme cofactors as well as transcriptional regulators has overturned the traditional assumption of biological inertia of these metals. However, so far, examples of such activities have been limited to alcohol dehydrogenases. Here we describe the physiological effects of a mutation in xoxG, a gene encoding a novel cytochrome, XoxG(4), and compare these to the effects of mutation in XoxF, a lanthanide-dependent methanol dehydrogenase, at the enzyme activity level and also at the community function level, using Methylomonas sp. strain LW13 as a model organism. Through comparative phenotypic characterization, we establish XoxG as the second protein directly involved in lanthanide-dependent metabolism, likely as a dedicated electron acceptor from XoxF. However, mutation in XoxG caused a phenotype that was dramatically different from the phenotype of the mutant in XoxF, suggesting a secondary function for this cytochrome, in metabolism of methane. We also purify XoxG(4) and demonstrate that this protein is a true cytochrome c, based on the typical absorption spectra, and we demonstrate that XoxG can be directly reduced by a purified XoxF, supporting one of its proposed physiological functions. Overall, our data continue to suggest the complex nature of the interplay between the calcium-dependent and lanthanide-dependent alcohol oxidation systems, while they also suggest that addressing the roles of these alternative systems is essential at the enzyme and community function level, in addition to the gene transcription level.IMPORTANCE The lanthanide-dependent biochemistry of living organisms remains a barely tapped area of knowledge. So far, only a handful of lanthanide-dependent alcohol dehydrogenases have been described, and their regulation by lanthanides has been demonstrated at the transcription level. Little information is available regarding the concentrations of lanthanides that could support sufficient enzymatic activities to support specific metabolisms, and so far, no other redox proteins involved in lanthanide-dependent methanotrophy have been demonstrated. The research presented here provides enzyme activity-level data on lanthanide-dependent methanotrophy in a model methanotroph. Additionally, we identify a second protein important for lanthanide-dependent metabolism in this organism, XoxG(4), a novel cytochrome. XoxG(4) appears to have multiple functions in methanotrophy, one function as an electron acceptor from XoxF and another function remaining unknown. On the basis of the dramatic phenotype of the XoxG(4) mutant, this function must be crucial for methanotrophy.

Ever since the discovery of the first rare earth element (REE)-dependent enzyme, the physiological role of lanthanides has become an emerging field of research due to the environmental implications and biotechnological opportunities. In Pseudomonas putida KT2440, the two pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) PedE and PedH are inversely regulated in response to REE availability. This transcriptional switch is orchestrated by a complex regulatory network that includes the PedR2/PedS2 two-component system and is important for efficient growth on several alcoholic volatiles. To study whether cellular responses beyond the REE switch exist, the differential proteomic responses that occur during growth on various model carbon sources were analyzed. Apart from the Ca2+-dependent enzyme PedE, the differential abundances of most identified proteins were conditional. During growth on glycerol-and concomitant with the proteomic changes-lanthanum (La3+) availability affected different growth parameters, including the onset of logarithmic growth and final optical densities. Studies with mutant strains revealed a novel metabolic route for glycerol utilization, initiated by PedE and/or PedH activity. Upon oxidation to glycerate via glyceraldehyde, phosphorylation by the glycerate kinase GarK most likely yields glycerate-2-phosphate, which is eventually channeled into the central metabolism of the cell. This new route functions in parallel with the main degradation pathway encoded by the glpFKRD operon and provides a growth advantage to the cells by allowing an earlier onset of growth with glycerol as the sole source of carbon and energy.IMPORTANCE The biological role of REEs has long been underestimated, and research has mainly focused on methanotrophic and methylotrophic bacteria. We have recently demonstrated that P. putida, a plant growth-promoting bacterium that thrives in the rhizosphere of various food crops, possesses a REE-dependent alcohol dehydrogenase (PedH), but knowledge about REE-specific effects on physiological traits in nonmethylotrophic bacteria is still scarce. This study demonstrates that the cellular response of P. putida to lanthanum (La3+) is mostly substrate specific and that La3+ availability highly affects the growth of cells on glycerol. Further, a novel route for glycerol metabolism is identified, which is initiated by PedE and/or PedH activity and provides a growth advantage to this biotechnologically relevant organism by allowing a faster onset of growth. Overall, these findings demonstrate that lanthanides can affect physiological traits in nonmethylotrophic bacteria and might influence their competitiveness in various environmental niches.

Mycofactocin is a new class of peptide-derived redox cofactors present in a selected group of bacteria including Mycobacterium tuberculosis. Mycofactocin biosynthesis requires at least six genes, including mftD, encoding putative lactate dehydrogenase, which catalyzes the penultimate biosynthetic step. Cellular functions remained unknown until recent reports on the significance of mycofactocin in primary alcohol metabolism. Here, we show that mftD transcript levels were increased in hypoxia-adapted M. tuberculosis; however, mftD functionality was found likely dispensable for l-lactate metabolism. Targeted deletion of mftD reduced the survival of M. tuberculosis in in vitro and in vivo hypoxia models but increased the bacterial growth in glucose-containing broth as well as in the lungs and spleens, albeit modestly, of aerosol-infected C57BL/6J mice. The cause of this growth advantage remains unestablished; however, the mftD-deficient M. tuberculosis strain had reduced NAD(H)/NADP(H) levels and glucose-6-phosphate dehydrogenase activity with no impairment in phthiocerol dimycocerosate lipid synthesis. An ultrastructural examination of parental and mycofactocin biosynthesis gene mutants in M. tuberculosis, M. marinum, and M. smegmatis showed no altered cell morphology and size except the presence of outer membrane-bound fibril-like features only in a mutant subpopulation. A cell surface-protein analysis of M. smegmatis mycofactocin biosynthesis mutants with trypsin revealed differential abundances of a subset of proteins that are known to interact with mycofactocin and their homologs that can enhance protein aggregation or amyloid-like fibrils in riboflavin-starved eukaryotic cells. In sum, phenotypic analyses of the mutant strain implicate the significance of MftD/mycofactocin in M. tuberculosis growth and persistence in its host. IMPORTANCE Characterization of proteins with unknown functions is a critical research priority as the intracellular growth and metabolic state of Mycobacterium tuberculosis, the causative agent of tuberculosis, remain poorly understood. Mycofactocin is a peptide-derived redox cofactor present in almost all mycobacterial species; however, its functional relevance in M. tuberculosis pathogenesis and host survival has never been studied experimentally. In this study, we examine the phenotypes of an M. tuberculosis mutant strain lacking a key mycofactocin biosynthesis gene in in vitro and disease-relevant mouse models. Our results pinpoint the multifaceted role of mycofactocin in M. tuberculosis growth, hypoxia adaptation, glucose metabolism, and redox homeostasis. This evidence strongly implies that mycofactocin could fulfill specialized biochemical functions that increase the survival fitness of mycobacteria within their specific niche.

Animals that are competent reservoirs of zoonotic pathogens commonly suffer little morbidity from the infections. To investigate mechanisms of this tolerance of infection, we used single-dose lipopolysaccharide (LPS) as an experimental model of inflammation and compared the responses of two rodents: Peromyscus leucopus, the white-footed deermouse and reservoir for the agents of Lyme disease and other zoonoses, and the house mouse Mus musculus Four hours after injection with LPS or saline, blood, spleen, and liver samples were collected and subjected to transcriptome sequencing (RNA-seq), metabolomics, and specific reverse transcriptase quantitative PCR (RT-qPCR). Differential expression analysis was at the gene, pathway, and network levels. LPS-treated deermice showed signs of sickness similar to those of exposed mice and had similar increases in corticosterone levels and expression of interleukin 6 (IL-6), tumor necrosis factor, IL-1β, and C-reactive protein. By network analysis, the M. musculus response to LPS was characterized as cytokine associated, while the P. leucopus response was dominated by neutrophil activity terms. In addition, dichotomies in the expression levels of arginase 1 and nitric oxide synthase 2 and of IL-10 and IL-12 were consistent with type M1 macrophage responses in mice and type M2 responses in deermice. Analysis of metabolites in plasma and RNA in organs revealed species differences in tryptophan metabolism. Two genes in particular signified the different phenotypes of deermice and mice: the Slpi and Ibsp genes. Key RNA-seq findings for P. leucopus were replicated in older animals, in a systemic bacterial infection, and with cultivated fibroblasts. The findings indicate that P. leucopus possesses several adaptive traits to moderate inflammation in its balancing of infection resistance and tolerance.IMPORTANCE Animals that are natural carriers of pathogens that cause human diseases commonly manifest little or no sickness as a consequence of infection. Examples include the deermouse, Peromyscus leucopus, which is a reservoir for Lyme disease and several other disease agents in North America, and some types of bats, which are carriers of viruses with pathogenicity for humans. Mechanisms of this phenomenon of infection tolerance and entailed trade-off costs are poorly understood. Using a single injection of lipopolysaccharide (LPS) endotoxin as a proxy for infection, we found that deermice differed from the mouse (Mus musculus) in responses to LPS in several diverse pathways, including innate immunity, oxidative stress, and metabolism. Features distinguishing the deermice cumulatively would moderate downstream ill effects of LPS. Insights gained from the P. leucopus model in the laboratory have implications for studying infection tolerance in other important reservoir species, including bats and other types of wildlife.

Microbial conversion of carbon dioxide to organic commodities via syngas metabolism or microbial electrosynthesis is an attractive option for production of renewable biocommodities. The recent development of an initial genetic toolbox for the acetogen Clostridium ljungdahlii has suggested that C. ljungdahlii may be an effective chassis for such conversions. This possibility was evaluated by engineering a strain to produce butyrate, a valuable commodity that is not a natural product of C. ljungdahlii metabolism. Heterologous genes required for butyrate production from acetyl-coenzyme A (CoA) were identified and introduced initially on plasmids and in subsequent strain designs integrated into the C. ljungdahlii chromosome. Iterative strain designs involved increasing translation of a key enzyme by modifying a ribosome binding site, inactivating the gene encoding the first step in the conversion of acetyl-CoA to acetate, disrupting the gene which encodes the primary bifunctional aldehyde/alcohol dehydrogenase for ethanol production, and interrupting the gene for a CoA transferase that potentially represented an alternative route for the production of acetate. These modifications yielded a strain in which ca. 50 or 70% of the carbon and electron flow was diverted to the production of butyrate with H2 or CO as the electron donor, respectively. These results demonstrate the possibility of producing high-value commodities from carbon dioxide with C. ljungdahlii as the catalyst. Importance: The development of a microbial chassis for efficient conversion of carbon dioxide directly to desired organic products would greatly advance the environmentally sustainable production of biofuels and other commodities. Clostridium ljungdahlii is an effective catalyst for microbial electrosynthesis, a technology in which electricity generated with renewable technologies, such as solar or wind, powers the conversion of carbon dioxide and water to organic products. Other electron donors for C. ljungdahlii include carbon monoxide, which can be derived from industrial waste gases or the conversion of recalcitrant biomass to syngas, as well as hydrogen, another syngas component. The finding that carbon and electron flow in C. ljungdahlii can be diverted from the production of acetate to butyrate synthesis is an important step toward the goal of renewable commodity production from carbon dioxide with this organism.

Pseudomonas savastanoi uses a type III secretion system (T3SS) to invade host plants. Our previous studies have demonstrated that a two-component system (TCS), RhpRS, enables P. savastanoi to coordinate the T3SS gene expression, which depends on the phosphorylation state of RhpR under different environmental conditions. Orthologues of RhpRS are distributed in a wide range of bacterial species, indicating a general regulatory mechanism. How RhpRS uses external signals and the phosphorylation state to exercise its regulatory functions remains unknown. We performed chromatin immunoprecipitation sequencing (ChIP-seq) assays to identify the specific binding sites of RhpR and RhpRD70A in either King's B medium (KB [a T3SS-inhibiting medium]) or minimal medium (MM [a T3SS-inducing medium]). We identified 125 KB-dependent binding sites and 188 phosphorylation-dependent binding sites of RhpR. In KB, RhpR directly and positively regulated cytochrome c550 production (via ccmA) and alcohol dehydrogenase activity (via adhB) but negatively regulated anthranilate synthase activity (via trpG) and protease activity (via hemB). In addition, phosphorylated RhpR (RhpR-P) directly and negatively regulated the T3SS (via hrpR and hopR1), swimming motility (via flhA), c-di-GMP levels (via PSPPH_2590), and biofilm formation (via algD). It positively regulated twitching motility (via fimA) and lipopolysaccharide production (via PSPPH_2653). Our transcriptome sequencing (RNA-seq) analyses identified 474 and 840 new genes that were regulated by RhpR in KB and MM, respectively. We showed nutrient-rich conditions allowed RhpR to directly regulate multiple metabolic pathways of P. savastanoi and phosphorylation enabled RhpR to specifically control virulence and the cell envelope. The action of RhpRS switched between virulence and regulation of multiple metabolic pathways by tuning its phosphorylation and sensing environmental signals in KB, respectively.IMPORTANCE The plant pathogen Pseudomonas savastanoi invades host plants through a type III secretion system, which is strictly regulated by a two-component system called RhpRS. The orthologues of RhpRS are widely distributed in the bacterial kingdom. The master regulator RhpR specifically depends on the phosphorylation state to regulate the majority of the virulence-related genes. Under nutrient-rich conditions, it modulates many important metabolic pathways, which consist of one-fifth of the genome. We propose that RhpRS uses phosphorylation- and nutrition-dependent mechanisms to switch between regulating virulence and metabolism, and this functionality is widely conserved among bacterial species.

L-cysteine is essential for virtually all living organisms, from bacteria to higher eukaryotes. Besides having a role in the synthesis of virtually all proteins and of taurine, cysteamine, glutathione, and other redox-regulating proteins, L-cysteine has important functions under anaerobic/microaerophilic conditions. In anaerobic or microaerophilic protozoan parasites, such as Entamoeba histolytica, L-cysteine has been implicated in growth, attachment, survival, and protection from oxidative stress. However, a specific role of this amino acid or related metabolic intermediates is not well understood. In this study, using stable-isotope-labeled L-cysteine and capillary electrophoresis-time of flight mass spectrometry, we investigated the metabolism of L-cysteine in E. histolytica. [U-(13)C3, (15)N]L-cysteine was rapidly metabolized into three unknown metabolites, besides L-cystine and L-alanine. These metabolites were identified as thiazolidine-4-carboxylic acid (T4C), 2-methyl thiazolidine-4-carboxylic acid (MT4C), and 2-ethyl-thiazolidine-4-carboxylic acid (ET4C), the condensation products of L-cysteine with aldehydes. We demonstrated that these 2-(R)-thiazolidine-4-carboxylic acids serve for storage of L-cysteine. Liberation of L-cysteine occurred when T4C was incubated with amebic lysates, suggesting enzymatic degradation of these L-cysteine derivatives. Furthermore, T4C and MT4C significantly enhanced trophozoite growth and reduced intracellular reactive oxygen species (ROS) levels when it was added to cultures, suggesting that 2-(R)-thiazolidine-4-carboxylic acids are involved in the defense against oxidative stress.