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ABSTRACT Aromatic compounds are an important source of commodity chemicals traditionally produced from fossil fuels. Aromatics derived from plant lignin can potentially be converted into commodity chemicals through depolymerization followed by microbial funneling of monomers and low molecular weight oligomers. This study investigates the catabolism of the β-5 linked aromatic dimer dehydrodiconiferyl alcohol (DC-A) by the bacterium Novosphingobium aromaticivorans. We used genome-wide screens to identify candidate genes involved in DC-A catabolism. Subsequent in vivo and in vitro analyses of these candidate genes elucidated a catabolic pathway composed of four required gene products and several partially redundant dehydrogenases that convert DC-A to aromatic monomers that can be funneled into the central aromatic metabolic pathway of N. aromaticivorans. Specifically, a newly identified γ-formaldehyde lyase, PcfL, opens the phenylcoumaran ring to form a stilbene and formaldehyde. A lignostilbene dioxygenase, LsdD, then cleaves the stilbene to generate the aromatic monomers vanillin and 5-formylferulate (5-FF). We also showed that the aldehyde dehydrogenase FerD oxidizes 5-FF before it is decarboxylated by LigW, yielding ferulic acid. We found that some enzymes involved in the β-5 catabolism pathway can act on multiple substrates and that some steps in the pathway can be mediated by multiple enzymes, providing new insights into the robust flexibility of aromatic catabolism in N. aromaticivorans. A comparative genomic analysis predicted that the newly discovered β-5 aromatic catabolic pathway is common within the order Sphingomonadales.IMPORTANCEIn the transition to a circular bioeconomy, the plant polymer lignin holds promise as a renewable source of industrially important aromatic chemicals. However, since lignin contains aromatic subunits joined by various chemical linkages, producing single chemical products from this polymer can be challenging. One strategy to overcome this challenge is using microbes to funnel a mixture of lignin-derived aromatics into target chemical products. This approach requires strategies to cleave the major inter-unit linkages of lignin to release monomers for funneling into valuable products. In this study, we report newly discovered aspects of a pathway by which the Novosphingobium aromaticivorans DSM12444 catabolizes aromatics joined by the second most common inter-unit linkage in lignin, the β-5 linkage. This work advances our knowledge of aromatic catabolic pathways, laying the groundwork for future metabolic engineering of this and other microbes for optimized conversion of lignin into products.

Bacterial cytoplasmic organelles are diverse and serve many varied purposes. Here, we employed Rhodobacter sphaeroides to investigate the accumulation of carbon and inorganic phosphate in the storage organelles, polyhydroxybutyrate (PHB) and polyphosphate (PP), respectively. Using cryo-electron tomography (cryo-ET), these organelles were observed to increase in size and abundance when growth was arrested by chloramphenicol treatment. The accumulation of PHB and PP was quantified from three-dimensional (3D) segmentations in cryo-tomograms and the analysis of these 3D models. The quantification of PHB using both segmentation analysis and liquid chromatography and mass spectrometry (LCMS) each demonstrated an over 10- to 20-fold accumulation of PHB. The cytoplasmic location of PHB in cells was assessed with fluorescence light microscopy using a PhaP-mNeonGreen fusion-protein construct. The subcellular location and enumeration of these organelles were correlated by comparing the cryo-ET and fluorescence microscopy data. A potential link between PHB and PP localization and possible explanations for co-localization are discussed. Finally, the study of PHB and PP granules, and their accumulation, is discussed in the context of advancing fundamental knowledge about bacterial stress response, the study of renewable sources of bioplastics, and highly energetic compounds.

Organic-rich industrial residues can serve as renewable feedstocks for the generation of useful products by microbial fermentation. We investigated fermenting communities enriched in a mixture of ultra-filtered milk permeate (UFMP) and acid whey from cottage cheese (CAW), two dairy coproducts rich in lactose. To evaluate how operational pH and temperature affect microbial communities and fermentation products, we operated 12 bioreactors for 140 days, each fed a 1:1 mixture of UFMP and CAW at either 35 °C or 50 °C and at either a pH of 4.8 or 5.5. The bioreactors operated at a pH of 4.8 resulted in the incomplete conversion of lactose, while those operated at a pH of 5.5 consistently fermented lactose, primarily into lactic, acetic, and hexanoic acids. The metagenomic analyses revealed that microbial communities obtained at a pH of 5.5 were dominated by lactic acid-producing organisms. Additionally, an inverse relationship was found between the abundance of chain elongating organisms and lactic acid accumulation, with 50 °C reducing the abundance of these organisms and enhancing lactic acid yields. We conclude that the pH and temperature are important determinants of the fermentation of dairy coproducts with a pH of 5.5 and 50 °C yielding the most promising results for lactic acid production. Additional research is required to better understand the factors affecting functional consistency of the process.

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

Change is an inevitable part of any organization if it wants to adapt and strive in a changing environment. That was what the American Academy of Microbiology (Academy) did from 2019-2023 when it transformed itself into a scientific think tank at ASM while maintaining the high standard of an honorific community of scholars. Here, we report on the recent history of the Academy and the changes that have taken place during this period. With the contribution of many thougtful leaders, the Academy refreshed its commitment to promote excellence and uphold its high values.

ABSTRACT The bacterial cell envelope is a key subcellular compartment with important roles in antibiotic resistance, nutrient acquisition, and cell morphology. We seek to gain a better understanding of proteins that contribute to the function of the cell envelope in Alphaproteobacteria. Using Rhodobacter sphaeroides, we show that a previously uncharacterized protein, RSP_1200, is an outer membrane (OM) lipoprotein that non-covalently binds peptidoglycan (PG). Using a fluorescently tagged version of this protein, we find that RSP_1200 undergoes a dynamic repositioning during the cell cycle and is enriched at the septum during cell division. We show that the position of RSP_1200 mirrors the location of FtsZ rings, leading us to propose that RSP_1200 is a newly identified component of the R. sphaeroides’ divisome. Additional support for this hypothesis includes the co-precipitation of RSP_1200 with FtsZ, the Pal protein, and several predicted PG L,D-transpeptidases. We also find that a ∆RSP_1200 mutation leads to defects in cell division, sensitivity to PG-active antibiotics, and results in the formation of OM protrusions at the septum during cell division. Based on these results, we propose to name RSP_1200 DalA (for division-associated lipoprotein A) and postulate that DalA serves as a scaffold to position or modulate the activity of PG transpeptidases that are needed to form envelope invaginations during cell division. We find that DalA homologs are present in members of the Rhodobacterales order within Alphaproteobacteria. Therefore, we propose that further analysis of this and related proteins will increase our understanding of the macromolecular machinery and proteins that participate in cell division in Gram-negative bacteria. IMPORTANCE Multi-protein complexes of the bacterial cell envelope orchestrate key processes like growth, division, biofilm formation, antimicrobial resistance, and production of valuable compounds. The subunits of these protein complexes are well studied in some bacteria, and differences in their composition and function are linked to variations in cell envelope composition, shape, and proliferation. However, some envelope protein complex subunits have no known homologs across the bacterial phylogeny. We find that Rhodobacter sphaeroides RSP_1200 is a newly identified lipoprotein (DalA) and that loss of this protein causes defects in cell division and changes the sensitivity to compounds, affecting cell envelope synthesis and function. We find that DalA forms a complex with proteins needed for cell division, binds the cell envelope polymer peptidoglycan, and colocalizes with enzymes involved in the assembly of this macromolecule. The analysis of DalA provides new information on the cell division machinery in this and possibly other Alphaproteobacteria.

ABSTRACT Cell elongation and division are essential aspects of the bacterial life cycle that must be coordinated for viability and replication. The impact of misregulation of these processes is not well understood as these systems are often not amenable to traditional genetic manipulation. Recently, we reported on the CenKR two-component system (TCS) in the Gram-negative bacterium Rhodobacter sphaeroides that is genetically tractable, widely conserved in α-proteobacteria, and directly regulates the expression of components crucial for cell elongation and division, including genes encoding subunit of the Tol-Pal complex. In this work, we show that overexpression of cenK results in cell filamentation and chaining. Using cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), we generated high-resolution two-dimensional (2D) images and three-dimensional (3D) volumes of the cell envelope and division septum of wild-type cells and a cenK overexpression strain finding that these morphological changes stem from defects in outer membrane (OM) and peptidoglycan (PG) constriction. By monitoring the localization of Pal, PG biosynthesis, and the bacterial cytoskeletal proteins MreB and FtsZ, we developed a model for how increased CenKR activity leads to changes in cell elongation and division. This model predicts that increased CenKR activity decreases the mobility of Pal, delaying OM constriction, and ultimately disrupting the midcell positioning of MreB and FtsZ and interfering with the spatial regulation of PG synthesis and remodeling. IMPORTANCE By coordinating cell elongation and division, bacteria maintain their shape, support critical envelope functions, and orchestrate division. Regulatory and assembly systems have been implicated in these processes in some well-studied Gram-negative bacteria. However, we lack information on these processes and their conservation across the bacterial phylogeny. In R. sphaeroides and other α-proteobacteria, CenKR is an essential two-component system (TCS) that regulates the expression of genes known or predicted to function in cell envelope biosynthesis, elongation, and/or division. Here, we leverage unique features of CenKR to understand how increasing its activity impacts cell elongation/division and use antibiotics to identify how modulating the activity of this TCS leads to changes in cell morphology. Our results provide new insight into how CenKR activity controls the structure and function of the bacterial envelope, the localization of cell elongation and division machinery, and cellular processes in organisms with importance in health, host-microbe interactions, and biotechnology.

The liquid residue resulting from various agroindustrial processes is both rich in organic material and an attractive source to produce a variety of chemicals. Using microbial communities to produce chemicals from these liquid residues is an active area of research, but it is unclear how to deploy microbial communities to produce specific products from the different agroindustrial residues. To address this, we fed anaerobic bioreactors one of several agroindustrial residues (carbohydrate-rich lignocellulosic fermentation conversion residue, xylose, dairy manure hydrolysate, ultra-filtered milk permeate, and thin stillage from a starch bioethanol plant) and inoculated them with a microbial community from an acid-phase digester operated at the wastewater treatment plant in Madison, WI, United States. The bioreactors were monitored over a period of months and sampled to assess microbial community composition and extracellular fermentation products. We obtained metagenome assembled genomes (MAGs) from the microbial communities in each bioreactor and performed comparative genomic analyses to identify common microorganisms, as well as any community members that were unique to each reactor. Collectively, we obtained a dataset of 217 non-redundant MAGs from these bioreactors. This metagenome assembled genome dataset was used to evaluate whether a specific microbial ecology model in which medium chain fatty acids (MCFAs) are simultaneously produced from intermediate products (e.g., lactic acid) and carbohydrates could be applicable to all fermentation systems, regardless of the feedstock. MAGs were classified using a multiclass classification machine learning algorithm into three groups, organisms fermenting the carbohydrates to intermediate products, organisms utilizing the intermediate products to produce MCFAs, and organisms producing MCFAs directly from carbohydrates. This analysis revealed common biological functions among the microbial communities in different bioreactors, and although different microorganisms were enriched depending on the agroindustrial residue tested, the results supported the conclusion that the microbial ecology model tested was appropriate to explain the MCFA production potential from all agricultural residues.

Fermentative microbial communities have the potential to serve as biocatalysts for the conversion of low-value dairy coproducts into renewable chemicals, contributing to a more sustainable global economy. To develop predictive tools for the design and operation of industrially relevant strategies that utilize fermentative microbial communities, there is a need to determine the genomic features of community members that are characteristic to the accumulation of different products. To address this knowledge gap, we performed a 282-day bioreactor experiment with a microbial community that was fed ultra-filtered milk permeate, a low-value coproduct from the dairy industry. The bioreactor was inoculated with a microbial community from an acid-phase digester. A metagenomic analysis was used to assess microbial community dynamics, construct metagenome-assembled genomes (MAGs), and evaluate the potential for lactose utilization and fermentation product synthesis of community members represented by the assembled MAGs. This analysis led us to propose that, in this reactor, members of the Actinobacteriota phylum are important in the degradation of lactose, via the Leloir pathway and the bifid shunt, and the production of acetic, lactic, and succinic acids. In addition, members of the Firmicutes phylum contribute to the chain-elongation-mediated production of butyric, hexanoic, and octanoic acids, with different microbes using either lactose, ethanol, or lactic acid as the growth substrate. We conclude that genes encoding carbohydrate utilization pathways, and genes encoding lactic acid transport into the cell, electron confurcating lactate dehydrogenase, and its associated electron transfer flavoproteins, are genomic features whose presence in Firmicutes needs to be established to infer the growth substrate used for chain elongation.

Abstract Background Lignocellulosic conversion residue (LCR) is the material remaining after deconstructed lignocellulosic biomass is subjected to microbial fermentation and treated to remove the biofuel. Technoeconomic analyses of biofuel refineries have shown that further microbial processing of this LCR into other bioproducts may help offset the costs of biofuel generation. Identifying organisms able to metabolize LCR is an important first step for harnessing the full chemical and economic potential of this material. In this study, we investigated the aerobic LCR utilization capabilities of 71 Streptomyces and 163 yeast species that could be engineered to produce valuable bioproducts. The LCR utilization by these individual microbes was compared to that of an aerobic mixed microbial consortium derived from a wastewater treatment plant as representative of a consortium with the highest potential for degrading the LCR components and a source of genetic material for future engineering efforts. Results We analyzed several batches of a model LCR by chemical oxygen demand (COD) and chromatography-based assays and determined that the major components of LCR were oligomeric and monomeric sugars and other organic compounds. Many of the Streptomyces and yeast species tested were able to grow in LCR, with some individual microbes capable of utilizing over 40% of the soluble COD. For comparison, the maximum total soluble COD utilized by the mixed microbial consortium was about 70%. This represents an upper limit on how much of the LCR could be valorized by engineered Streptomyces or yeasts into bioproducts. To investigate the utilization of specific components in LCR and have a defined media for future experiments, we developed a synthetic conversion residue (SynCR) to mimic our model LCR and used it to show lignocellulose-derived inhibitors (LDIs) had little effect on the ability of the Streptomyces species to metabolize SynCR. Conclusions We found that LCR is rich in carbon sources for microbial utilization and has vitamins, minerals, amino acids and other trace metabolites necessary to support growth. Testing diverse collections of Streptomyces and yeast species confirmed that these microorganisms were capable of growth on LCR and revealed a phylogenetic correlation between those able to best utilize LCR. Identification and quantification of the components of LCR enabled us to develop a synthetic LCR (SynCR) that will be a useful tool for examining how individual components of LCR contribute to microbial growth and as a substrate for future engineering efforts to use these microorganisms to generate valuable bioproducts.

Background: Plastics have conveyed great benefits to humanity and made possible some of the most significant advances of modern civilization in fields as diverse as medicine, electronics, aerospace, construction, food packaging, and sports. It is now clear, however, that plastics are also responsible for significant harms to human health, the economy, and the earth’s environment. These harms occur at every stage of the plastic life cycle, from extraction of the coal, oil, and gas that are its main feedstocks through to ultimate disposal into the environment. The extent of these harms not been systematically assessed, their magnitude not fully quantified, and their economic costs not comprehensively counted. Goals: The goals of this Minderoo-Monaco Commission on Plastics and Human Health are to comprehensively examine plastics’ impacts across their life cycle on: (1) human health and well-being; (2) the global environment, especially the ocean; (3) the economy; and (4) vulnerable populations—the poor, minorities, and the world’s children. On the basis of this examination, the Commission offers science-based recommendations designed to support development of a Global Plastics Treaty, protect human health, and save lives. Report Structure: This Commission report contains seven Sections. Following an Introduction, Section 2 presents a narrative review of the processes involved in plastic production, use, and disposal and notes the hazards to human health and the environment associated with each of these stages. Section 3 describes plastics’ impacts on the ocean and notes the potential for plastic in the ocean to enter the marine food web and result in human exposure. Section 4 details plastics’ impacts on human health. Section 5 presents a first-order estimate of plastics’ health-related economic costs. Section 6 examines the intersection between plastic, social inequity, and environmental injustice. Section 7 presents the Commission’s findings and recommendations. Plastics: Plastics are complex, highly heterogeneous, synthetic chemical materials. Over 98% of plastics are produced from fossil carbon- coal, oil and gas. Plastics are comprised of a carbon-based polymer backbone and thousands of additional chemicals that are incorporated into polymers to convey specific properties such as color, flexibility, stability, water repellence, flame retardation, and ultraviolet resistance. Many of these added chemicals are highly toxic. They include carcinogens, neurotoxicants and endocrine disruptors such as phthalates, bisphenols, per- and poly-fluoroalkyl substances (PFAS), brominated flame retardants, and organophosphate flame retardants. They are integral components of plastic and are responsible for many of plastics’ harms to human health and the environment. Global plastic production has increased almost exponentially since World War II, and in this time more than 8,300 megatons (Mt) of plastic have been manufactured. Annual production volume has grown from under 2 Mt in 1950 to 460 Mt in 2019, a 230-fold increase, and is on track to triple by 2060. More than half of all plastic ever made has been produced since 2002. Single-use plastics account for 35–40% of current plastic production and represent the most rapidly growing segment of plastic manufacture. Explosive recent growth in plastics production reflects a deliberate pivot by the integrated multinational fossil-carbon corporations that produce coal, oil and gas and that also manufacture plastics. These corporations are reducing their production of fossil fuels and increasing plastics manufacture. The two principal factors responsible for this pivot are decreasing global demand for carbon-based fuels due to increases in ‘green’ energy, and massive expansion of oil and gas production due to fracking. Plastic manufacture is energy-intensive and contributes significantly to climate change. At present, plastic production is responsible for an estimated 3.7% of global greenhouse gas emissions, more than the contribution of Brazil. This fraction is projected to increase to 4.5% by 2060 if current trends continue unchecked. Plastic Life Cycle: The plastic life cycle has three phases: production, use, and disposal. In production, carbon feedstocks—coal, gas, and oil—are transformed through energy-intensive, catalytic processes into a vast array of products. Plastic use occurs in every aspect of modern life and results in widespread human exposure to the chemicals contained in plastic. Single-use plastics constitute the largest portion of current use, followed by synthetic fibers and construction. Plastic disposal is highly inefficient, with recovery and recycling rates below 10% globally. The result is that an estimated 22 Mt of plastic waste enters the environment each year, much of it single-use plastic and are added to the more than 6 gigatons of plastic waste that have accumulated since 1950. Strategies for disposal of plastic waste include controlled and uncontrolled landfilling, open burning, thermal conversion, and export. Vast quantities of plastic waste are exported each year from high-income to low-income countries, where it accumulates in landfills, pollutes air and water, degrades vital ecosystems, befouls beaches and estuaries, and harms human health—environmental injustice on a global scale. Plastic-laden e-waste is particularly problematic. Environmental Findings: Plastics and plastic-associated chemicals are responsible for widespread pollution. They contaminate aquatic (marine and freshwater), terrestrial, and atmospheric environments globally. The ocean is the ultimate destination for much plastic, and plastics are found throughout the ocean, including coastal regions, the sea surface, the deep sea, and polar sea ice. Many plastics appear to resist breakdown in the ocean and could persist in the global environment for decades. Macro- and micro-plastic particles have been identified in hundreds of marine species in all major taxa, including species consumed by humans. Trophic transfer of microplastic particles and the chemicals within them has been demonstrated. Although microplastic particles themselves (>10 µm) appear not to undergo biomagnification, hydrophobic plastic-associated chemicals bioaccumulate in marine animals and biomagnify in marine food webs. The amounts and fates of smaller microplastic and nanoplastic particles (MNPs

This article details a correction to: Landrigan PJ, Raps H, Cropper M, et al. The Minderoo-Monaco Commission on Plastics and Human Health. Annals of Global Health. 2023; 89(1): 23. DOI: https://doi.org/10.5334/aogh.4056.

The economic and environmental sustainability of lignocellulosic biomass biorefineries is predicated on generating biofuels and bioproducts from cell-wall polysaccharide and lignin polymers. Historical efforts in plant genetic engineering have focused on the development of strategies that facilitate biomass deconstruction, with more recently efforts including the synthesis of high-value chemicals in planta. One such genetic modification is the expression of the bacterial quinate and shikimate utilization B (qsuB) gene that increases the accumulation of protocatechuic acid in lignocellulosic biomass. Herein, we evaluated the effectiveness of an alkaline pretreatment process to extract phenolics directly from wild-type and QsuB-transgenic lines of Arabidopsis, poplar, and sorghum, and then upgrade them to the polyester precursor 2-pyrone-4,6-dicarboxylic acid (PDC) with an engineered strain of Novosphingobium aromaticivorans. Protocatechuic acid extracted from all QsuB transgenic lines was found to be mostly in the glycosylated form. Glycosylated protocatechuic acid and other plant-derived phenolics were effectively metabolized by N. aromaticivorans, and PDC production was greatest using extracts from an Arabidopsis QsuB transgenic line (∼5% w/w), followed by QsuB sorghum (∼1.1% w/w), and QsuB poplar (∼0.4% w/w) lines. The comparison of PDC production from wild-type and QsuB transgenic lines of Arabidopsis, poplar, and sorghum demonstrates the utility of a mild alkaline pretreatment to liberate phenolics from plant biomass that are either naturally present or that accumulate as a consequence of genetic engineering strategies. All QsuB transgenic lines outperformed their wild-type counterparts with respect to observed PDC yields. In addition, microbial funneling to PDC was effective even when most of the protocatechuic acid extracted was in glycosylated form, clearly demonstrating that this bacterium can metabolize these aromatic conjugates. These findings illustrate the benefits of combining plant and microbial engineering for bioproduct formation from phenolics in lignocellulosic biorefineries.

Abstract Background Nontuberculous mycobacteria (NTM) are environmental bacterium that may cause and/or compound respiratory diseases in humans. There are over a hundred NTM species with varying pathogenicity’s Therefore, it is necessary to characterize the populations at risk for each species. Methods Demographic (age, sex, and state of residence) and microbiological data from 2014 were extracted from Mississippi, Missouri, and Ohio disease surveillance systems. NTM species with > 50 reports were included in the analysis. Patient sex, age, and incidence rates were generated for each of the following NTM species: M. abscessus, M. avium complex (MAC), M. chelonae, M. fortuitum, M. gordonae, M. kansasii, M. mucogenicum, and M. peregrinum. Results Analysis by sex showed that M. chelonae,M. fortuitum, M. gordonae,and M. kansasii had significantly higher rates in males than females. Age was not associated with patient rates for several specific NTM species e.g., M. chelonae. Mississippi had the highest patient’ rates for M. avium, M. gordonae, M. kansasii, and M. chelonae. Ohio had the highest patient’ rates for M. abscessus, M. mucogenicum, and M. peregrinum. The highest patient’s rate for M. fortuitum was observed in Missouri. Conclusion This study showed that NTM infection occurred more frequently in males. The highest rates were observed in Mississippi for most of the NTMs studied. Age was not a strong risk factor for some of the NTM species.

Bacterial two-component systems (TCSs) often function through the detection of an extracytoplasmic stimulus and the transduction of a signal by a transmembrane sensory histidine kinase. This kinase then initiates a series of reversible phosphorylation modifications to regulate the activity of a cognate, cytoplasmic response regulator as a transcription factor. Several TCSs have been implicated in the regulation of cell cycle dynamics, cell envelope integrity, or cell wall development in Escherichia coli and other well-studied Gram-negative model organisms. However, many α-proteobacteria lack homologs to these regulators, so an understanding of how α-proteobacteria orchestrate extracytoplasmic events is lacking. In this work we identify an essential TCS, CenKR (Cell envelope Kinase and Regulator), in the α-proteobacterium Rhodobacter sphaeroides and show that modulation of its activity results in major morphological changes. Using genetic and biochemical approaches, we dissect the requirements for the phosphotransfer event between CenK and CenR, use this information to manipulate the activity of this TCS in vivo, and identify genes that are directly and indirectly controlled by CenKR in Rb. sphaeroides. Combining ChIP-seq and RNA-seq, we show that the CenKR TCS plays a direct role in maintenance of the cell envelope, regulates the expression of subunits of the Tol-Pal outer membrane division complex, and indirectly modulates the expression of peptidoglycan biosynthetic genes. CenKR represents the first TCS reported to directly control the expression of Tol-Pal machinery genes in Gram-negative bacteria, and we predict that homologs of this TCS serve a similar function in other closely related organisms. We propose that Rb. sphaeroides genes of unknown function that are directly regulated by CenKR play unknown roles in cell envelope biosynthesis, assembly, and/or remodeling in this and other α-proteobacteria.

Abstract The observed spatial and temporal dynamics in landscape and ecosystem resources are the net effects of natural processes and management activities. Monitoring the impact that humans have on these resources requires that these two sources of variability be partitioned, removing the natural variability to reveal variability due to management activities. Here, we present Compere, a relative benchmarking framework for monitoring the management‐driven impacts on ecosystem resources. The framework identifies locations in a region that share similar biophysical properties to a target location. Taking an attribute of interest, it then compares the value of the target location with those of all its biophysically equivalent locations, with any differences being attributed to variations in management. We provide an example application of Compere, using satellite‐derived vegetation cover data to examine the impact of hydrocarbon extraction activities on land condition (as described by vegetation cover) across the 490,000‐km2 Cooper Creek region in arid central Australia. Validation was performed by comparing land condition estimates to known disturbance patterns across the region. We found that the establishment of well sites was associated with between 12% and 41% decreases in land condition but that condition recovered to original levels by 6 years after establishment. We also found that fires had much larger and longer lasting effects on land condition across these landscapes, indicating that mining‐related activities that change fire patterns are likely to affect land condition most. Compere is a generic, flexible framework, providing a new capability for monitoring management impacts on multiple types of ecosystem resources.

Microbiomes are important contributors to many ecosystems, including ones where nutrient cycling is stimulated by aeration control. Optimizing cyclic aeration helps reduce energy needs and maximize microbiome performance during wastewater treatment; however, little is known about how most microbial community members respond to these alternating conditions.

Transcription of DNA to RNA by RNA polymerase is essential for cells to grow, develop, and respond to stress. Understanding the process and control of transcription is important for health, disease, the environment, and biotechnology.

Dairy manure (DM) is an abundant agricultural residue that is largely composed of lignocellulosic biomass. The aim of this study was to investigate if carbon derived from DM fibers can be recovered as medium-chain fatty acids (MCFAs), which are mixed culture fermentation products of economic interest. DM fibers were subjected to combinations of physical, enzymatic, chemical, and thermochemical pretreatments to evaluate the possibility of producing carbohydrate-rich hydrolysates suitable for microbial fermentation by mixed cultures. Among the pretreatments tested, decrystalization dilute acid pretreatment (DCDA) produced the highest concentrations of glucose and xylose, and was selected for further experiments. Bioreactors fed DCDA hydrolysate were operated. Acetic acid and butyric acid comprised the majority of end products during operation of the bioreactors. MCFAs were transiently produced at a maximum concentration of 0.17 mg CODMCFAs/mg CODTotal. Analyses of the microbial communities in the bioreactors suggest that lactic acid bacteria, Megasphaera, and Caproiciproducens were involved in MCFA and C4 production during DCDA hydrolysate metabolism.

The economy of biorefineries is influenced not only by biofuel production from carbohydrates but also by the production of valuable compounds from largely underutilized industrial residues. Currently, the demand for many chemicals that could be made in a biorefinery, such as succinic acid (SA), medium-chain fatty acids (MCFAs), and lactic acid (LA), is fulfilled using petroleum, palm oil, or pure carbohydrates as raw materials, respectively. Thin stillage (TS), the residual liquid material following distillation of ethanol, is an underutilized coproduct from the starch biofuel industry. This carbon-rich material has the potential for chemical upgrading by microorganisms. Here, we explored the formation of different fermentation products by microbial communities grown on TS using different bioreactor conditions. At the baseline operational condition (6-day retention time, pH 5.5, 35°C), we observed a mixture of MCFAs as the principal fermentation products. Operation of a bioreactor with a 1-day retention time induced an increase in SA production, and a temperature increase to 55°C resulted in the accumulation of lactic and propionic acids. In addition, a reactor operated with a 1-day retention time at 55°C conditions resulted in LA accumulation as the main fermentation product. The prominent members of the microbial community in each reactor were assessed by 16S rRNA gene amplicon sequencing and phylogenetic analysis. Under all operating conditions, members of the Lactobacillaceae family within Firmicutes and the Acetobacteraceae family within Proteobacteria were ubiquitous. Members of the Prevotellaceae family within Bacteroidetes and Lachnospiraceae family within the Clostridiales order of Firmicutes were mostly abundant at 35°C and not abundant in the microbial communities of the TS reactors incubated at 55°C. The ability to adjust bioreactor operating conditions to select for microbial communities with different fermentation product profiles offers new strategies to explore and compare potentially valuable fermentation products from TS and allows industries the flexibility to adapt and switch chemical production based on market prices and demands.

Objectives:. Triaging patients at admission to determine subsequent deterioration risk can be difficult. This is especially true of coronavirus disease 2019 patients, some of whom experience significant physiologic deterioration due to dysregulated immune response following admission. A well-established acuity measure, the Rothman Index, is evaluated for stratification of patients at admission into high or low risk of subsequent deterioration. Design:. Multicenter retrospective study. Setting:. One academic medical center in Connecticut, and three community hospitals in Connecticut and Maryland. Patients:. Three thousand four hundred ninety-nine coronavirus disease 2019 and 14,658 noncoronavirus disease 2019 adult patients admitted to a medical service between January 1, 2020, and September 15, 2020. Interventions:. None. Measurements and Main Results:. Performance of the Rothman Index at admission to predict in-hospital mortality or ICU utilization for both general medical and coronavirus disease 2019 populations was evaluated using the area under the curve. Precision and recall for mortality prediction were calculated, high- and low-risk thresholds were determined, and patients meeting threshold criteria were characterized. The Rothman Index at admission has good to excellent discriminatory performance for in-hospital mortality in the coronavirus disease 2019 (area under the curve, 0.81–0.84) and noncoronavirus disease 2019 (area under the curve, 0.90–0.92) populations. We show that for a given admission acuity, the risk of deterioration for coronavirus disease 2019 patients is significantly higher than for noncoronavirus disease 2019 patients. At admission, Rothman Index–based thresholds segregate the majority of patients into either high- or low-risk groups; high-risk groups have mortality rates of 34–45% (coronavirus disease 2019) and 17–25% (noncoronavirus disease 2019), whereas low-risk groups have mortality rates of 2–5% (coronavirus disease 2019) and 0.2–0.4% (noncoronavirus disease 2019). Similarly large differences in ICU utilization are also found. Conclusions:. Acuity level at admission may support rapid and effective risk triage. Notably, in-hospital mortality risk associated with a given acuity at admission is significantly higher for coronavirus disease 2019 patients than for noncoronavirus disease 2019 patients. This insight may help physicians more effectively triage coronavirus disease 2019 patients, guiding level of care decisions and resource allocation.

ABSTRACT Heme is an essential metabolite for most life on earth. Bacterial pathogens almost universally require iron to infect a host, often acquiring this nutrient in the form of heme. The Gram-negative pathogen Pseudomonas aeruginosa is no exception, where heme acquisition and metabolism are known to be crucial for both chronic and acute infections. To unveil unknown genes and pathways that could play a role with heme metabolic flux in this pathogen, we devised an omic-based approach we dubbed “Met-Seq,” for metabolite-coupled transposon sequencing. Met-Seq couples a biosensor with fluorescence-activated cell sorting (FACS) and massively parallel sequencing, allowing for direct identification of genes associated with metabolic changes. In this work, we first construct and validate a heme biosensor for use with P. aeruginosa and exploit Met-Seq to identify 188 genes that potentially influence intracellular heme levels. Identified genes largely consisted of metabolic pathways not previously associated with heme, including many secreted virulence effectors, as well as 11 predicted small RNAs (sRNAs) and riboswitches whose functions are not currently understood. We verify that five Met-Seq hits affect intracellular heme levels; a predicted extracytoplasmic function (ECF) factor, a phospholipid acquisition system, heme biosynthesis regulator Dnr, and two predicted antibiotic monooxygenase (ABM) domains of unknown function (PA0709 and PA3390). Finally, we demonstrate that PA0709 and PA3390 are novel heme-binding proteins. Our data suggest that Met-Seq could be extrapolated to other biological systems and metabolites for which there is an available biosensor, and provides a new template for further exploration of iron/heme regulation and metabolism in P. aeruginosa and other pathogens. IMPORTANCE The ability to simultaneously and more directly correlate genes with metabolite levels on a global level would provide novel information for many biological platforms yet has thus far been challenging. Here, we describe a method to help address this problem, which we dub “Met-Seq” (metabolite-coupled Tn sequencing). Met-Seq uses the powerful combination of fluorescent biosensors, fluorescence-activated cell sorting (FACS), and next-generation sequencing (NGS) to rapidly identify genes that influence the levels of specific intracellular metabolites. For proof of concept, we create and test a heme biosensor and then exploit Met-Seq to identify novel genes involved in the regulation of heme in the pathogen Pseudomonas aeruginosa. Met-Seq-generated data were largely comprised of genes which have not previously been reported to influence heme levels in this pathogen, two of which we verify as novel heme-binding proteins. As heme is a required metabolite for host infection in P. aeruginosa and most other pathogens, our studies provide a new list of targets for potential antimicrobial therapies and shed additional light on the balance between infection, heme uptake, and heme biosynthesis.

ABSTRACT Multispecies microbial communities determine the fate of materials in the environment and can be harnessed to produce beneficial products from renewable resources. In a recent example, fermentations by microbial communities have produced medium-chain fatty acids (MCFAs). Tools to predict, assess, and improve the performance of these communities, however, are limited. To provide such tools, we constructed two metabolic models of MCFA-producing microbial communities based on available genomic, transcriptomic, and metabolomic data. The first model is a unicellular model (iFermCell215), while the second model (iFermGuilds789) separates fermentation activities into functional guilds. Ethanol and lactate are fermentation products known to serve as substrates for MCFA production, while acetate is another common cometabolite during MCFA production. Simulations with iFermCell215 predict that low molar ratios of acetate to ethanol favor MCFA production, whereas the products of lactate and acetate coutilization are less dependent on the acetate-to-lactate ratio. In simulations of an MCFA-producing community fed a complex organic mixture derived from lignocellulose, iFermGuilds789 predicted that lactate was an extracellular cometabolite that served as a substrate for butyrate (C4) production. Extracellular hexanoic (C6) and octanoic (C8) acids were predicted by iFermGuilds789 to be from community members that directly metabolize sugars. Modeling results provide several hypotheses that can improve our understanding of microbial roles in an MCFA-producing microbiome and inform strategies to increase MCFA production. Further, these models represent novel tools for exploring the role of mixed microbial communities in carbon recycling in the environment, as well as in beneficial reuse of organic residues. IMPORTANCE Microbiomes are vital to human health, agriculture, and protecting the environment. Predicting behavior of self-assembled or synthetic microbiomes, however, remains a challenge. In this work, we used unicellular and guild-based metabolic models to investigate production of medium-chain fatty acids by a mixed microbial community that is fed multiple organic substrates. Modeling results provided insights into metabolic pathways of three medium-chain fatty acid-producing guilds and identified potential strategies to increase production of medium-chain fatty acids. This work demonstrates the role of metabolic models in augmenting multi-omic studies to gain greater insights into microbiome behavior.

ABSTRACT Activity of the NtrYX two-component system has been associated with important processes in diverse bacteria, ranging from symbiosis to nitrogen and energy metabolism. In the facultative alphaproteobacterium Rhodobacter sphaeroides, loss of the two-component system NtrYX results in increased lipid production and sensitivity to some known cell envelope-active compounds. In this study, we show that NtrYX directly controls multiple properties of the cell envelope. We find that the response regulator NtrX binds upstream of cell envelope genes, including those involved in peptidoglycan biosynthesis and modification and in cell division. We show that loss of NtrYX impacts the cellular levels of peptidoglycan precursors and lipopolysaccharide and alters cell envelope structure, increasing cell length and the thickness of the periplasm. Cell envelope function is also disrupted in the absence of NtrYX, resulting in increased outer membrane permeability. Based on the properties of R. sphaeroides cells lacking NtrYX and the target genes under direct control of this two-component system, we propose that NtrYX plays a previously undescribed, and potentially conserved, role in the assembly, structure, and function of the cell envelope in a variety of bacteria. IMPORTANCE The bacterial cell envelope provides many important functions. It protects cells from harsh environments, serves as a selective permeability barrier, houses bioenergetic functions, defines sensitivity to antibacterial agents, and plays a crucial role in biofilm formation, symbiosis, and virulence. Despite the important roles of this cellular compartment, we lack a detailed understanding of the biosynthesis and remodeling of the cell envelope. Here, we report that the R. sphaeroides two-component signaling system NtrYX is a previously undescribed regulator of cell envelope processes, providing evidence that it is directly involved in controlling transcription of genes involved in cell envelope assembly, structure, and function in this and possibly other bacteria. Thus, our data report on a newly discovered process used by bacteria to assemble and remodel the cell envelope.

Lignin is an abundant renewable source of aromatics and precursors for the production of other organic chemicals. However, lignin is a heterogeneous polymer, so the mixture of aromatics released during its depolymerization can make its conversion to chemicals challenging. Microbes are a potential solution to this challenge, as some can catabolize multiple aromatic substrates into one product. Novosphingobium aromaticivorans has this ability, and its use as a bacterial chassis for lignin valorization could be improved by the ability to predict product yields based on thermodynamic and metabolic inputs. In this work, we built a genome-scale metabolic model of N. aromaticivorans, iNovo479, to guide the engineering of strains for aromatic conversion into products. iNovo479 predicted product yields from single or multiple aromatics, and the impact of combinations of aromatic and non-aromatic substrates on product yields. We show that enzyme reactions from other organisms can be added to iNovo479 to predict the feasibility and profitability of producing additional products by engineered strains. Thus, we conclude that iNovo479 can help guide the design of bacteria to convert lignin aromatics into valuable chemicals.

Abstract Background Lignocellulosic biomass is seen as an abundant renewable source of liquid fuels and chemicals that are currently derived from petroleum. When lignocellulosic biomass is used for ethanol production, the resulting liquid residue (stillage) contains large amounts of organic material that could be further transformed into recoverable bioproducts, thus enhancing the economics of the biorefinery. Results Here we test the hypothesis that a bacterial community could transform the organics in stillage into valuable bioproducts. We demonstrate the ability of this microbiome to convert stillage organics into medium-chain fatty acids (MCFAs), identify the predominant community members, and perform a technoeconomic analysis of recovering MCFAs as co-products of ethanol production. Steady-state operation of a stillage-fed bioreactor showed that 18% of the organic matter in stillage was converted to MCFAs. Xylose and complex carbohydrates were the primary substrates transformed. During the MCFA production period, the five major genera represented more than 95% of the community, including Lactobacillus, Roseburia, Atopobium, Olsenella, and Pseudoramibacter. To assess the potential benefits of producing MCFAs from stillage, we modeled the economics of ethanol and MCFA co-production, at MCFA productivities observed during reactor operation. Conclusions The analysis predicts that production of MCFAs, ethanol, and electricity could reduce the minimum ethanol selling price from $2.15 to $1.76 gal−1 ($2.68 gal−1 gasoline equivalents) when compared to a lignocellulosic biorefinery that produces only ethanol and electricity.

Abstract Background Nontuberculous Mycobacteria (NTM) are environmental microorganisms that can affect human health. A 2009–2010 occurrence survey of NTM in potable tap water samples indicated an increased recovery rate for many clinically significant species such as M. avium (30%) and M. abscessus (12%). To determine if these trends by species were mirrored in human infections, isolation rates of NTM species identified in clinical laboratory reports from four states were evaluated. Method Clinical laboratory reports from the Mississippi, Missouri, Ohio, and Wisconsin Health Departments were used to investigate the species of NTM isolated from human specimens in 2014. The NTM positive specimen reports were tabulated for each species and complex/group. The number of reports by month were used to investigate seasonal trends. The 2014 isolation rates were compared to historic values to examine longitudinal trends. Results The positive rate of NTM specimens increased from 8.2 per 100,000 persons in 1994 to 16 per 100,000 persons in 2014 (or 13.3 per 100,000 after excluding Mycobacterium gordonae). Changes in NTM diversity were observed in complex/groups known to be clinically significant. Between 1994 and 2014 the rate implicating M. abscesses-chelonae group and M. avium complex increased by 322 and 149%, respectively. Conclusions Based on public health data supplied by the four State’s Health Departments and the 2014 U.S. population, 50,976 positive NTM specimen reports per year were projected for the nation; serving as an indicator for the national potential disease burden that year.

The onus for monitoring crop growth from space is its ability to be applied anytime and anywhere, to produce crop yield estimates that are consistent at both the subfield scale for farming management strategies and the country level for national crop yield assessment. Historically, the requirements for satellites to successfully monitor crop growth and yield differed depending on the extent of the area being monitored. Diverging imaging capabilities can be reconciled by blending images from high-temporal-frequency (HTF) and high-spatial-resolution (HSR) sensors to produce images that possess both HTF and HSR characteristics across large areas. We evaluated the relative performance of Moderate Resolution Imaging Spectroradiometer (MODIS), Landsat, and blended imagery for crop yield estimates (2009–2015) using a carbon-turnover yield model deployed across the Australian cropping area. Based on the fraction of missing Landsat observations, we further developed a parsimonious framework to inform when and where blending is beneficial for nationwide crop yield prediction at a finer scale (i.e., the 25-m pixel resolution). Landsat provided the best yield predictions when no observations were missing, which occurred in 17% of the cropping area of Australia. Blending was preferred when

ABSTRACT Biomanufacturing from renewable feedstocks can offset fossil fuel-based chemical production. One potential biomanufacturing strategy is production of medium-chain fatty acids (MCFA) from organic feedstocks using either pure cultures or microbiomes. While the set of microbes in a microbiome can often metabolize organic materials of greater diversity than a single species can and while the role of specific species may be known, knowledge of the carbon and energy flow within and between organisms in MCFA-producing microbiomes is only now starting to emerge. Here, we integrated metagenomic, metatranscriptomic, and thermodynamic analyses to predict and characterize the metabolic network of an anaerobic microbiome producing MCFA from organic matter derived from lignocellulosic ethanol fermentation conversion residue. A total of 37 high-quality (>80% complete,

ABSTRACT Lipids from microbes offer a promising source of renewable alternatives to petroleum-derived compounds. In particular, oleaginous microbes are of interest because they accumulate a large fraction of their biomass as lipids. In this study, we analyzed genetic changes that alter lipid accumulation in Rhodobacter sphaeroides. By screening an R. sphaeroides Tn5 mutant library for insertions that increased fatty acid content, we identified 10 high-lipid (HL) mutants for further characterization. These HL mutants exhibited increased sensitivity to drugs that target the bacterial cell envelope and changes in shape, and some had the ability to secrete lipids, with two HL mutants accumulating ~60% of their total lipids extracellularly. When one of the highest-lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to that of oleaginous microbes, with the majority of the lipids secreted into the medium. Based on the properties of these HL mutants, we conclude that alterations of the cell envelope are a previously unreported approach to increase microbial lipid production. We also propose that this approach may be combined with knowledge about biosynthetic pathways, in this or other microbes, to increase production of lipids and other chemicals. IMPORTANCE This paper reports on experiments to understand how to increase microbial lipid production. Microbial lipids are often cited as one renewable replacement for petroleum-based fuels and chemicals, but strategies to increase the yield of these compounds are needed to achieve this goal. While lipid biosynthesis is often well understood, increasing yields of these compounds to industrially relevant levels is a challenge, especially since genetic, synthetic biology, or engineering approaches are not feasible in many microbes. We show that altering the bacterial cell envelope can be used to increase microbial lipid production. We also find that the utility of some of these alterations can be enhanced by growing cells in bioreactor configurations that can be used industrially. We propose that our findings can inform current and future efforts to increase production of microbial lipids, other fuels, or chemicals that are currently derived from petroleum.

ABSTRACT Rhodobacter sphaeroides is one of the best-studied alphaproteobacteria from biochemical, genetic, and genomic perspectives. To gain a better systems-level understanding of this organism, we generated a large transposon mutant library and used transposon sequencing (Tn-seq) to identify genes that are essential under several growth conditions. Using newly developed Tn-seq analysis software (TSAS), we identified 493 genes as essential for aerobic growth on a rich medium. We then used the mutant library to identify conditionally essential genes under two laboratory growth conditions, identifying 85 additional genes required for aerobic growth in a minimal medium and 31 additional genes required for photosynthetic growth. In all instances, our analyses confirmed essentiality for many known genes and identified genes not previously considered to be essential. We used the resulting Tn-seq data to refine and improve a genome-scale metabolic network model (GEM) for R. sphaeroides. Together, we demonstrate how genetic, genomic, and computational approaches can be combined to obtain a systems-level understanding of the genetic framework underlying metabolic diversity in bacterial species. IMPORTANCE Knowledge about the role of genes under a particular growth condition is required for a holistic understanding of a bacterial cell and has implications for health, agriculture, and biotechnology. We developed the Tn-seq analysis software (TSAS) package to provide a flexible and statistically rigorous workflow for the high-throughput analysis of insertion mutant libraries, advanced the knowledge of gene essentiality in R. sphaeroides, and illustrated how Tn-seq data can be used to more accurately identify genes that play important roles in metabolism and other processes that are essential for cellular survival. Author Video: An author video summary of this article is available.