Biology subjects

Harwood, C. S.

Publications and source records attributed to Harwood, C. S..

7 recordsLinked to original sources

bioRxiv2024

A Rhodopseudomonas strain with a substantially smaller genome retains the core metabolic versatility of its genus

Rhodopseudomonas are a group of phototrophic microbes with a marked metabolic versatility and flexibility that underpins their potential use in production of value-added products, bioremediation and plant growth promotion. Members of this group have an average genome size of about 5.5 Mb, but two closely related strains have genome sizes of about 4.0 Mb. To identify the types of genes missing in a reduced genome strain, we compared strain DSM127 with other Rhodopseudomonas isolates at the genomic and phenotypic levels. We found that DSM127 can grow as well as other members of the Rhodopseudomonas genus and retains most of their metabolic versatility, but it has many fewer genes associated with high affinity transport of nutrients, iron uptake, nitrogen metabolism and biodegradation of aromatic compounds. This analysis indicates genes that can be deleted in genome reduction campaigns and suggests that DSM127 could be a favorable choice for biotechnology applications using Rhodopseudomonas or as a strain that can be engineered further to reside in a specialized natural environment. IMPORTANCERhodopseudomonas are a cohort of phototrophic bacteria with broad metabolic versatility. Members of this group are present in diverse soil and water environments and some strains are found associated with plants and have plant growth promoting activity. Motivated by the idea that it may be possible to design bacteria with reduced genomes that can survive well only in a specific environment or that may be more metabolically efficient, we compared Rhodopseudomonas strains with typical genome sizes of about 5.5 Mb to a strain with a reduced genome size of 4.0 Mb. From this we concluded that metabolic versatility is part of the identity of the Rhodopseudomonas group, but high-affinity transport genes and genes of apparent redundant function can be dispensed with.

microbiology↗

bioRxiv2023

A purine salvage bottleneck leads to bacterial adenine cross-feeding

Diverse ecosystems host microbial relationships that are stabilized by nutrient cross-feeding. Cross-feeding can involve metabolites that should hold value for the producer. Externalization of such communally valuable metabolites is often unexpected and difficult to predict. Previously, we fortuitously discovered purine externalization by Rhodopseudomonas palustris by its ability to rescue growth of an Escherichia coli purine auxotroph. Here we found that an E. coli purine auxotroph can stably coexist with R. palustris due to purine cross-feeding. We identified the cross-fed purine as adenine. Adenine was externalized by R. palustris under diverse growth conditions. Computational models suggested that adenine externalization occurs via passive diffusion across the cytoplasmic membrane. RNAseq analysis led us to hypothesize that accumulation and externalization of adenine stems from an adenine salvage bottleneck at the enzyme encoded by apt. Ectopic expression of apt eliminated adenine externalization, supporting our hypothesis. A comparison of 49 R. palustris strains suggested that purine externalization is relatively common, with 15 of the strains exhibiting the trait. Purine externalization was correlated with the genomic orientation of apt orientation, but apt orientation alone could not explain adenine externalization in some strains. Our results provide a mechanistic understanding of how a communally valuable metabolite can participate in cross-feeding. Our findings also highlight the challenge in identifying genetic signatures for metabolite externalization.

microbiology↗

bioRxiv2023

Sorgoleone degradation by sorghum-associated bacteria; an opportunity for enforcing plant growth promotion

Metabolite exchange between plant roots and their associated rhizosphere microbiomes underpins plant growth promotion by microbes. Sorghum bicolor is a cereal crop that feeds animals and humans and is used for bioethanol production. Its root tips exude large amounts of a lipophilic benzoquinone called sorgoleone. Sorgoleone is an allelochemical that suppresses the growth of competing plant seedlings and is mineralized by microbes in soil. As an avenue to understand how sorghum and its root microbiome may be connected through root exudates, we identified the molecular determinants of microbial sorgoleone degradation and the distribution of this trait among microbes. We isolated and studied from sorghum-associated soils, three bacterial strains classified as Acinetobacter, Burkholderia, and Pseudomonas species that grow with sorgoleone as a sole carbon and energy source. The genomes of these strains were sequenced and subjected to transcriptomic and gene fitness analyses to identify candidate sorgoleone degradation genes. Follow up mutational analysis showed that sorgoleone catabolism is dependent on four contiguous genes that are conserved among the species we sequenced. Phylogenetic analysis of the sorgoleone degradation gene cluster showed that sorgoleone catabolism is enriched in sorghum-associated Streptomyces strains. The discovery of bacteria that grow on a compound like sorgoleone that is plant specific and not widely distributed in the environment, provides an opportunity to study how a plant exudate can enforce the development of a rhizosphere specific microbiome for the mutual benefit of plant and microbe. SignificanceThe grain crop sorghum exudes an herbicidal compound called sorgoleone from its root tips, which inhibits the growth of other plants. We isolated bacteria that grow on sorogleone and identified a cluster of bacterial genes required for sorogleone degradation that can be used as a biomarker for this trait. An approach to improve the production of crops in stressful conditions such as drought, is to encourage their association with plant growth promoting bacteria. Our discovery of sorgoleone degradation genes opens the door to engineering bacteria that receive benefit from sorghum in the form of a plant-specific growth substrate, and in return promote the growth of this crop.

microbiology↗

bioRxiv2023

ATP is a major determinant of phototrophic bacterial longevity in growth arrest.

How bacteria transition into growth arrest as part of stationary phase has been well-studied, but our knowledge of features that help cells to stay alive in the following days and weeks is incomplete. Most studies have used heterotrophic bacteria that are growth-arrested by depletion of substrates used for both biosynthesis and energy generation, making is difficult to disentangle the effects of the two. In contrast, when grown anaerobically in light, the phototrophic bacterium Rhodopseudomonas palustris generates ATP from light via cyclic photophosphorylation and builds biomolecules from organic substrates such as acetate. As such, energy generation and carbon utilization are independent from one another. Here we compared the physiological and molecular responses of R. palustris to growth arrest caused by carbon source depletion in light (energy-replete) and dark (energy-depleted) conditions. Both sets of cells remained viable for six to ten days, at which point dark-incubated cells lost viability whereas light-incubated cells remained fully viable for 60 days. Dark-incubated cells were depleted in intracellular ATP prior to losing viability, suggesting that ATP depletion is a cause of cell death. Dark-incubated cells also shut down measurable protein synthesis, whereas light-incubated cells continued to synthesize proteins at low levels. Cells incubated in both conditions continued to transcribe genes. We suggest that R. palustris may completely shut down protein synthesis in dark, energy-depleted, conditions as a strategy to survive the nighttime hours of day/night cycles it experiences in nature, where there is a predictable source of energy in the form of sunlight during days. IMPORTANCEThe molecular and physiological basis of bacterial longevity in growth arrest is important to investigate for several reasons. Such investigations could improve treatment of chronic infections, advance use of non-growing bacteria as biocatalysts to make high yields of value-added products, and improve estimates of microbial activities in natural habitats, where cells are often growing slowly or not at all. Here we compared survival of the phototrophic bacterium Rhodopseudomonas palustris under conditions where it generates ATP (incubation in light) and where it does not generate ATP (incubation in dark) to directly assess effects of energy depletion on long-term viability. We found that ATP is important for long-term survival over weeks. However, R. palustris survives 12h periods of ATP depletion without loss of viability, apparently in anticipation of sunrise and restoration of its ability to generate ATP. Our work suggests that cells respond to ATP depletion by shutting down protein synthesis.

microbiology↗

bioRxiv2022

Non-catalyzable denitrification intermediates induce nitrous oxide reduction in two purple nonsulfur bacteria

Denitrification is a form of anaerobic respiration wherein nitrate (NO3-) is sequentially reduced via nitrite (NO2-), nitric oxide, and nitrous oxide (N2O) to dinitrogen gas (N2) by four reductase enzymes. Partial denitrifying bacteria possess only one, or some, of these four reductases and use them as independent respiratory modules. However, it is unclear if partial denitrifiers sense and respond to denitrification intermediates outside of their reductase repertoire. Here we tested the denitrifying capabilities of two purple nonsulfur bacteria, Rhodopseudomonas palustris CGA0092 and Rhodobacter capsulatus SB1003. Each had denitrifying capabilities that matched their genome annotation; CGA0092 reduced NO2- to N2 and SB1003 reduced N2O to N2. For each bacterium, N2O reduction could be used for both electron balance during growth on electron-rich organic compounds in light and for energy transformation via respiration in the dark. However, N2O reduction required supplementation with a denitrification intermediate, including those for which there was no associated denitrification enzyme. For CGA0092, NO3- served as a stable, non-catalyzable molecule that was sufficient to activate N2O reduction. Using a {beta}-galactosidase reporter we found that NO3- acted, at least in part, by stimulating N2O reductase gene expression. In SB1003, NO2-, but not NO3-, activated N2O reduction but NO2- was slowly removed, likely by a promiscuous enzyme activity. Our findings reveal that partial denitrifiers can still be subject to regulation by denitrification intermediates that they cannot use. ImportanceDenitrification is a form of microbial respiration wherein nitrate is converted via several nitrogen oxide intermediates into harmless dinitrogen gas. Partial denitrifying bacteria, which individually have some but not all denitrifying enzymes, can achieve complete denitrification as a community by cross-feeding nitrogen oxide intermediates. However, the last intermediate, nitrous oxide (N2O), is a potent greenhouse gas that often escapes, motivating efforts to understand and improve the efficiency of denitrification. Here we found that at least some partial denitrifying N2O reducers can sense and respond to nitrogen oxide intermediates that they cannot otherwise use. The regulatory effects of nitrogen oxides on partial denitrifiers are thus an important consideration in understanding and applying denitrifying bacterial communities to combat greenhouse gas emissions.

microbiology↗

bioRxiv2021

CsrA-controlled proteins important for Acinetobacter baumannii desiccation tolerance

Hospital environments are excellent reservoirs for the opportunistic pathogen Acinetobacter baumannii in part because it is exceptionally tolerant to desiccation. We found that relative to other A. baumannii strains, the virulent strain AB5075 was strikingly desiccation resistant at 2% relative humidity (RH), suggesting that its a good model for studies of the functional basis of this trait. Consistent with results from other A. baumannii strains at 30% RH, we found the global post-transcriptional regulator CsrA to be critically important for desiccation tolerance of AB5075 at 2% RH. To identify CsrA-controlled proteins that may contribute to desiccation tolerance we used proteomics to identify proteins that were differentially present in wild type and csrA mutant cells. Subsequent mutant analysis revealed nine genes that were required for wild type levels of desiccation tolerance, five of which had modest phenotypes. Catalase and a universal stress protein gene were moderately important for desiccation tolerance and two genes of unknown function had very strong desiccation phenotypes. The predicted amino acid sequence of one of these genes predicts an intrinsically disordered protein. This category of proteins is widespread in eukaryotes but less so in prokaryotes. Our results suggest there may be mechanisms responsible for desiccation tolerance that have not previously been explored in bacteria. IMPORTANCEAcinetobacter baumannii is commonly found in terrestrial environments but can cause nosocomial infections in very sick patients. A factor that contributes to the prevalence of A. baumannii in hospital settings is that it is intrinsically resistant to dry conditions. Here, we established the virulent strain A. baumannii AB5075 as a model for studies of desiccation tolerance at very low relative humidity. Our results show that this trait depends on two proteins of unknown function, one of which is predicted to be an intrinsically disordered protein. This category of protein is critical for the small animals named tardigrades to survive desiccation. Our results suggest that A. baumannii may have novel strategies to survive desiccation that have not previously been seen in bacteria.

microbiology↗

bioRxiv2019

Longevity of growth-arrested bacteria requires protein synthesis and a stringent response

Gram-negative bacteria in infections, biofilms and industrial settings often stop growing due to nutrient depletion, immune responses or environmental stresses. Bacteria in this state tend to be tolerant to antibiotics and are often referred to as dormant. Rhodopseudomonas palustris, a phototrophic -proteobacterium, can remain fully viable for more than four months when growth is arrested. Here, we show that protein synthesis, specific proteins involved in translation and a stringent response are required for this remarkable longevity. Because it can generate ATP from light during growth arrest, R. palustris is an extreme example of a bacterial species that will stay alive for long periods of time as a relatively homogeneous population of cells and it is thus an excellent model organism for studies of bacterial longevity. There is evidence that other Gram-negative species also continue to synthesize proteins during growth arrest and that a stringent response is required for their longevity as well. Our observations challenge the notion that growth-arrested cells are necessarily dormant and metabolically inactive, and suggest that such bacteria may have a level of metabolic activity that is higher than many would have assumed. Our results also expand our mechanistic understanding of a crucial but understudied phase of the bacterial life cycle.\n\nIMPORTANCEWe are surrounded by bacteria; but they do not completely dominate our planet despite the ability of many to grow extremely rapidly in the laboratory. This has been interpreted to mean that bacteria in nature are often in a dormant state. We investigated life in growth arrest of Rhodopseudomonas palustris, a proteobacterium that stays alive for months when it is not growing. We found that cells were metabolically active; they continued to synthesize proteins and mounted a stringent response, both of which were required for their longevity. Our results suggest that long-lived bacteria are not necessarily inactive but have an active metabolism that is well adjusted to life without growth.

microbiology↗