Integrated bacterial transcriptome and host metabolome analysis reveals insights into "Candidatus Liberibacter asiaticus" population dynamics in the fruit pith of three citrus cultivars with different tolerance.

"Candidatus Liberibacter asiaticus" (CLas), the causal agent of citrus Huanglongbing (HLB), is able to multiply to a high abundance in citrus fruit pith. However, little is known about the biological processes and phytochemical substances that are vital for CLas colonization and growth in fruit pith. In this study, CLas-infected fruit pith of three citrus cultivars ("Shatangju" mandarin, "Guanxi" pomelo, and "Shatian" pomelo) exhibiting different tolerance to CLas were collected and used for dual RNA-Seq and untargeted metabolome analysis. Comparative transcriptome analysis found that the activation of the CLas noncyclic TCA pathway and pathogenic-related effectors could contribute to the colonization and growth of CLas in fruit pith. The pre-established Type 2 prophage in the CLas genome and the induction of its CRISPR/cas system could enhance the phage resistance of CLas and, in turn, facilitate CLas population growth in fruit pith. CLas infection caused the accumulation of amino acids that were correlated with tolerance to CLas. The accumulation of most sugars and organic acids in CLas-infected fruit pith, which could be due to the phloem blockage caused by CLas infection, was thought to be beneficial for CLas growth in localized phloem tissue. The higher levels of flavonoids and terpenoids in the fruit pith of CLas-tolerant cultivars, particularly those known for their antimicrobial properties, could hinder the growth of CLas. This study advances our understanding of CLas multiplication in fruit pith and offers novel insight into metabolites that could be responsible for tolerance to CLas or essential to CLas population growth.IMPORTANCECitrus Huanglongbing (HLB, also called citrus greening disease) is a highly destructive disease currently threatening citrus production worldwide. HLB is caused by an unculturable bacterial pathogen, "Candidatus Liberibacter asiaticus" (CLas). However, the mechanism of CLas colonization and growth in citrus hosts is poorly understood. In this study, we utilized the fruit pith tissue, which was able to maintain the CLas at a high abundance, as the materials for dual RNA-Seq and untargeted metabolome analysis, aiming to reveal the biological processes and phytochemical substances that are vital for CLas colonization and growth. We provided a genome-wide CLas transcriptome landscape in the fruit pith of three citrus cultivars with different tolerance and identified the important genes/pathways that contribute to CLas colonization and growth in the fruit pith. Metabolome profiling identified the key metabolites, which were mainly affected by CLas infection and influenced the population dynamic of CLas in fruit pith.

Functional features of a novel Sb(III)- and As(III)-oxidizing bacterium: Implications for the interactions between bacterial Sb(III) and As(III) oxidation pathways.

Antimony (Sb) and arsenic (As) share similar chemical characteristics and commonly coexist in contaminated environments. It has been reported that the biogeochemical cycles of antimony and arsenic affect each other. However, there is limited understanding regarding microbial coupling between the biogeochemical processes of antimony and arsenic. Here, we aimed to solve this issue. We successfully isolated a novel bacterium, Shinella sp. SbAsOP1, which possesses both Sb(III) and As(III) oxidase, and can effectively oxidize both Sb(III) and As(III) under aerobic and anaerobic conditions. SbAsOP1 exhibits greater aerobic oxidation activity for the oxidation of As(III) or Sb(III) compared to its anaerobic activity. SbAsOP1 also significantly catalyzes the oxidative mobilization of solid-phase Sb(III) under aerobic conditions. The activity of SbAsOP1 in oxidizing solid Sb(III) is 3 times lower than its activity in oxidizing soluble form. It is noteworthy that, in the presence of both Sb(III) and As(III) under aerobic conditions, either As(III) or Sb(III) significantly inhibits the oxidation of Sb(III) or As(III), respectively. In comparison, under anaerobic conditions and in the coexistence of Sb(III) and As(III), As(III) significantly inhibits Sb(III) oxidation, whereas Sb(III) almost completely inhibits As(III) oxidation. These findings suggest that under both aerobic and anaerobic conditions, SbAsOP1 demonstrates a partial preference for Sb(III) oxidation. Additionally, bacterial oxidations of Sb(III) and As(III) mutually inhibit each other to varying degrees. These observations gain a novel understanding of the interplay between the biogeochemical processes of antimony and arsenic.

Tetracycline hydrochloride degradation in polarity inverted microbial fuel cells: Performance, mechanisms and microbiology.

Tetracycline antibiotics are widely used in veterinary medicine, human therapy and agriculture, and their presence in natural water raises environmental concerns. In this study, more than 94% of tetracycline hydrochloride (TCH) could be rapidly degraded within 48 h in polarity-inverted microbial fuel cells. The electrochemically active bacteria had the best electrochemical performance at 1 mg/L of TCH with the minimum internal resistance of 77.38 Ω. The electron-rich functional groups of TCH were continuously attacked and finally degradated into small molecules in three possible degradation pathways. Microbial community structure analysis showed that Comamonas and Shinella were enriched at the electrode as polarity-inverted bacteria. Genomic analysis showed that both direct and indirect electron transfer participated in the degradation of TCH in polarity-inverted microbial fuel cell (MFC) and the functional genes related to electrical conductivity in polarity-inverted MFC were more enriched on the electrode surface than non-polarity-inverted MFC. This study can facilitate further investigations about the biodegradation of TCH in polarity-inverted microbial fuel cell.

An effector of 'Candidatus Liberibacter asiaticus' manipulates autophagy to promote bacterial infection.

Autophagy functions in plant host immunity responses to pathogen infection. The molecular mechanisms and functions used by the citrus Huanglongbing (HLB)-associated intracellular bacterium 'Candidatus Liberibacter asiaticus' (CLas) to manipulate autophagy are unknown. We identified a CLas effector, SDE4405 (CLIBASIA_04405), which contributes to HLB progression. 'Wanjincheng' orange (Citrus sinensis) transgenic plants expressing SDE4405 promotes CLas proliferation and symptom expression via suppressing host immunity responses. SDE4405 interacts with the ATG8-family of proteins (ATG8s), and their interactions activate autophagy in Nicotiana benthamiana. The occurrence of autophagy is also significantly enhanced in SDE4405-transgenic citrus plants. Interrupting NbATG8s-SDE4405 interaction by silencing of NbATG8c reduces Pseudomonas syringae pv. tomato strain DC3000ΔhopQ1-1 (Pst DC3000ΔhopQ1-1) proliferation in N. benthamiana, and transient overexpression of CsATG8c and SDE4405 in citrus promotes Xanthomonas citri subsp. citri (Xcc) multiplication, suggesting that SDE4405-ATG8s interaction negatively regulates plant defense. These results demonstrate the role of the CLas effector protein in manipulating autophagy, and provide new molecular insights into the interaction between CLas and citrus hosts.

Biochemical characterization and structure-based in silico screening of potent inhibitor molecules against the 1 cys peroxiredoxin of bacterioferritin comigratory protein family from Candidatus Liberibacter asiaticus.

Bacterioferritin comigratory protein family 1 Cys peroxiredoxin from Candidatus Liberibacter asiaticus (CLaBCP) is an important antioxidant defense protein that participates in the reduction of ROS, free radicals, and peroxides. In the present study, we report the biochemical studies and in silico screening of potent antibacterial molecules against CLaBCP. The CLaBCP showed enzymatic activity with the Km value 54.43, 94.34, 120.6 µM, and Vmax of 59.37, 69.37, 70.0 µM min-1 for H2O2, TBHP, CHP respectively. The residual peroxidase activity of CLaBCP was analyzed at different ranges of pH and temperatures. The CLaBCP showed structural changes and unfolding in the presence of its substrates and guanidinium chloride by CD and fluorescence. The structure-based drug design method was employed to screen and identify the more efficient molecule against CLaBCP. The validated CLaBCP model was used for the virtual screening of potent antibacterial molecules. The docking was performed at CLaBCP active site to evaluate the binding energy of the top five molecules (LAS 34150849, BDE 33184869, LAS 51497689, BDE 33672484, and LAS 34150966). All identified molecule has a higher binding affinity than adenanthin analyzed by molecular docking. Molecular dynamics studies such as RMSD, Rg, SASA, and PCA results showed that the CLaBCP inhibitor(s) complex is more stable than the CLaBCP-adenanthin complex. MMPBSA results suggested that the identified molecule could form a lower energy CLaBCP-inhibiter(s) complex than the CLaBCP-adenanthin complex. The screened molecules may pave the route for the development of potent antibacterial molecules against CLa.Communicated by Ramaswamy H. Sarma.

Isolation and identification of acetaminophen degrading strain Shinella sp. HZA2.

Acetaminophen (APP), frequently used as analgesic and antipyretic drug in our life, is potentially toxic to both animals and humans. A novel acetaminophen degrading strain HZA2, was isolated from the activated sludge, and identified as Shinella sp. based on its 16S rRNA gene sequence analysis, morphological, physiological, and biochemical characterizations. This strain could degrade 100 mg L-1 acetaminophen completely within 12 h, and it was also a very effective strain for the degradation of high concentration of acetaminophen below 3000 mg L-1 under the optimal condition. The optimal degrading conditions of acetaminophen by HZA2 were pH 7.5 and 32.7 °C by the analysis of response surface methodology. Exogenous carbon source could enhance the biodegradation of acetaminophen. During the process, the intermediate metabolites were identified as 4-aminophenol and hydroquinone via gas chromatography-mass spectrometry analysis. The results indicated that strain HZA2 may be a promising bacterium for the bioremediation of acetaminophen pollutions.

In-silico screening and identification of potential inhibitors against 2Cys peroxiredoxin of Candidatus Liberibacter asiaticus.

Huanglongbing (HLB) is a worldwide citrus plant disease-related to non-culturable and fastidious α-proteobacteria Candidatus Liberibacter asiaticus (CLas). In CLas, Peroxiredoxin (Prx) plays a major role in the reduction of the level of reactive species such as reactive oxygen species (ROS), free radicals and peroxides, etc. Here, we have used structure-based drug designing approach was used to screen and identify the potent molecules against 2Cys Prx. The virtual screening of fragments library was performed against the three-dimensional validated model of Prx. To evaluate the binding affinity, the top four molecules (N-Boc-2-amino isobutyric acid (B2AI), BOC-L-Valine (BLV), 1-(boc-amino) cyclobutane carboxylic acid (1BAC), and N-Benzoyl-DL-alanine (BDLA)) were docked at the active site of Prx. The molecular docking results revealed that all the identified molecules had a higher binding affinity than Tert butyl hydroperoxide (TBHP), a substrate of Prx. Molecular dynamics analysis such as RMSD, Rg, SASA, hydrogen bonds, and PCA results indicated that Prx-inhibitor(s) complexes had lesser fluctuations and were more stable and compact than Prx-TBHP complex. MMPBSA results confirmed that the identified compounds could bind at the active site of Prx to form a lower energy Prx-inhibitor(s) complex than Prx-TBHP complex. The identified potent molecules may pave the path for the development of antimicrobial agents against CLA.Communicated by Ramaswamy H. Sarma.

'Candidatus Liberibacter asiaticus'-Encoded BCP Peroxiredoxin Suppresses Lipopolysaccharide-Mediated Defense Signaling and Nitrosative Stress In Planta.

The lipopolysaccharides (LPS) of gram-negative bacteria trigger a nitrosative and oxidative burst in both animals and plants during pathogen invasion. Liberibacter crescens strain BT-1 is a surrogate for functional genomic studies of the uncultured pathogenic 'Candidatus Liberibacter' spp. that are associated with severe diseases such as citrus greening and potato zebra chip. Structural determination of L. crescens LPS revealed the presence of a very long chain fatty acid modification. L. crescens LPS pretreatment suppressed growth of Xanthomonas perforans on nonhost tobacco (Nicotiana benthamiana) and X. citri subsp. citri on host orange (Citrus sinensis), confirming bioactivity of L. crescens LPS in activation of systemic acquired resistance (SAR). L. crescens LPS elicited a rapid burst of nitric oxide (NO) in suspension cultured tobacco cells. Pharmacological inhibitor assays confirmed that arginine-utilizing NO synthase (NOS) activity was the primary source of NO generation elicited by L. crescens LPS. LPS treatment also resulted in biological markers of NO-mediated SAR activation, including an increase in the glutathione pool, callose deposition, and activation of the salicylic acid and azelaic acid (AzA) signaling networks. Transient expression of 'Ca. L. asiaticus' bacterioferritin comigratory protein (BCP) peroxiredoxin in tobacco compromised AzA signaling, a prerequisite for LPS-triggered SAR. Western blot analyses revealed that 'Ca. L. asiaticus' BCP peroxiredoxin prevented peroxynitrite-mediated tyrosine nitration in tobacco. 'Ca. L. asiaticus' BCP peroxiredoxin (i) attenuates NO-mediated SAR signaling and (ii) scavenges peroxynitrite radicals, which would facilitate repetitive cycles of 'Ca. L. asiaticus' acquisition and transmission by fecund psyllids throughout the limited flush period in citrus.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Plants Saline Environment in Perception with Rhizosphere Bacteria Containing 1-Aminocyclopropane-1-Carboxylate Deaminase.

Soil salinity stress has become a serious roadblock for food production worldwide since it is one of the key factors affecting agricultural productivity. Salinity and drought are predicted to cause considerable loss of crops. To deal with this difficult situation, a variety of strategies have been developed, including plant breeding, plant genetic engineering, and a wide range of agricultural practices, including the use of plant growth-promoting rhizobacteria (PGPR) and seed biopriming techniques, to improve the plants' defenses against salinity stress, resulting in higher crop yields to meet future human food demand. In the present review, we updated and discussed the negative effects of salinity stress on plant morphological parameters and physio-biochemical attributes via various mechanisms and the beneficial roles of PGPR with 1-Aminocyclopropane-1-Carboxylate(ACC) deaminase activity as green bio-inoculants in reducing the impact of saline conditions. Furthermore, the applications of ACC deaminase-producing PGPR as a beneficial tool in seed biopriming techniques are updated and explored. This strategy shows promise in boosting quick seed germination, seedling vigor and plant growth uniformity. In addition, the contentious findings of the variation of antioxidants and osmolytes in ACC deaminase-producing PGPR treated plants are examined.

Malate Transport and Metabolism in Nitrogen-Fixing Legume Nodules.

Legumes form a symbiosis with rhizobia, a soil bacterium that allows them to access atmospheric nitrogen and deliver it to the plant for growth. Biological nitrogen fixation occurs in specialized organs, termed nodules, that develop on the legume root system and house nitrogen-fixing rhizobial bacteroids in organelle-like structures termed symbiosomes. The process is highly energetic and there is a large demand for carbon by the bacteroids. This carbon is supplied to the nodule as sucrose, which is broken down in nodule cells to organic acids, principally malate, that can then be assimilated by bacteroids. Sucrose may move through apoplastic and/or symplastic routes to the uninfected cells of the nodule or be directly metabolised at the site of import within the vascular parenchyma cells. Malate must be transported to the infected cells and then across the symbiosome membrane, where it is taken up by bacteroids through a well-characterized dct system. The dicarboxylate transporters on the infected cell and symbiosome membranes have been functionally characterized but remain unidentified. Proteomic and transcriptomic studies have revealed numerous candidates, but more work is required to characterize their function and localise the proteins in planta. GABA, which is present at high concentrations in nodules, may play a regulatory role, but this remains to be explored.

Biodegradation of flonicamid by Ensifer adhaerens CGMCC 6315 and enzymatic characterization of the nitrile hydratases involved.

BACKGROUND: Flonicamid (N-cyanomethyl-4-trifluoromethylnicotinamide, FLO) is a new type of pyridinamide insecticide that regulates insect growth. Because of its wide application in agricultural production and high solubility in water, it poses potential risks to aquatic environments and food chain. RESULTS: In the present study, Ensifer adhaerens CGMCC 6315 was shown to efficiently transform FLO into N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM) via a hydration pathway mediated by two nitrile hydratases, PnhA and CnhA. In pure culture, resting cells of E. adhaerens CGMCC 6315 degraded 92% of 0.87 mmol/L FLO within 24 h at 30 °C (half-life 7.4 h). Both free and immobilized (by gel beads, using calcium alginate as a carrier) E. adhaerens CGMCC 6315 cells effectively degraded FLO in surface water. PnhA has, to our knowledge, the highest reported degradation activity toward FLO, Vmax = 88.7 U/mg (Km = 2.96 mmol/L). Addition of copper ions could increase the enzyme activity of CnhA toward FLO by 4.2-fold. Structural homology modeling indicated that residue ß-Glu56 may be important for the observed significant difference in enzyme activity between PnhA and CnhA. CONCLUSIONS: Application of E. adhaerens may be a good strategy for bioremediation of FLO in surface water. This work furthers our understanding of the enzymatic mechanisms of biodegradation of nitrile-containing insecticides and provides effective transformation strategies for microbial remediation of FLO contamination.

Comprehensive characterization of stress tolerant bacteria with plant growth-promoting potential isolated from glyphosate-treated environment.

The application of plant growth-promoting bacteria in agricultural systems is an efficient and environment-friendly strategy to improve crop yields and maintain soil quality. However, as different soils have diverse and specific ecological characteristics and may represent adverse abiotic conditions, in vivo application requires the careful selection of the desired beneficial microorganisms. In this study we report Ensifer adhaerens SZMC 25856 and Pseudomonas resinovorans SZMC 25875 isolates recovered from glyphosate-treated soil to possess yet undiscovered plant growth-enhancing potential. The strains were found to promote the growth of tomato seedlings significantly, to have the ability of synthesizing indole-3-acetic acid and siderophores, to tolerate pH in the range of 6.59-7.96, salinity up to 12.5 g L-1 NaCl and drought up to 125 g L-1 polyethylene glycol 6000, as well as to survive in the presence of various pesticides including glyphosate, diuron, chlorotoluron, carbendazim and thiabendazole, and heavy metals such as Al, Fe, Mn, Zn, Pb and Cu. The plant growth-promoting traits of the examined E. adhaerens and P. resinovorans isolates and their tolerance to numerous abiotic stress factors make them promising candidates for application in different agricultural environments, including soils polluted with glyphosate.

ACC deaminase-producing Ensifer adhaerens KS23 enhances proximate nutrient of Pisum sativum L. cultivated in high altitude.

A phytohormone producing, N2-fixing and 1-aminocyclopropane-1-carboxylate (ACC) deaminase synthesizing bacterium Ensifer adhaerens KS23 effectively increased the yield and nutritional contents of Pisum sativum var. Arkel. The isolate KS23 showed positive ACC deaminase activity with 174.2 (nmol of α-ketobutyrate/g-1 biomass½ h-1) a 9.7-fold increase in glutathione S-transferase activity. The proximate analysis exhibited an increased yield of protein (21.45%), carbohydrate (38.90%), sulphur (29.94%) starch (27.52%), total ash (35.57%), fat content (27.5%), nitrogen (24.06%) and hydrogen (17.91%) in treated seeds of P. sativum as compared to untreated crop seeds in field trials at Srikot village, Srinagar-246,174 (Garhwal) India. The most desirable essential and non-essential amino-acids content was also enhanced simultaneously by E. adhaerens KS23 as compared to non-treated crop seeds. This study revealed the enhancement of various nutritional contents resulting in quality improvement and an increase in growth productivity of pea. This study provides an encouraging result that may benefit the marginal income of farmers belonging mainly to hilly regions who are dependent on traditional methods of farming and thus improving their economy.

Isolation and characterization of bacteria from activated sludge capable of degrading 17α-ethinylestradiol, a contaminant of high environmental concern.

The compound 17α-ethinylestradiol (EE2) is a synthetic oestrogen which is classified as a group 1 carcinogen by the World Health Organization. Together with other endocrine disruptor compounds, EE2 has been included in the surface water Watch List by the European Commission, since it causes severe adverse effects in ecosystems. Thus, it became a high priority to find or improve processes such as biodegradation of EE2 to completely remove this drug from the wastewater treatment plants (WWTPs). The present study aimed at the isolation of bacteria capable of degrading EE2 using environmental samples, namely a sludge from the Faro Northwest WWTP. Four isolates with ability to grow in the presence of 50 mg l-1 EE2 were obtained. The analysis of 16SrRNA gene sequences identified the isolated bacteria as Acinetobacter bouvetii, Acinetobacter kookii, Pantoea agglomerans and Shinella zoogloeoides. The results of biodegradation assays showed that Acinetobacter bouvetii, Acinetobacter kookii, Pantoea agglomerans and Shinella zoogloeoides were able to degrade 47±4 %, 55±3 %, 64±4% and 35±4 %, respectively of 13 mg l-1 EE2 after 168 h at 28 °C. To the best of our knowledge, these bacterial isolates were identified as EE2 degraders for the first time. In a preliminary experiment on the identification of metabolic products resulting from EE2 degradation products such as estrone (E1), γ-lactone compounds, 2-pentanedioic acid and 2-butenedioic acid an intermediate metabolite of the TCA cycle, were detected.

Phylogenetic diversity analysis reveals Bradyrhizobium yuanmingense and Ensifer aridi as major symbionts of mung bean (Vigna radiata L.) in Pakistan.

The present study was carried out to evaluate the diversity of rhizobia associated with nodules of mung bean in Pakistan, because this information is necessary for inoculum development. Based on sequence analysis of 16S rRNA gene of thirty-one bacteria, 11 were assigned to genus Bradyrhizobium, 17 to Ensifer, and 3 to Rhizobium. Phylogenetic analyses on the basis of 16S-23S ITS region, atpD, recA, nifH, and nodA of representative strains revealed that B. yuanmingense is the predominant species distributed throughout different mung bean-growing areas. Among the fast-growing rhizobia, Ensifer aridi was predominant in Faisalabad, Layyah, and Rawalpindi, while E. meliloti in Thal desert. Sequence variations and phylogeny of nifH and nodA genes suggested that these genes might have been co-evolved with the housekeeping genes and maintained by vertical gene transfer in rhizobia detected in the present study. Host infectivity assay revealed the successful nodulation of host by rhizobia related to genera Bradyrhizobium, Ensifer and Rhizobium. Among all, Bradyrhizobium and Ensifer spp. inoculation exhibited a significantly higher number of nodules (11-34 nodules plant-1) and nitrogenase activity (nodule ARA 60-110 µmol g-1 h-1). Contrary to the previous studies, our data reveal that B. yuanmingense and E. aridi are predominant species forming effective nodules in mung bean in Pakistan. Furthermore, to the best of our knowledge, this is the first report showing the effective symbiosis of E. aridi, E. meliloti, and Rhizobium pusense with mung bean. The diversity of rhizobia in different habitats revealed in the present study will contribute towards designing site-specific inocula for mung bean.

Promotion of growth and phytoextraction of cadmium and lead in Solanum nigrum L. mediated by plant-growth-promoting rhizobacteria.

Plant growth-promoting rhizobacteria (PGPR) are a specific category of microbes that improve plant growth and promote greater tolerance to metal stress through their interactions with plant roots. We evaluated the effects of phytoremediation combining the cadmium accumulator Solanum nigrum L. and two Cd- and Pb-resistant bacteria isolates. To understand the interaction between PGPR and their host plant, we conducted greenhouse experiments with inoculation treatments at Nanjing Agricultural University (Jiangsu Province, China), in June 2018. Two Cd- and Pb-resistant PGPR with various growth-promoting properties were isolated from heavy metal-contaminated soil. 16S rRNA analyses indicated that the two isolates were Bacillus genus, and they were named QX8 and QX13. Pot experiments demonstrated that inoculation may improve the rhizosphere soil environment and promote absorption of Fe and P by plants. Inoculation with QX8 and QX13 also enhanced the dry weight of shoots (1.36- and 1.7-fold, respectively) and roots (1.42- and 1.96-fold) of plants growing in Cd- and Pb-contaminated soil, and significantly increased total Cd (1.28-1.81 fold) and Pb (1.08-1.55 fold) content in aerial organs, compared to non-inoculated controls. We also detected increases of 23% and 22% in the acid phosphatase activity of rhizosphere soils inoculated with QX8 and QX13, respectively. However, we did not detect significant differences between inoculated and non-inoculated treatments in Cd and Pb concentrations in plants and available Cd and Pb content in rhizosphere soils. We demonstrated that PGPR-assisted phytoremediation is a promising technique for remediating heavy metal-contaminated soils, with the potential to enhance phytoremediation efficiency and improve soil quality.

Detoxification and bioaugmentation potential for acetaminophen and its derivatives using Ensifer sp. isolated from activated sludge.

Acetaminophen (APAP), a widely used analgesic-antipyretic drug, is frequently detected in the environment and may pose ecological risks to aquatic communities. In this work, an APAP-degrading organism, designated as Ensifer sp. POKHU, was isolated from activated sludge (AS) enriched with APAP. POKHU degraded up to 630 mg/L of APAP without substrate inhibition. The bacterium metabolized APAP to hydroquinone (HQ) via 4-aminophenol (4-AP). APAP derivatives, 4AP, HQ, and 1,4-benzoquinone (BQ), frequently detected in the environment, were found to inhibit nitrogen metabolism (ammonium oxidation) to a greater extent than APAP. POKHU had the ability to degrade varying levels (0.4-40 mg/L) of 4-AP, HQ, and BQ, which indicated a great potential for detoxification in environments contaminated with both APAP and its derivatives. The addition of POKHU to fresh AS samples taken from a wastewater treatment plant greatly increased the biotransformation rates of APAP from 5.6 d-1 (no POKHU augmentation) to >20.0 d-1 (5% POKHU). Bioaugmentation with POKHU reduced 400 µg/L of APAP to levels below its ecotoxicity threshold within 4 h, which is shorter than the typical hydraulic retention times for full-scale AS processing. Overall, this study identified a new auxiliary biological agent for APAP detoxification, which could degrade both APAP and its metabolic derivatives (those that can be more toxic than the parent contaminant, APAP). The results have practical implications for developing a biological means (detoxification and bioaugmentation) of treating high-strength pharmaceutical waste streams, such as wastewater from hospitals and drug manufactures, and of landfill leachates.

3-Hydroxypyridine Dehydrogenase HpdA Is Encoded by a Novel Four-Component Gene Cluster and Catalyzes the First Step of 3-Hydroxypyridine Catabolism in Ensifer adhaerens HP1.

3-Hydroxypyridine (3HP) is an important natural pyridine derivative. Ensifer adhaerens HP1 can utilize 3HP as its sole sources of carbon, nitrogen, and energy to grow, but the genes responsible for the degradation of 3HP remain unknown. In this study, we predicted that a gene cluster, designated 3hpd, might be responsible for the degradation of 3HP. The analysis showed that the initial hydroxylation of 3HP in E. adhaerens HP1 was catalyzed by a four-component dehydrogenase (HpdA1A2A3A4) and led to the formation of 2,5-dihydroxypyridine (2,5-DHP). In addition, the SRPBCC component in HpdA existed as a separate subunit, which is different from other SRPBCC-containing molybdohydroxylases acting on N-heterocyclic aromatic compounds. Moreover, the results demonstrated that the phosphoenolpyruvate (PEP)-utilizing protein and pyruvate-phosphate dikinase were involved in the HpdA activity, and the presence of the gene cluster 3hpd was discovered in the genomes of diverse microbial strains. Our findings provide a better understanding of the microbial degradation of pyridine derivatives in nature and indicated that further research on the origin of the discovered four-component dehydrogenase with a separate SRPBCC domain and the function of PEP-utilizing protein and pyruvate-phosphate dikinase might be of great significance.IMPORTANCE 3-Hydroxypyridine is an important building block for the synthesis of drugs, herbicides, and antibiotics. Although the microbial degradation of 3-hydroxypyridine has been studied for many years, the molecular mechanisms remain unclear. Here, we show that 3hpd is responsible for the catabolism of 3-hydroxypyridine. The 3hpd gene cluster was found to be widespread in Actinobacteria, Rubrobacteria, Thermoleophilia, and Alpha-, Beta-, and Gammaproteobacteria, and the genetic organization of the 3hpd gene clusters in these bacteria shows high diversity. Our findings provide new insight into the catabolism of 3-hydroxypyridine in bacteria.

Raman Spectroscopy vs Quantitative Polymerase Chain Reaction In Early Stage Huanglongbing Diagnostics.

Raman spectroscopy (RS) is an emerging analytical technique that can be used to develop and deploy precision agriculture. RS allows for confirmatory diagnostic of biotic and abiotic stresses on plants. Specifically, RS can be used for Huanglongbing (HLB) diagnostics on both orange and grapefruit trees, as well as detection and identification of various fungal and viral diseases. The questions that remain to be answered is how early can RS detect and identify the disease and whether RS is more sensitive than qPCR, the "golden standard" in pathogen diagnostics? Using RS and HLB as case study, we monitored healthy (qPCR-negative) in-field grown citrus trees and compared their spectra to the spectra collected from healthy orange and grapefruit trees grown in a greenhouse with restricted insect access and confirmed as HLB free by qPCR. Our result indicated that RS was capable of early prediction of HLB and that nearly all in-field qPCR-negative plants were infected by the disease. Using advanced multivariate statistical analysis, we also showed that qPCR-negative plants exhibited HLB-specific spectral characteristics that can be distinguished from unrelated nutrition deficit characteristics. These results demonstrate that RS is capable of much more sensitive diagnostics of HLB compared to qPCR.

Biochemical, functional and molecular characterization of pigeon pea rhizobia isolated from semi-arid regions of India.

Pigeon pea (Cajanus cajan (L.) Millspaugh) is among the top ten legumes grown globally not only having high tolerance to environmental stresses along, but also has the high biomass and productivity with optimal nutritional profiles. In the present study, 55 isolates of rhizobia were identified from 22 nodule samples of pigeon pea collected from semi-arid regions of India on the basis of morphological, biochemical, plant growth promoting activities and their ability to tolerate the stress conditions viz. pH, salt, temperature and drought stress. Amongst all the 55 isolates, 37 isolates showed effective nodulation under in vitro conditions in pigeon pea. Further, five isolates having multiple PGP activities and high in vitro symbiotic efficiency were subjected to 16S rRNA sequencing and confirmed their identities as Rhizobium, Mesorhizobium, Sinorhizobium sp. Further these 37 isolates were characterized at molecular level using ARDRA and revealed significant molecular diversity. Based on UPGMA clustering analysis, these isolates showed significant molecular diversity. The high degree of molecular diversity is due to mixed cropping of legumes in the region. The assessment of genetic diversity and molecular characterization of novel strains is a very important tool for the replacement of ineffective rhizobial strains with the efficient strains for the improvement in the nodulation and pigeon pea quality. The pigeon pea isolates with multiple PGPR activities could be further used for commercial production.