Explore the vast range of titles available.

Microbial communities mediating anaerobic ammonium oxidation (anammox) represent one of the most energy-efficient environmental biotechnologies for nitrogen removal from wastewater. However, little is known about the functional role heterotrophic bacteria play in anammox granules. Here, we use genome-centric metagenomics to recover 17 draft genomes of anammox and heterotrophic bacteria from a laboratory-scale anammox bioreactor. We combine metabolic network reconstruction with metatranscriptomics to examine the gene expression of anammox and heterotrophic bacteria and to identify their potential interactions. We find that Chlorobi-affiliated bacteria may be highly active protein degraders, catabolizing extracellular peptides while recycling nitrate to nitrite. Other heterotrophs may also contribute to scavenging of detritus and peptides produced by anammox bacteria, and potentially use alternative electron donors, such as H 2 , acetate and formate. Our findings improve the understanding of metabolic activities and interactions between anammox and heterotrophic bacteria and offer the first transcriptional insights on ecosystem function in anammox granules. The use of anammox microbiomes to treat wastewater is an escalating biotechnology, yet the functional role heterotrophic bacteria play in these systems remains poorly understood. Here, Lawson et al . use metagenomics and metatranscriptomics to reveal that heterotrophs degrade free peptides, while recycling nitrate to nitrite.

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 primarysubstrates transformed. During the MCFA production period, the five major genera represented more than 95% of the community, including Lactobacillus, Roseburia, Atopobium, Olsenella, andPseudoramibacter. 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.

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 ( C ell en velope K inase and R egulator), 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.

Nitrospira -like bacteria are among the most diverse and widespread nitrifiers in natural ecosystems and the dominant nitrite oxidizers in wastewater treatment plants (WWTPs). The recent discovery of comammox-like Nitrospira strains, capable of complete oxidation of ammonia to nitrate, raises new questions about specific traits responsible for the functional versatility and adaptation of this genus to a variety of environments. The availability of new Nitrospira genome sequences from both nitrite-oxidizing and comammox bacteria offers a way to analyze traits in different Nitrospira functional groups. Our comparative genomics analysis provided new insights into the adaptation of Nitrospira strains to specific lifestyles and environmental niches. The recently discovered comammox bacteria have the potential to completely oxidize ammonia to nitrate. These microorganisms are part of the Nitrospira genus and are present in a variety of environments, including biological nutrient removal (BNR) systems. However, the physiological traits within and between comammox and nitrite-oxidizing bacterium (NOB)-like Nitrospira species have not been analyzed in these ecosystems. In this study, we identified Nitrospira strains dominating the nitrifying community of a sequencing batch reactor (SBR) performing BNR under microaerobic conditions. We recovered metagenome-derived draft genomes from two Nitrospira strains: (i) Nitrospira sp. strain UW-LDO-01, a comammox-like organism classified as “ Candidatus Nitrospira nitrosa,” and (ii) Nitrospira sp. strain UW-LDO-02, a nitrite-oxidizing strain belonging to the Nitrospira defluvii species. A comparative genomic analysis of these strains with other Nitrospira -like genomes identified genomic differences in “ Ca . Nitrospira nitrosa” mainly attributed to each strain’s niche adaptation. Traits associated with energy metabolism also differentiate comammox from NOB-like genomes. We also identified several transcriptionally regulated adaptive traits, including stress tolerance, biofilm formation, and microaerobic metabolism, which might explain survival of Nitrospira under multiple environmental conditions. Overall, our analysis expanded our understanding of the genetic functional features of “ Ca . Nitrospira nitrosa” and identified genomic traits that further illuminate the phylogenetic diversity and metabolic plasticity of the Nitrospira genus. IMPORTANCE Nitrospira -like bacteria are among the most diverse and widespread nitrifiers in natural ecosystems and the dominant nitrite oxidizers in wastewater treatment plants (WWTPs). The recent discovery of comammox-like Nitrospira strains, capable of complete oxidation of ammonia to nitrate, raises new questions about specific traits responsible for the functional versatility and adaptation of this genus to a variety of environments. The availability of new Nitrospira genome sequences from both nitrite-oxidizing and comammox bacteria offers a way to analyze traits in different Nitrospira functional groups. Our comparative genomics analysis provided new insights into the adaptation of Nitrospira strains to specific lifestyles and environmental niches. Author Video : An author video summary of this article is available.

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.

Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis.

Sensor driven aeration control strategies have recently been developed as a means to efficiently carry out biological nutrient removal (BNR) and reduce aeration costs in wastewater treatment plants. Under load-based aeration control, often implemented as ammonia-based aeration control (ABAC), airflow is regulated to meet desired effluent standards without specifically setting dissolved oxygen (DO) targets. Another approach to reduce aeration requirements is to constantly maintain low DO conditions and allow the microbial community to adapt to the low-DO environment. In this study, we compared the performance of two pilot-scale BNR treatment trains that simultaneously used ABAC and low-DO operation to evaluate the combination of these two strategies. One pilot plant was operated with continuous ABAC while the other one used intermittent ABAC. Both processes achieved greater than 90% total Kjehldal nitrogen (TKN) removal, 60% total nitrogen removal, and nearly 90% total phosphorus removal. Increasing the solids retention time (SRT) during the period of cold (∼12 °C) water temperatures helped maintain ammonia removal performance under low-DO conditions. However, both processes experienced poor solids settling characteristics during winter. While settling was recovered under warmer temperatures, improving settling quality remains a challenge under low-DO operation.

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. 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.

Transcriptional regulatory networks (TRNs) program cells to dynamically alter their gene expression in response to changing internal or environmental conditions. In this study, we develop a novel workflow for generating large-scale TRN models that integrates comparative genomics data, global gene expression analyses, and intrinsic properties of transcription factors (TFs). An assessment of this workflow using benchmark datasets for the well-studied γ-proteobacterium Escherichia coli showed that it outperforms expression-based inference approaches, having a significantly larger area under the precision-recall curve. Further analysis indicated that this integrated workflow captures different aspects of the E. coli TRN than expression-based approaches, potentially making them highly complementary. We leveraged this new workflow and observations to build a large-scale TRN model for the α-Proteobacterium Rhodobacter sphaeroides that comprises 120 gene clusters, 1211 genes (including 93 TFs), 1858 predicted protein-DNA interactions and 76 DNA binding motifs. We found that ~67% of the predicted gene clusters in this TRN are enriched for functions ranging from photosynthesis or central carbon metabolism to environmental stress responses. We also found that members of many of the predicted gene clusters were consistent with prior knowledge in R. sphaeroides and/or other bacteria. Experimental validation of predictions from this R. sphaeroides TRN model showed that high precision and recall was also obtained for TFs involved in photosynthesis (PpsR), carbon metabolism (RSP_0489) and iron homeostasis (RSP_3341). In addition, this integrative approach enabled generation of TRNs with increased information content relative to R. sphaeroides TRN models built via other approaches. We also show how this approach can be used to simultaneously produce TRN models for each related organism used in the comparative genomics analysis. Our results highlight the advantages of integrating comparative genomics of closely related organisms with gene expression data to assemble large-scale TRN models with high-quality predictions.

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. 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.