Integrated Hfq-interacting RNAome and transcriptomic analysis reveals complex regulatory networks of nitrogen fixation in root-associated Pseudomonas stutzeri A1501.

The RNA chaperone Hfq acts as a global regulator of numerous biological processes, such as carbon/nitrogen metabolism and environmental adaptation in plant-associated diazotrophs; however, its target RNAs and the mechanisms underlying nitrogen fixation remain largely unknown. Here, we used enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing to identify hundreds of Hfq-binding RNAs probably involved in nitrogen fixation, carbon substrate utilization, biofilm formation, and other functions. Collectively, these processes endow strain A1501 with the requisite capabilities to thrive in the highly competitive rhizosphere. Our findings revealed a previously uncharted landscape of Hfq target genes. Notable among these is nifM, encoding an isomerase necessary for nitrogenase reductase solubility; amtB, encoding an ammonium transporter; oprB, encoding a carbohydrate porin; and cheZ, encoding a chemotaxis protein. Furthermore, we identified more than 100 genes of unknown function, which expands the potential direct regulatory targets of Hfq in diazotrophs. Our data showed that Hfq directly interacts with the mRNA of regulatory proteins (RsmA, AlgU, and NifA), regulatory ncRNA RsmY, and other potential targets, thus revealing the mechanistic links in nitrogen fixation and other metabolic pathways. IMPORTANCE: Numerous experimental approaches often face challenges in distinguishing between direct and indirect effects of Hfq-mediated regulation. New technologies based on high-throughput sequencing are increasingly providing insight into the global regulation of Hfq in gene expression. Here, enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing was employed to identify the Hfq-binding sites and potential targets in the root-associated Pseudomonas stutzeri A1501 and identify hundreds of novel Hfq-binding RNAs that are predicted to be involved in metabolism, environmental adaptation, and nitrogen fixation. In particular, we have shown Hfq interactions with various regulatory proteins' mRNA and their potential targets at the posttranscriptional level. This study not only enhances our understanding of Hfq regulation but, importantly, also provides a framework for addressing integrated regulatory network underlying root-associated nitrogen fixation.

Biodegradation of Imazethapyr by Bacterial Strain IM9601 Isolated from Agricultural Soil.

The widespread utilization of the herbicide imazethapyr presents significant challenges to crop rotation and results in detrimental soil degradation issues. Bacterial biodegradation has emerged as a promising and eco-friendly approach for mitigating pesticide residues contamination in the environment. In this study, a novel bacterium, identified as Brevibacterium sp. IM9601, was isolated and characterized based on morphological, physiological, and biochemical characteristics, as well as 16S rRNA gene sequence. This strain exhibited the ability to utilize imazethapyr as its sole carbon source for growth. Response surface methodology (RSM) was applied to optimize the degradation conditions. The most favorable conditions were determined to be a temperature of 27 °C, pH of 6.0, and an initial inoculum with a final OD600 of 0.15. Under these optimized condition, bacterial strain IM9601 exhibited substantial imazethapyr degradation, with removal rates of 90.08 and 87.05% for initial imazethapyr concentrations of 50 and 100 mg L-1, respectively, achieved within a 5-day incubation period. This investigation highlights imazethapyr-degrading capabilities of the Brevibacterium genus bacterial strain IM9601, marking it as a potentially novel and effective solution for addressing the environmental pollution resulting from the usage of imazethapyr. The study contributes to the growing body of research on bioremediation approaches, offering a sustainable and environmentally friendly method for mitigating the adverse impacts of herbicide contamination in agricultural settings.

Regulation of hierarchical carbon substrate utilization, nitrogen fixation, and root colonization by the Hfq/Crc/CrcZY genes in Pseudomonas stutzeri.

Bacteria of the genus Pseudomonas consume preferred carbon substrates in nearly reverse order to that of enterobacteria, and this process is controlled by RNA-binding translational repressors and regulatory ncRNA antagonists. However, their roles in microbe-plant interactions and the underlying mechanisms remain uncertain. Here we show that root-associated diazotrophic Pseudomonas stutzeri A1501 preferentially catabolizes succinate, followed by the less favorable substrate citrate, and ultimately glucose. Furthermore, the Hfq/Crc/CrcZY regulatory system orchestrates this preference and contributes to optimal nitrogenase activity and efficient root colonization. Hfq has a central role in this regulatory network through different mechanisms of action, including repressing the translation of substrate-specific catabolic genes, activating the nitrogenase gene nifH posttranscriptionally, and exerting a positive effect on the transcription of an exopolysaccharide gene cluster. Our results illustrate an Hfq-mediated mechanism linking carbon metabolism to nitrogen fixation and root colonization, which may confer rhizobacteria competitive advantages in rhizosphere environments.

The Sigma Factor AlgU Regulates Exopolysaccharide Production and Nitrogen-Fixing Biofilm Formation by Directly Activating the Transcription of pslA in Pseudomonas stutzeri A1501.

Pseudomonas stutzeri A1501, a plant-associated diazotrophic bacterium, prefers to conform to a nitrogen-fixing biofilm state under nitrogen-deficient conditions. The extracytoplasmic function (ECF) sigma factor AlgU is reported to play key roles in exopolysaccharide (EPS) production and biofilm formation in the Pseudomonas genus; however, the function of AlgU in P. stutzeri A1501 is still unclear. In this work, we mainly investigated the role of algU in EPS production, biofilm formation and nitrogenase activity in A1501. The algU mutant ΔalgU showed a dramatic decrease both in the EPS production and the biofilm formation capabilities. In addition, the biofilm-based nitrogenase activity was reduced by 81.4% in the ΔalgU mutant. The transcriptional level of pslA, a key Psl-like (a major EPS in A1501) synthesis-related gene, was almost completely inhibited in the algU mutant and was upregulated by 2.8-fold in the algU-overexpressing strain. A predicted AlgU-binding site was identified in the promoter region of pslA. The DNase I footprinting assays indicated that AlgU could directly bind to the pslA promoter, and ß-galactosidase activity analysis further revealed mutations of the AlgU-binding boxes drastically reduced the transcriptional activity of the pslA promoter; moreover, we also demonstrated that AlgU was positively regulated by RpoN at the transcriptional level and negatively regulated by the RNA-binding protein RsmA at the posttranscriptional level. Taken together, these data suggest that AlgU promotes EPS production and nitrogen-fixing biofilm formation by directly activating the transcription of pslA, and the expression of AlgU is controlled by RpoN and RsmA at different regulatory levels.

Master regulator NtrC controls the utilization of alternative nitrogen sources in Pseudomonas stutzeri A1501.

Pseudomonas stutzeri A1501 is a model strain used to study associative nitrogen fixation, and it possesses the nitrogen regulatory NtrC protein in the core genome. Nitrogen sources represent one of the important factors affecting the efficiency of biological nitrogen fixation in the natural environment. However, the regulation of NtrC during nitrogen metabolism in P. stutzeri A1501 has not been clarified. In this work, a phenotypic analysis of the ntrC mutant characterized the roles of NtrC in nitrogen metabolism and the oxidative stress response of P. stutzeri A1501. To systematically identify NtrC-controlled gene expression, RNA-seq was performed to further analyse the gene expression differences between the wild-type strain and the ∆ntrC mutant under nitrogen fixation conditions. A total of 1431 genes were found to be significantly altered by ntrC deletion, among which 147 associative genes had NtrC-binding sites, and the pathways for nitrogen fixation regulation, nitrogenous compound acquisition and catabolism and nitrate assimilation were discussed. Furthermore, the oxidative stress-related gene (katB), which was upregulated by ntrC deletion, was suggested to be a potential target gene of NtrC, thus highlighting the importance of NtrC in nitrogenase protection against oxygen damage. Based on these findings, we propose that NtrC is a high-ranking element in the regulatory network of P. stutzeri A1501 that controls a variety of nitrogen metabolic and oxidative stress responsive traits required for adaptation to complex rhizosphere environments.

NfiR, a New Regulatory Noncoding RNA (ncRNA), Is Required in Concert with the NfiS ncRNA for Optimal Expression of Nitrogenase Genes in Pseudomonas stutzeri A1501.

Expression of nitrogenase genes (nifHDK) is strictly regulated at both transcriptional and posttranscriptional levels. Efficient nitrogenase activity requires maintaining sufficient levels of nif mRNAs, yet the underlying mechanism is not fully understood due to its complexity. We have previously shown that a novel regulatory noncoding RNA (ncRNA), NfiS, optimizes nitrogen fixation through targeting nifK mRNA in Pseudomonas stutzeri A1501. Here, we report the identification and characterization of a second ncRNA inducible under nitrogen fixation conditions (nitrogen-free and microaerobic conditions), termed NfiR (for nitrogen fixation condition-inducible ncRNA), the expression of which is dependent on two global regulators, NtrC and Hfq. Comparative phenotypic and proteomic analyses of an nfiR mutant identify a role of NfiR in regulating the expression of nitrogenase genes. Further microscale thermophoresis and genetic complementation showed that an 11-nucleotide (nt) sequence in the stem-loop structure of NfiR (nucleotides 12 to 22) pairs with its counterpart in the coding region of nifD mRNA (nucleotides 1194 to 1207) by eight nucleotides. Significantly, deletion of nfiR caused a 60% reduction of nitrogenase activity, and the half-life of nifD mRNA was reduced from 20 min for the wild type to 15 min for the ΔnfiR mutant. With regard to nitrogenase activity and stability of the nifD and nifK transcripts, phenotypes were more severe for the double deletion mutant lacking nfiR and nfiS, suggesting that NfiR, in concert with NfiS, optimizes nitrogenase production at the posttranscriptional level.IMPORTANCE Biological nitrogen fixation is an energy-expensive process requiring the hydrolysis of 16 ATPs. Consequently, the expression of nif genes is highly regulated at both transcriptional and posttranscriptional levels through complex regulatory networks. Global regulation involves a number of regulatory proteins, such as the nif-specific activator NifA and the global nitrogen regulator NtrC, as well as various regulatory ncRNAs. We show that the two P. stutzeri ncRNAs, namely NfiS and NfiR (for nitrogen fixation condition-inducible ncRNA), optimize nitrogen fixation and environmental stress responses. NfiS and NfiR respond differently to various environmental signals and differ in their secondary structures. In addition, the two ncRNAs target the mRNAs of nifK and nifD, respectively. Such ncRNA-based posttranscriptional regulation of nitrogenase expression might be an evolved survival strategy, particularly in nitrogen-limiting environments. This study not only highlights the significant roles of regulatory ncRNAs in the coordination and fine tuning of various physiological processes but also provides a new paradigm for posttranscriptional regulation in nitrogen-fixing bacteria.

Rhizobium wenxiniae sp. nov., an endophytic bacterium isolated from maize root.

A novel Gram-stain-negative, aerobic, rod-shaped strain designated 166T was isolated from surface-sterilized root tissue of maize planted in the Fangshan District of Beijing, PR China. The 16S rRNA gene sequence analysis indicated that strain 166T belongs to the genus Rhizobium and is closely related to Rhizobium cellulosilyticum ALA10B2T and Rhizobium yantingense H66T with sequence similarities of 98.8 and 98.3 %, respectively. According to atpD and recA sequence analysis, the highest sequence similarity between strain 166T and R. cellulosilyticum ALA10B2T is 93.8 and 84.7 %, respectively. However, the new isolate exhibited relatively low levels of DNA-DNA relatedness with respect to R. cellulosilyticum DSM 18291T (20.8±2.3 %) and Rhizobium yantingense CCTCC AB 2014007T (47.2±1.4 %). The DNA G+C content of strain 166T was 59.8 mol%. The main polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, diphosphatidylglycerol, an unidentified aminophospholipid and an unidentified aminolipid. The major fatty acids of strain 166T were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The results of the physiological and biochemical tests and minor differences in the fatty acid profiles allowed a clear phenotypic differentiation of strain 166T from the type strains of closely related species, R. cellulosilyticum DSM 18291T and R. yantingense CCTCC AB 2014007T. Strain 166T represents a novel species within the genus Rhizobium, for which the name Rhizobium wenxiniae sp. nov. is proposed, with the type strain 166T (=CGMCC 1.15279T=DSM 100734T).

Microbacterium zeae sp. nov., an endophytic bacterium isolated from maize stem.

A novel Gram-stain positive, aerobic, non-motile, non-spore-forming and rod-shaped strain designated 1204T was isolated from surface-sterilised stem tissue of maize planted in Fangshan District of Beijing, People's Republic of China. A polyphasic taxonomic study was performed on the new isolate. On the basis of 16S rRNA gene sequence similarity studies, this isolate belongs to the genus Microbacterium. High levels of 16S rRNA gene sequence similarity were found between strain 1204T and Microbacterium enclense NIO-1002T (98.8%) and Microbacterium proteolyticum RZ36T (98.4%) respectively. However, the DNA-DNA hybridization values between strain 1204T and its closely related species M. proteolyticum DSM 27100T and M. enclense DSM 25125T were 53.9 ± 1.6 and 20.9 ± 1.5% respectively. The DNA G+C content of strain 1204T was determined to be 68.0 mol%. The major fatty acids were found to consist of anteiso-C15:0 (37.6%), iso-C16:0 (28.6%) and anteiso-C17:0 (16.6%). The predominant menaquinone was MK-11 and the polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, an unidentified glycolipid and an unidentified lipid. The results of physiological and biochemical tests and minor differences in the fatty acid profiles allowed a clear phenotypic differentiation of strain 1204T from the closely related species in the genus Microbacterium. Thus, it was concluded that strain 1204T represents a novel species within the genus Microbacterium, for which the name Microbacterium zeae sp. nov. is proposed, with the type strain 1204T (= CGMCC 1.15289 = DSM 100750).

Dyadobacter endophyticus sp. nov., an endophytic bacterium isolated from maize root.

A novel Gram-stain-negative, aerobic and rod-shaped bacterial strain, designated 65T, was isolated from surface-sterilized root tissue of maize, collected from Fangshan District of Beijing, People's Republic of China, and was subjected to a taxonomic study by using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 65T belonged to the genus Dyadobacter and had highest 16S rRNA gene sequence similarity to Dyadobacter jiangsuensis CGMCC 1.12969T (99.1 %), Dyadobacter beijingensis CGMCC 1.6375T (98.8 %), Dyadobacter fermentans DSM 18053T (98.6 %) and Dyadobacter soli KCTC 22481T (98.6 %). However, the new isolate exhibited relatively low levels of DNA-DNA relatedness with respect to D. jiangsuensis CGMCC 1.12969T (18.2±1.3 %), D. beijingensis CGMCC 1.6375T (14.2±2.0 %), D. fermentans DSM 18053T (14.1±2.0 %) and D. soli KCTC 22481T (13.8±0.6 %). The predominant respiratory quinone was menaquinone-7 (MK-7) and the major cellular fatty acids were summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH), iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 1ω5c, iso-C15 : 0 3-OH, C16 : 0 3-OH and C16 : 0. The polar lipid profile of strain 65T revealed the presence of phosphatidylethanolamine, four aminolipids and two unidentified phospholipids. The DNA G+C content was 46.6 mol%. The results of physiological and biochemical tests and the differences in the fatty acid profiles allowed the clear phenotypic differentiation of strain 65T from closely related species of the genus Dyadobacter. Strain 65T thus represents a novel species within the genus Dyadobacter, for which the name Dyadobacterendophyticus sp. nov. is proposed. The type strain is 65T (=CGMCC 1.15288T=DSM 100786T).

Sphingomonaszeicaulis sp. nov., an endophytic bacterium isolated from maize root.

A novel Gram-staining-negative, aerobic and rod-shaped strain designated 541T was isolated from surface-sterilized root tissue of maize, collected from the Fangshan District of Beijing, People's Republic of China, and was subjected to a taxonomic study using a polyphasic approach. According to a phylogenetic tree based on 16S rRNA gene sequences, strain 541T represented a member of the genus Sphingomonas and clustered with Sphingomonas sanxanigenens DSM 19645T, with which it shared the highest 16S rRNA gene sequence similarity (98.8 %). The predominant respiratory quinone was ubiquinone-10 (Q-10), the major polyamine was sym-homospermidine and the major cellular fatty acids were C18 : 1ω7c (50.9 %), C16 : 0 (22.0 %) and C14 : 0 2-OH (11.4 %). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and sphingoglycolipid. The DNA G+C content was 64.7 mol%. DNA-DNA relatedness between strain 541T and its closest phylogenetic relative Sphingomonas sanxanigenens DSM 19645T was 50.8 %. The results of physiological and biochemical tests and the differences in the fatty acid profiles allowed a clear phenotypic differentiation of strain 541T from closely related species of the genus Sphingomonas. Strain 541T represents a novel species within the genus Sphingomonas, for which the nameSphingomonas zeicaulis sp. nov. is proposed, with the type strain 541T (=CGMCC 1.15008T=DSM 100587T).

Filimonas zeae sp. nov., an endophytic bacterium isolated from maize root.

A novel Gram-stain-negative, aerobic, motile by gliding and filamentous strain, designated 772T,was isolated from surface-sterilized root tissue of maize planted in the Fangshan District of Beijing, China. 16S rRNA gene sequence analysis indicated that strain 772T was closely related to Filimonas endophytica SR2-06T andFilimonas lacunae YT21T of the family Chitinophagaceae with sequence similarities of 99.0 and 96.9 %, respectively. However, the new isolate exhibited relatively low levels of DNA-DNA relatedness with respect to Filimonas. endophytica KCTC 42060T (18.7±1.8 %) and Filimonas. lacunae DSM 21054T (17.9±2.0%). The DNA G+C content of strain 772T was 44.9 mol%. The respiratory quinone was menaquinone-7 and the polar lipid profile consisted of phosphatidylethanolamine, two unidentified aminophospholipids, two unidentified phospholipids and one unidentified lipid. The major fatty acids were iso-C15 : 0 and iso-C15 : 1 G. The results of the physiological and biochemical tests and minor differences in the fatty acid profiles allowed the clear phenotypic differentiation of strain 772T from the closely related species Filimonas. endophytica andF. lacunae. Strain 772T thus represents a novel species within the genus Filimonas, for which the name Filimonas zeae sp. nov. is proposed. The type strain is 772T (=CGMCC 1.15290T=DSM 100760T).

Paenibacillus radicis sp. nov., an endophytic bacterium isolated from maize root.

A novel Gram-stain-positive, aerobic, endospore-forming, and rod-shaped strain designated 694T was isolated from surface-sterilized root tissue of a maize planted in the Fangshan District of Beijing, People's Republic of China. A polyphasic taxonomic study was performed on the new isolate. On the basis of 16S rRNA gene sequence similarity studies, this isolate belongs to the genus Paenibacillus. High levels of 16S rRNA gene sequence similarity were found between strain 694T and Paenibacillus xinjiangensis DSM 30034T (98.5 %) and Paenibacillus glycanilyticus (98.1 %), respectively. However, the DNA-DNA hybridization values between strain 694T and its close relatives P. xinjiangensis 16970T and Paenibacillus algorifonticola CGMCC 1.10223T were 30.0 % and 36.7 % respectively. The DNA G+C content of strain 694T was determined to be 46.9 mol%. The predominant respiratory quinone was identified as menaquinone-7 and the polar lipid profile was found to be composed of the major lipids diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids were found to be anteiso-C15 : 0 (42.1 %), iso-C15 : 0 (18.4 %), iso-C16 : 0 (11.2 %) and C16 : 0 (12.1 %). The results of physiological and biochemical tests and minor differences in the fatty acid profiles allowed a clear phenotypic differentiation of strain 694T from the closely related species in the genus Paenibacillus. Strain 694T is concluded to represent a novel species within the genus Paenibacillus, for which the name Paenibacillus radicis sp. nov. is proposed, with the type strain 694T ( = CGMCC 1.15286T = DSM 100762T).

Paenibacillus wenxiniae sp. nov., a nifH gene -harbouring endophytic bacterium isolated from maize.

A novel Gram-positive, aerobic, motile, endospore-forming, rod-shaped bacterium, designated 373(T) was isolated from surface-sterilised root tissue of a maize planted in Fangshan District of Beijing, Peopole's Republic of China. A polyphasic taxonomic study was performed on the new isolate. On the basis of 16S rRNA gene sequence similarity studies, this isolate belongs to the genus Paenibacillus. The highest 16S rRNA gene sequence similarity was found between strain 373(T) and Paenibacillus hunanensis (98.1%), meanwhile the 16S rRNA gene sequence similarity between strain 373(T) and the type strains of other recognised members of the genus Paenibacillus were all below 95.6%. However, the DNA-DNA hybridization values between strain 373(T) and the type strain P. hunanensis DSM 22170(T) was 30.2%. The DNA G+C content of strain 373(T) was determined to be 46.0 mol%. The predominant respiratory quinone was identified as menaquinone-7 and the polar lipid profile was found to be composed of the major lipids diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids were found to consist of anteiso-C15: 0 (59.6%), anteiso-C17: 0 (12.8%) and C16: 0 (6.7%). The results of physiological and biochemical tests and minor differences in the fatty acid profiles allowed a clear phenotypic differentiation of strain 373(T) from the closely related species in this genus Paenibacillus. Strain 373(T) is concluded to represent a novel species within the genus Paenibacillus, for which the name Paenibacillus wenxiniae sp. nov. is proposed, with the type strain 373(T) (= CGMCC 1.15007 (T) = DSM100576 ).

Flavobacterium endophyticum sp. nov., a nifH gene-harbouring endophytic bacterium isolated from maize root.

A novel Gram-stain-negative, aerobic, rod-shaped bacterium, designated strain 522T, was isolated from surface-sterilized root tissue of maize planted in Fangshan District of Beijing, China. A polyphasic taxonomic study was performed on the new isolate. On the basis of 16S rRNA gene sequence similarity studies, this isolate belonged to the genus Flavobacterium and showed less than 93.9 % similarity to the type strains of all recognized species of the genus Flavobacterium. The predominant respiratory quinone was menaquinone-6 and the polar lipid profile was composed of the major lipids phosphatidylethanolamine, phosphatidylserine and two unidentified amino lipids. The major fatty acids were C15 : 0, iso-C15 : 0, iso-C15 : 1 G and iso-C16 : 0.The G+C content of the DNA was 37.7 mol%. The results of physiological and biochemical tests and the differences in fatty acid profiles allowed the clear phenotypic differentiation of strain 522T from closely related species of the genus Flavobacterium. Strain 522T therefore represents a novel species within the genus Flavobacterium, for which the name Flavobacterium endophyticum sp. nov. is proposed. The type strain is 522T ( = ACCC 19708T = DSM 29537T).