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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., Importance: In 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., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors declare no conflict of interest.

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., (Copyright © 2020 Lemmer et al.)

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 Tn 5 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., (Copyright © 2017 Lemmer et al.)

Unlabelled: The proton-translocating NADH:quinone oxidoreductase (complex I) is a multisubunit integral membrane enzyme found in the respiratory chains of both bacteria and eukaryotic organelles. Although much research has focused on the enzyme's central role in the mitochondrial respiratory chain, comparatively little is known about its role in the diverse energetic lifestyles of different bacteria. Here, we used a phylogenomic approach to better understand the distribution of complex I across bacteria, the evolution of this enzyme, and its potential roles in shaping the physiology of different bacterial groups. By surveying 970 representative bacterial genomes, we predict complex I to be present in ~50% of bacteria. While this includes bacteria with a wide range of energetic schemes, the presence of complex I is associated with specific lifestyles, including aerobic respiration and specific types of phototrophy (bacteria with only a type II reaction center). A phylogeny of bacterial complex I revealed five main clades of enzymes whose evolution is largely congruent with the evolution of the bacterial groups that encode complex I. A notable exception includes the gammaproteobacteria, whose members encode one of two distantly related complex I enzymes predicted to participate in different types of respiratory chains (aerobic versus anaerobic). Comparative genomic analyses suggest a broad role for complex I in reoxidizing NADH produced from various catabolic reactions, including the tricarboxylic acid (TCA) cycle and fatty acid beta-oxidation. Together, these findings suggest diverse roles for complex I across bacteria and highlight the importance of this enzyme in shaping diverse physiologies across the bacterial domain., Importance: Living systems use conserved energy currencies, including a proton motive force (PMF), NADH, and ATP. The respiratory chain enzyme, complex I, connects these energy currencies by using NADH produced during nutrient breakdown to generate a PMF, which is subsequently used for ATP synthesis. Our goal is to better understand the role of complex I in bacteria, whose energetic diversity allows us to view its function in a range of biological contexts. We analyzed sequenced bacterial genomes to predict the presence, evolution, and function of complex I in bacteria. We identified five main classes of bacterial complex I and predict that different classes participate in different types of respiratory chains (aerobic and anaerobic). We also predict that complex I helps maintain a cellular redox state by reoxidizing NADH produced from central metabolism. Our findings suggest diverse roles for complex I in bacterial physiology, highlighting the need for future laboratory-based studies., (Copyright © 2015 Spero et al.)

Unlabelled: Many pathways of carbon and energy metabolism are conserved across the phylogeny, but the networks that regulate their expression or activity often vary considerably among organisms. In this work, we show that two previously uncharacterized transcription factors (TFs) are direct regulators of genes encoding enzymes of central carbon and energy metabolism in the alphaproteobacterium Rhodobacter sphaeroides. The LacI family member CceR (RSP_1663) directly represses genes encoding enzymes in the Entner-Doudoroff pathway, while activating those encoding the F1F0 ATPase and enzymes of the tricarboxylic acid (TCA) cycle and gluconeogenesis, providing a direct transcriptional network connection between carbon and energy metabolism. We identified bases that are important for CceR DNA binding and showed that DNA binding by this TF is inhibited by 6-phosphogluconate. We also showed that the GntR family TF AkgR (RSP_0981) directly activates genes encoding several TCA cycle enzymes, and we identified conditions where its activity is increased. The properties of single and double ΔCceR and ΔAkgR mutants illustrate that these 2 TFs cooperatively regulate carbon and energy metabolism. Comparative genomic analysis indicates that CceR and AkgR orthologs are found in other alphaproteobacteria, where they are predicted to have a conserved function in regulating central carbon metabolism. Our characterization of CceR and AkgR has provided important new insight into the networks that control central carbon and energy metabolism in alphaproteobacteria that can be exploited to modify or engineer new traits in these widespread and versatile bacteria., Importance: To extract and conserve energy from nutrients, cells coordinate a set of metabolic pathways into integrated networks. Many pathways that conserve energy or interconvert metabolites are conserved across cells, but the networks regulating these processes are often highly variable. In this study, we characterize two previously unknown transcriptional regulators of carbon and energy metabolism that are conserved in alphaproteobacteria, a group of abundant, environmentally and biotechnologically important organisms. We identify the genes they regulate, the DNA sequences they recognize, the metabolite that controls the activity of one of the regulators, and conditions where they are required for growth. We provide important new insight into conserved cellular networks that can also be used to improve a variety of hosts for converting feedstock into valuable products., (Copyright © 2015 Imam et al.)

ABSTRACT DksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the "stringent response" to nutrient starvation in the gammaproteobacterium Escherichia coli and for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly related Rhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length to E. coli DksA but lacks the Zn finger motif of the E. coli DksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of an E. coli strain lacking the dksA gene and modulates transcription in vitro with E. coli RNA polymerase (RNAP) similarly to E. coli DksA. RSP2654 reduces RNAP-promoter complex stability in vitro with RNAPs from E. coli or R. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity to E. coli DksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsp has distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp. IMPORTANCE The role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis in Rhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

Unlabelled: Singlet oxygen ((1)O(2)) is a reactive oxygen species generated by energy transfer from one or more excited donors to molecular oxygen. Many biomolecules are prone to oxidation by (1)O(2), and cells have evolved systems to protect themselves from damage caused by this compound. One way that the photosynthetic bacterium Rhodobacter sphaeroides protects itself from (1)O(2) is by inducing a transcriptional response controlled by ChrR, an anti-σ factor which releases an alternative sigma factor, σ(E), in the presence of (1)O(2). Here we report that induction of σ(E)-dependent gene transcription is decreased in the presence of (1)O(2) when two conserved genes in the σ(E) regulon are deleted, including one encoding a cyclopropane fatty acid synthase homologue (RSP2144) or one encoding a protein of unknown function (RSP1091). Thus, we conclude that RSP2144 and RSP1091 are each necessary to increase σ(E) activity in the presence of (1)O(2). In addition, we found that unlike in wild-type cells, where ChrR is rapidly degraded when (1)O(2) is generated, turnover of this anti-σ factor is slowed when cells lacking RSP2144, RSP1091, or both of these proteins are exposed to (1)O(2). Further, we demonstrate that the organic hydroperoxide tert-butyl hydroperoxide promotes ChrR turnover in both wild-type cells and mutants lacking RSP2144 or RSP1091, suggesting differences in the ways different types of oxidants increase σ(E) activity., Importance: Oxygen serves many crucial functions on Earth; it is produced during photosynthesis and needed for other pathways. While oxygen is relatively inert, it can be converted to reactive oxygen species (ROS) that destroy biomolecules, cause disease, or kill cells. When energy is transferred to oxygen, the ROS singlet oxygen is generated. To understand how singlet oxygen impacts cells, we study the stress response to this ROS in Rhodobacter sphaeroides, a bacterium that, like plants, generates this compound as a consequence of photosynthesis. This paper identifies proteins that activate a stress response to singlet oxygen and shows that they act in a specific response to this ROS. The identified proteins are found in many free-living, symbiotic, or pathogenic bacteria that can encounter singlet oxygen in nature. Thus, our findings provide new information about a stress response to a ROS of broad biological, agricultural, and biomedical importance.