The effects of chemical fungicides and salicylic acid on the apple microbiome and fungal disease incidence under changing environmental conditions.

Epiphytic and endophytic micro-organisms associated with plants form complex communities on or in their host plant. These communities influence physiological traits, development, and host susceptibility to abiotic and biotic stresses, and these communities are theorized to have evolved alongside their hosts, forming a unit of selection known as the holobiont. The microbiome is highly variable and can be influenced by abiotic factors, including applied exogenous agents. In this study, we compared the impact of chemical fungicide and salicylic acid treatments on the fungal communities of "Honeycrisp" apples at harvest over two consecutive growing years. We demonstrated variations in fungal community structure and composition by tissue type, growing season, and treatment regimes and that fungicide treatments were associated with reduced network complexity. Finally, we show that the inclusion of salicylic acid with 50% less chemical fungicides in an integrated spray program allowed a reduction in fungicide use while maintaining effective control of disease at harvest and following storage.

Why Do We Need Alternative Methods for Fungal Disease Management in Plants?

Fungal pathogens pose a major threat to food production worldwide. Traditionally, chemical fungicides have been the primary means of controlling these pathogens, but many of these fungicides have recently come under increased scrutiny due to their negative effects on the health of humans, animals, and the environment. Furthermore, the use of chemical fungicides can result in the development of resistance in populations of phytopathogenic fungi. Therefore, new environmentally friendly alternatives that provide adequate levels of disease control are needed to replace chemical fungicides-if not completely, then at least partially. A number of alternatives to conventional chemical fungicides have been developed, including plant defence elicitors (PDEs); biological control agents (fungi, bacteria, and mycoviruses), either alone or as consortia; biochemical fungicides; natural products; RNA interference (RNAi) methods; and resistance breeding. This article reviews the conventional and alternative methods available to manage fungal pathogens, discusses their strengths and weaknesses, and identifies potential areas for future research.

Role of Bacteria-Derived Flavins in Plant Growth Promotion and Phytochemical Accumulation in Leafy Vegetables.

Sinorhizobium meliloti 1021 bacteria secretes a considerable amount of flavins (FLs) and can form a nitrogen-fixing symbiosis with legumes. This strain is also associated with non-legume plants. However, its role in plant growth promotion (PGP) of non-legumes is not well understood. The present study evaluated the growth and development of lettuce (Lactuca sativa) and kale (Brassica oleracea var. acephala) plants inoculated with S. meliloti 1021 (FL+) and its mutant 1021ΔribBA, with a limited ability to secrete FLs (FL-). The results from this study indicated that inoculation with 1021 significantly (p < 0.05) increased the lengths and surface areas of the roots and hypocotyls of the seedlings compared to 1021ΔribBA. The kale and lettuce seedlings recorded 19% and 14% increases in total root length, respectively, following inoculation with 1021 compared to 1021ΔribBA. A greenhouse study showed that plant growth, photosynthetic rate, and yield were improved by 1021 inoculation. Moreover, chlorophylls a and b, and total carotenoids were more significantly (p < 0.05) increased in kale plants associated with 1021 than non-inoculated plants. In kale, total phenolics and flavonoids were significantly (p < 0.05) increased by 6% and 23%, respectively, and in lettuce, the increments were 102% and 57%, respectively, following 1021 inoculation. Overall, bacterial-derived FLs enhanced kale and lettuce plant growth, physiological indices, and yield. Future investigation will use proteomic approaches combined with plant physiological responses to better understand host-plant responses to bacteria-derived FLs.

Apple Root Microbiome as Indicator of Plant Adaptation to Apple Replant Diseased Soils.

The tree fruit industry in Nova Scotia, Canada, is dominated by the apple (Malus domestica) sector. However, the sector is faced with numerous challenges, including apple replant disease (ARD), which is a well-known problem in areas with intensive apple cultivation. A study was performed using 16S rRNA/18S rRNA and 16S rRNA/ITS2 amplicon sequencing to assess soil- and root-associated microbiomes, respectively, from mature apple orchards and soil microbiomes alone from uncultivated soil. The results indicated significant (p < 0.05) differences in soil microbial community structure and composition between uncultivated soil and cultivated apple orchard soil. We identified an increase in the number of potential pathogens in the orchard soil compared to uncultivated soil. At the same time, we detected a significant (p < 0.05) increase in relative abundances of several potential plant-growth-promoting or biocontrol microorganisms and non-fungal eukaryotes capable of promoting the proliferation of bacterial biocontrol agents in orchard soils. Additionally, the apple roots accumulated several potential PGP bacteria from Proteobacteria and Actinobacteria phyla, while the relative abundances of fungal taxa with the potential to contribute to ARD, such as Nectriaceae and plant pathogenic Fusarium spp., were decreased in the apple root microbiome compared to the soil microbiome. The results suggest that the health of a mature apple tree can be ascribed to a complex interaction between potential pathogenic and plant growth-promoting microorganisms in the soil and on apple roots.

Changes to Soil Microbiome Resulting from Synergetic Effects of Fungistatic Compounds Pyrimethanil and Fluopyram in Lowbush Blueberry Agriculture, with Nine Fungicide Products Tested.

Lowbush blueberries (Vaccinium spp.) are a crop of economic significance to Atlantic Canada, Quebec, and Maine. The fruit is produced by the management of naturally occurring plant populations. The plants have an intimate relationship with the soil microbiome and depend on it for their health and productivity. Fungicides are an important tool in combatting disease pressure but pose a potential risk to soil health. In this study, amplicon sequencing was used to determine the effects of six fungistatic compounds both alone and in combination via nine commercially available fungicide products on the bacterial and fungal microbiomes associated with lowbush blueberries and to study whether these effects are reflected in crop outcomes and plant phenotypes. One fungicide, Luna Tranquility, a combination of fluopyram and pyrimethanil, was found to impart significant effects to fungal and bacterial community structure, fungal taxonomic abundances, and bacterial functions relative to control. The two fungicides which contained fluopyram and pyrimethanil as single ingredients (Velum Prime and Scala, respectively) did not induce significant changes in any of these regards. These results suggest the possibility that these microbiome changes are the result of the synergistic effect of fluopyram and pyrimethanil on soil microbiomes. While these results suggest a significant disruption to the soil microbiome, no corresponding changes to crop development and outcomes were noted. Ultimately, the majority of the fungicides analysed in this trial did not produce significant changes to the soil microbiome relative to the untreated group (UTG). However, one of the fungicide treatments, Luna Tranquility, did produce significant changes to the soil ecosystem that could have longer-term effects on soil health and its future use may merit additional investigation onto its ecotoxicological properties.

Impacts of abiotic factors on the fungal communities of 'Honeycrisp' apples in Canada.

The maintenance of the beneficial plant microbiome to control plant pathogens is an emerging concept of disease management, and necessitates a clear understanding of these microbial communities and the environmental factors that affect their diversity and compositional structure. As such, studies investigating the microbiome of economically significant cultivars within each growing region are necessary to develop adequate disease management strategies. Here, we assessed the relative impacts of growing season, management strategy, and geographical location on the fungal microbiome of 'Honeycrisp' apples from seven different orchard locations in the Atlantic Maritime Ecozone for two consecutive growing years. Though apple fruit tissue was dominated by relatively few fungal genera, significant changes in their fungal communities were observed as a result of environmental factors, including shifts in genera with plant-associated lifestyles (symbionts and pathogens), such as Aureobasidium, Alternaria, Penicillium, Diplodia, and Mycosphaerella. Variation in fungal composition between different tissues of fruit was also observed. We demonstrate that growing season is the most significant factor affecting fungal community structure and diversity of apple fruit, suggesting that future microbiome studies should take place for multiple growing seasons to better represent the host-microbiome of perennial crops under different environmental conditions.

Changes in Soil Characteristics, Microbial Metabolic Pathways, TCA Cycle Metabolites and Crop Productivity following Frequent Application of Municipal Solid Waste Compost.

The benefit sof municipal solid waste (MSW) compost on soil health and plant productivity are well known, but not its long-term effect on soil microbial and plant metabolic pathways. A 5-year study with annual (AN), biennial (BI) and no (C, control) MSW compost application were carried out to determine the effect on soil properties, microbiome function, and plantgrowth and TCA cycle metabolites profile of green beans (Phaseolus vulgaris), lettuce (Latuca sativa) and beets (Beta vulgaris). MSW compost increased soil nutrients and organic matter leading to a significant (p < 0.05) increase in AN-soil water-holding capacity followed by BI-soil compared to C-soil. Estimated nitrogen release in the AN-soil was ca. 23% and 146% more than in BI-soil and C-soil, respectively. Approximately 44% of bacterial community due to compost. Deltaproteobacteria, Bacteroidetes Bacteroidia, and Chloroflexi Anaerolineae were overrepresented in compost amended soils compared to C-soil. A strong positive association existed between AN-soil and 18 microbial metabolic pathways out of 205. Crop yield in AN-soil were increased by 6−20% compared to the BI-soil, and by 35−717% compared to the C-soil. Plant tricarboxylic acid cycle metabolites were highly (p < 0.001) influenced by compost. Overall, microbiome function and TCA cycle metabolites and crop yield were increased in the AN-soil followed by the BI-soil and markedly less in C-soil. Therefore, MSW compost is a possible solution to increase soil health and plants production in the medium to long term. Future study must investigate rhizosphere metabolic activities.

A novel formamidase is required for riboflavin biosynthesis in invasive bacteria.

Biosynthesis of riboflavin (RF), the precursor of the redox cofactors FMN and FAD, was thought to be well understood in bacteria, with all the pathway enzymes presumed to be known and essential. Our previous research has challenged this view by showing that, in the bacterium Sinorhizobium meliloti, deletion of the ribBA gene encoding the enzyme that catalyzes the initial steps on the RF biosynthesis pathway only causes a reduction in flavin secretion rather than RF auxotrophy. This finding led us to hypothesize that RibBA participates in the biosynthesis of flavins destined for secretion, whereas S. meliloti has another enzyme that performs this function for internal cellular metabolism. Here, we identify and biochemically characterize a novel formamidase (SMc02977) involved in the production of RF for intracellular functions in S. meliloti. This catalyst, which we named Sm-BrbF, releases formate from the early RF precursor 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate to yield 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate. We show that homologs of this enzyme are present in many bacteria, are highly abundant in the Rhizobiales order, and that sequence homologs from Brucella abortus and Liberobacter solanacearum complement the RF auxotrophy of the Sm1021ΔSMc02977 mutant. Furthermore, we show that the B. abortus enzyme (Bab2_0247, Ba-BrbF) is also an 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate formamidase, and that the bab2_0247 mutant is a RF auxotroph exhibiting a lower level of intracellular infection than the wildtype strain. Finally, we show that Sm-BrbF and Ba-BrbF directly interact with other RF biosynthesis pathway enzymes. Together, our results provide novel insight into the intricacies of RF biosynthesis in bacteria.

Microbial Consortium Associated with Crustacean Shells Composting.

Soil microbes play an essential role in the biodegradation of crustacean shells, which is the process of sustainable bioconversion to chitin derivatives ultimately resulting in the promotion of plant growth properties. While a number of microorganisms with chitinolytic properties have been characterized, little is known about the microbial taxa that participate in this process either by active chitin degradation or by facilitation of this activity through nutritional cooperation and composting with the chitinolytic microorganisms. In this study, we evaluated the transformation of the soil microbiome triggered by close approximation to the green crab shell surface. Our data indicate that the microbial community associated with green crab shell matter undergoes significant specialized changes, which was reflected in a decreased fungal and bacterial Shannon diversity and evenness and in a dramatic alteration in the community composition. The relative abundance of several bacterial and fungal genera including bacteria Flavobacterium, Clostridium, Pseudomonas, and Sanguibacter and fungi Mortierella, Mycochlamys, and Talaromyces were increased with approximation to the shell surface. Association with the shell triggered significant changes in microbial cooperation that incorporate microorganisms that were previously reported to be involved in chitin degradation as well as ones with no reported chitinolytic activity. Our study indicates that the biodegradation of crab shells in soil incorporates a consortium of microorganisms that might provide a more efficient way for bioconversion.

Response of Plant-Associated Microbiome to Plant Root Colonization by Exogenous Bacterial Endophyte in Perennial Crops.

The application of bacterial inoculums for improving plant growth and production is an important component of sustainable agriculture. However, the efficiency of perennial crop inoculums depends on the ability of the introduced endophytes to exert an impact on the host-plant over an extended period of time. This impact might be evaluated by the response of plant-associated microbiome to the inoculation. In this study, we monitored the effect of a single bacterial strain inoculation on the diversity, structure, and cooperation in plant-associated microbiome over 1-year period. An endophyte (RF67) isolated from Vaccinium angustifolium (wild blueberry) roots and annotated as Rhizobium was used for the inoculation of 1-year-old Lonicera caerulea (Haskap) plants. A significant level of bacterial community perturbation was detected in plant roots after 3 months post-inoculation. About 23% of root-associated community variation was correlated with an application of the inoculant, which was accompanied by increased cooperation between taxa belonging to Proteobacteria and Actinobacteriota phyla and decreased cooperation between Firmicutes in plant roots. Additionally, a decrease in bacterial Shannon diversity and an increase in the relative abundances of Rhizobiaceae and Enterobacteriaceae were detected in the roots of inoculated plants relative to the non-inoculated control. A strong effect of the inoculation on the bacterial cooperation was also detected after 1 year of plant field growth, whereas no differences in bacterial community composition and also alpha and beta diversities were detected between bacterial communities from inoculated and non-inoculated roots. These findings suggest that while exogenous endophytes might have a short-term effect on the root microbiome structure and composition, they can boost cooperation between plant-growth-promoting endophytes, which can exist for the extended period of time providing the host-plant with long-lasting beneficial effects.

The Sinorhizobium meliloti Nitrogen Stress Response Changes Radically in the Face of Concurrent Phosphate Stress.

Expression of hundreds of S. meliloti genes changed more than two-fold in response to either nitrogen or phosphate limitation. When these two stresses were applied together, stress responsive gene expression shifted dramatically. In particular, the nitrogen stress response in the presence of phosphate stress had only 30 of about 350 genes in common with the 280 genes that responded to nitrogen stress with adequate phosphate. Expression of sRNAs was also altered in response to these stresses. 82% of genes that responded to nitrogen stress also responded to phosphate stress, including 20 sRNAs. A subset of these sRNAs is known to be chaperoned by the RNA binding protein, Hfq. Hfq had previously been shown to influence about a third of the genes that responded to both nitrogen and phosphate stresses. Phosphate limitation influenced changes in gene expression more than nitrogen limitation and, when both stresses were present, phosphate stress sometimes reversed the direction of some of the changes induced by nitrogen stress. These nutrient stress responses are therefore context dependent.

Specialization in a Nitrogen-Fixing Symbiosis: Proteome Differences Between Sinorhizobium medicae Bacteria and Bacteroids.

Using tandem mass spectrometry (MS/MS), we analyzed the proteome of Sinorhizobium medicae WSM419 growing as free-living cells and in symbiosis with Medicago truncatula. In all, 3,215 proteins were identified, over half of the open reading frames predicted from the genomic sequence. The abundance of 1,361 proteins displayed strong lifestyle bias. In total, 1,131 proteins had similar levels in bacteroids and free-living cells, and the low levels of 723 proteins prevented statistically significant assignments. Nitrogenase subunits comprised approximately 12% of quantified bacteroid proteins. Other major bacteroid proteins included symbiosis-specific cytochromes and FixABCX, which transfer electrons to nitrogenase. Bacteroids had normal levels of proteins involved in amino acid biosynthesis, glycolysis or gluconeogenesis, and the pentose phosphate pathway; however, several amino acid degradation pathways were repressed. This suggests that bacteroids maintain a relatively independent anabolic metabolism. Tricarboxylic acid cycle proteins were highly expressed in bacteroids and no other catabolic pathway emerged as an obvious candidate to supply energy and reductant to nitrogen fixation. Bacterial stress response proteins were induced in bacteroids. Many WSM419 proteins that are not encoded in S. meliloti Rm1021 were detected, and understanding the functions of these proteins might clarify why S. medicae WSM419 forms a more effective symbiosis with M. truncatula than S. meliloti Rm1021.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Effect of Fungicide Application on Lowbush Blueberries Soil Microbiome.

Lowbush blueberries (Vaccinium sp.) are perennial crops produced throughout eastern Canada and Maine through management of wild populations. Given the constraints of this cropping system, the application of fungicides is critical to reducing disease pressure and ensuring consistent yields. However, as plant health is intertwined with soil health, it is important to consider the impact of fungicides on microbial communities. To understand the effects of fungicides in this context, bacterial and fungal microbial communities from fungicide-treated plots, as well as untreated control plots (UTG) were analyzed using amplicon sequencing. The fungicides, considered collectively as a combined treatment group (CTG), lead to a loss in fungal richness. One family, Clavariaceae, had an increased abundance under prothioconazole relative to UTG. This finding may be significant as taxa in Clavariaceae have been thought to potentially form ericoid mycorrhizae with Vaccinium. Five functional pathways and 74 enzymes differed significantly in relative abundance between CTG and UTG including enzymes associated with soil nutrient cycles. Most notably, enzymes corresponding to the breakdown of halogen-organic compounds had an increased abundance in CTG, suggesting bacterial fungicide degradation. Some enzymes associated with soil nutrient cycles differed significantly, possibly implying changes to nutrient pathways due to fungicide treatment.

Sinorhizobium medicae WSM419 Genes That Improve Symbiosis between Sinorhizobium meliloti Rm1021 and Medicago truncatula Jemalong A17 and in Other Symbiosis Systems.

Some soil bacteria, called rhizobia, can interact symbiotically with legumes, in which they form nodules on the plant roots, where they can reduce atmospheric dinitrogen to ammonia, a form of nitrogen that can be used by growing plants. Rhizobium-plant combinations can differ in how successful this symbiosis is: for example, Sinorhizobium meliloti Rm1021 forms a relatively ineffective symbiosis with Medicago truncatula Jemalong A17, but Sinorhizobium medicae WSM419 is able to support more vigorous plant growth. Using proteomic data from free-living and symbiotic S. medicae WSM419, we previously identified a subset of proteins that were not closely related to any S. meliloti Rm1021 proteins and speculated that adding one or more of these proteins to S. meliloti Rm1021 would increase its effectiveness on M. truncatula A17. Three genes, Smed_3503, Smed_5985, and Smed_6456, were cloned into S. meliloti Rm1021 downstream of the E. coli lacZ promoter. Strains with these genes increased nodulation and improved plant growth, individually and in combination with one another. Smed_3503, renamed iseA (increased symbiotic effectiveness), had the largest impact, increasing M. truncatula biomass by 61%. iseA homologs were present in all currently sequenced S. medicae strains but were infrequent in other Sinorhizobium isolates. Rhizobium leguminosarum bv. viciae 3841 containing iseA led to more nodules on pea and lentil. Split-root experiments with M. truncatula A17 indicated that S. meliloti Rm1021 carrying the S. medicae iseA is less sensitive to plant-induced resistance to rhizobial infection, suggesting an interaction with the plant's regulation of nodule formation. IMPORTANCE Legume symbiosis with rhizobia is highly specific. Rhizobia that can nodulate and fix nitrogen on one legume species are often unable to associate with a different species. The interaction can be more subtle. Symbiotically enhanced growth of the host plant can differ substantially when nodules are formed by different rhizobial isolates of a species, much like disease severity can differ when conspecific isolates of pathogenic bacteria infect different cultivars. Much is known about bacterial genes essential for a productive symbiosis, but less is understood about genes that marginally improve performance. We used a proteomic strategy to identify Sinorhizobium genes that contribute to plant growth differences that are seen when two different strains nodulate M. truncatula A17. These genes could also alter the symbiosis between R. leguminosarum bv. viciae 3841 and pea or lentil, suggesting that this approach identifies new genes that may more generally contribute to symbiotic productivity.

Microbial co-occurrence network analysis of soils receiving short- and long-term applications of alkaline treated biosolids.

Agricultural soils are inherently disturbed systems where organic matter additions are considered to enhance microbial community structure and resilience. High-throughput sequencing of community was applied to soils receiving annual applications of an alkaline stabilized biosolid (ATB), at four increasing rates over 10 years, as an environmental stressor in contrast to a one-time application of ATB ten years prior. Bacterial community structure was more greatly influenced by annual ATB applications relative to fungi and eukaryotes. Specifically, higher relative abundances of Proteobacteria, Acidobacteria, Bacteroidetes, and Chloroflexi were measured in annual ATB rates relative to the single ATB rates and the control. High rates of annual ATB applications resulted in lower bacterial alpha-diversity, as well as fungal and eukaryotic Shannon diversity, but single ATB or lower rates of ATB applied annually showed increased alpha -diversity relative to the control. Soil microbiome responses to annual ATB and single ATB rates were also examined using co-occurrence network analysis. High rates and frequency of ATB application resulted in a decrease in network interactions, lower average number of neighbors, and reduced network density compared to control soils. A concomitant increase in network diameter and characteristic path length further suggests annual additions of ATB led to a more adapted, but less cooperative, state in the microbiome. The data suggest a more universal functional response of microbiomes to the stressors compared to community structure and local diversity. In particular, beta-analysis and network analysis were both able to resolve significant effects on soil microbiomes 10 years post-application of low rates of ATB. Community complexity and stability were increased by single low rate of ATB additions and decreased by single high rate and annual moderate rates of ATB additions. These results provide insights into the effects that ATB additions have on soil community after only one-time use and after annual additions over a decade.

Metagenomic Functional Shifts to Plant Induced Environmental Changes.

The Vaccinium angustifolium (wild blueberry) agricultural system involves transformation of the environment surrounding the plant to intensify plant propagation and to improve fruit yield, and therefore is an advantageous model to study the interaction between soil microorganisms and plant-host interactions. We studied this system to address the question of a trade-off between microbial adaptation to a plant-influenced environment and its general metabolic capabilities. We found that many basic metabolic functions were similarly represented in bulk soil and rhizosphere microbiomes overall. However, we identified a niche-specific difference in functions potentially beneficial for microbial survival in the rhizosphere but that might also reduce the ability of microbes to withstand stresses in bulk soils. These functions could provide the microbiome with additional capabilities to respond to environmental fluctuations in the rhizosphere triggered by changes in the composition of root exudates. Based on our analysis we hypothesize that the rhizosphere-specific pathways involved in xenobiotics biodegradation could provide the microbiome with functional flexibility to respond to plant stress status.

Dissecting Community Structure in Wild Blueberry Root and Soil Microbiome.

A complex network of functions and symbiotic interactions between a eukaryotic host and its microbiome is a the foundation of the ecological unit holobiont. However, little is known about how the non-fungal eukaryotic microorganisms fit in this complex network of host-microbiome interactions. In this study, we employed a unique wild blueberry ecosystem to evaluate plant-associated microbiota, encompassing both eukaryotic and bacterial communities. We found that, while soil microbiome serves as a foundation for root microbiome, plant-influenced species sorting had stronger effect on eukaryotes than on bacteria. Our study identified several fungal and protist taxa, which are correlated with decreased fruit production in wild blueberry agricultural ecosystems. The specific effect of species sorting in root microbiome resulted in an increase in relative abundance of fungi adapted to plant-associated life-style, while the relative abundance of non-fungal eukaryotes was decreased along the soil-endosphere continuum in the root, probably because of low adaptation of these microorganisms to host-plant defense responses. Analysis of community correlation networks indicated that bacterial and eukaryotic interactions became more complex along the soil-endosphere continuum and, in addition to extensive mutualistic interactions, co-exclusion also played an important role in shaping wild blueberry associated microbiome. Our study identified several potential hub taxa with important roles in soil fertility and/or plant-microbe interaction, suggesting the key role of these taxa in the interconnection between soils and plant health and overall microbial community structure. This study also provides a comprehensive view of the role of non-fungal eukaryotes in soil ecosystem.

Interaction between Nitrogen and Phosphate Stress Responses in Sinorhizobium meliloti.

Bacteria have developed various stress response pathways to improve their assimilation and allocation of limited nutrients, such as nitrogen and phosphate. While both the nitrogen stress response (NSR) and phosphate stress response (PSR) have been studied individually, there are few experiments reported that characterize effects of multiple stresses on one or more pathways in Sinorhizobium meliloti, a facultatively symbiotic, nitrogen-fixing bacteria. The PII proteins, GlnB and GlnK, regulate the NSR activity, but analysis of global transcription changes in a PII deficient mutant suggest that the S. meliloti PII proteins may also regulate the PSR. PII double deletion mutants grow very slowly and pseudoreversion of the slow growth phenotype is common. To understand this phenomenon better, transposon mutants were isolated that had a faster growing phenotype. One mutation was in phoB, the response regulator for a two component regulatory system that is important in the PSR. phoB::Tn5 mutants had different phenotypes in the wild type compared to a PII deficient background. This led to the hypothesis that phosphate stress affects the NSR and conversely, that nitrogen stress affects the PSR. Our results show that phosphate availability affects glutamine synthetase activity and expression, which are often used as indicators of NSR activity, but that nitrogen availability did not affect alkaline phosphatase activity and expression, which are indicators of PSR activity. We conclude that the NSR is co-regulated by nitrogen and phosphate, whereas the PSR does not appear to be co-regulated by nitrogen in addition to its known phosphate regulation.

A directed-overflow and damage-control N-glycosidase in riboflavin biosynthesis.

Plants and bacteria synthesize the essential human micronutrient riboflavin (vitamin B2) via the same multi-step pathway. The early intermediates of this pathway are notoriously reactive and may be overproduced in vivo because riboflavin biosynthesis enzymes lack feedback controls. In the present paper, we demonstrate disposal of riboflavin intermediates by COG3236 (DUF1768), a protein of previously unknown function that is fused to two different riboflavin pathway enzymes in plants and bacteria (RIBR and RibA respectively). We present cheminformatic, biochemical, genetic and genomic evidence to show that: (i) plant and bacterial COG3236 proteins cleave the N-glycosidic bond of the first two intermediates of riboflavin biosynthesis, yielding relatively innocuous products; (ii) certain COG3236 proteins are in a multi-enzyme riboflavin biosynthesis complex that gives them privileged access to riboflavin intermediates; and (iii) COG3236 action in Arabidopsis thaliana and Escherichia coli helps maintain flavin levels. COG3236 proteins thus illustrate two emerging principles in chemical biology: directed overflow metabolism, in which excess flux is diverted out of a pathway, and the pre-emption of damage from reactive metabolites.