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IScience Dec 2022Bacteria of the genus consume preferred carbon substrates in nearly reverse order to that of enterobacteria, and this process is controlled by RNA-binding translational...
Bacteria of the genus 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 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 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.
PubMed: 36505936
DOI: 10.1016/j.isci.2022.105663 -
Metabolites May 2023Plant growth-promoting rhizobacteria (PGPR) can colonize plant root surfaces or form biofilms to promote plant growth and enhance plant resistance to harsh external...
Plant growth-promoting rhizobacteria (PGPR) can colonize plant root surfaces or form biofilms to promote plant growth and enhance plant resistance to harsh external environments. However, plant-PGPR interactions, especially chemical signaling molecules, are poorly understood. This study aimed to gain an in-depth understanding of the rhizosphere interaction mechanisms between PGPR and tomato plants. This study found that inoculation with a certain concentration of significantly promoted tomato growth and induced significant changes in tomato root exudates. Furthermore, the root exudates significantly induced NRCB010 growth, swarming motility, and biofilm formation. In addition, the composition of the root exudates was analyzed, and four metabolites (methyl hexadecanoate, methyl stearate, 2,4-di-tert-butylphenol, and n-hexadecanoic acid) significantly related to the chemotaxis and biofilm formation of NRCB010 were screened. Further assessment showed that these metabolites positively affected the growth, swarming motility, chemotaxis, or biofilm formation of strain NRCB010. Among these, n-hexadecanoic acid induced the most remarkable growth, chemotactic response, biofilm formation, and rhizosphere colonization. This study will help develop effective PGPR-based bioformulations to improve PGPR colonization and crop yields.
PubMed: 37233705
DOI: 10.3390/metabo13050664 -
Molecules (Basel, Switzerland) Jul 2023Various host systems have been employed to increase the yield of recombinant proteins. However, some recombinant proteins were successfully produced at high yields but...
Various host systems have been employed to increase the yield of recombinant proteins. However, some recombinant proteins were successfully produced at high yields but with no functional activities. To achieve both high protein yield and high activities, molecular biological strategies have been continuously developed. This work describes the effect of signal peptide (SP) and co-expression of molecular chaperones on the production of active recombinant protein in . Extracellular enzymes from , including β-1,4-xylanase, β-1,4-glucanase, and β-mannanase constructed with and without their signal peptides and intracellular enzymes from ST201, including benzoylformate decarboxylase (BFDC), benzaldehyde dehydrogenase (BADH), and d-phenylglycine aminotransferase (d-PhgAT) were cloned and overexpressed in BL21(DE3). Co-expression of molecular chaperones with all enzymes studied was also investigated. Yields of β-1,4-xylanase (Xyn), β-1,4-glucanase (Cel), and β-mannanase (Man), when constructed without their N-terminal signal peptides, increased 1112.61-, 1.75-, and 1.12-fold, respectively, compared to those of spXyn, spCel, and spMan, when constructed with their signal peptides. For the natural intracellular enzymes, the chaperones, GroEL-GroES complex, increased yields of active BFDC, BADH, and d-PhgAT, up to 1.31-, 4.94- and 37.93-fold, respectively, and also increased yields of Man and Xyn up to 1.53- and 3.46-fold, respectively, while other chaperones including DnaK-DnaJ-GrpE and Trigger factor (Tf) showed variable effects with these enzymes. This study successfully cloned and overexpressed extracellular and intracellular enzymes in BL21(DE3). When the signal peptide regions of the secretory enzymes were removed, yields of active enzymes were higher than those with intact signal peptides. In addition, a higher yield of active enzymes was obtained, in general, when these enzymes were co-expressed with appropriate chaperones. Therefore, can produce cytoplasmic and secretory enzymes effectively if only the enzyme coding sequence without its signal peptide is used and appropriate chaperones are co-expressed to assist in correct folding.
Topics: Humans; Escherichia coli; beta-Mannosidase; Molecular Chaperones; Escherichia coli Proteins; Recombinant Proteins
PubMed: 37513466
DOI: 10.3390/molecules28145594 -
MSphere Jun 2024The RNA chaperone Hfq acts as a global regulator of numerous biological processes, such as carbon/nitrogen metabolism and environmental adaptation in plant-associated...
UNLABELLED
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 , encoding an isomerase necessary for nitrogenase reductase solubility; , encoding an ammonium transporter; , encoding a carbohydrate porin; and , 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 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.
Topics: Pseudomonas stutzeri; Host Factor 1 Protein; Nitrogen Fixation; Gene Expression Regulation, Bacterial; Plant Roots; RNA, Bacterial; Gene Expression Profiling; Gene Regulatory Networks; Bacterial Proteins; High-Throughput Nucleotide Sequencing; Transcriptome; Rhizosphere
PubMed: 38747590
DOI: 10.1128/msphere.00762-23 -
Microbiology Spectrum Mar 2023One of the major challenges for the bioremediation application of microbial nitrous oxide (NO) reduction is its oxygen sensitivity. While a few strains were reported...
One of the major challenges for the bioremediation application of microbial nitrous oxide (NO) reduction is its oxygen sensitivity. While a few strains were reported capable of reducing NO under aerobic conditions, the NO reduction kinetics of phylogenetically diverse NO reducers are not well understood. Here, we analyzed and compared the kinetics of clade I and clade II NO-reducing bacteria in the presence or absence of oxygen (O) by using a whole-cell assay with NO and O microsensors. Among the seven strains tested, NO reduction of Stutzerimonas stutzeri TR2 and ZoBell was not inhibited by oxygen (i.e., oxygen tolerant). Paracoccus denitrificans, Azospirillum brasilense, and Gemmatimonas aurantiaca reduced NO in the presence of O but slower than in the absence of O (i.e., oxygen sensitive). NO reduction of Pseudomonas aeruginosa and Dechloromonas aromatica did not occur when O was present (i.e., oxygen intolerant). Amino acid sequences and predicted structures of NosZ were highly similar among these strains, whereas oxygen-tolerant NO reducers had higher oxygen consumption rates. The results suggest that the mechanism of O tolerance is not directly related to NosZ structure but is rather related to the scavenging of O in the cells and/or accessory proteins encoded by the cluster. Some bacteria can reduce NO in the presence of O, whereas others cannot. It is unclear whether this trait of aerobic NO reduction is related to the phylogeny and structure of NO reductase. The understanding of aerobic NO reduction is critical for guiding emission control, due to the common concurrence of NO and O in natural and engineered systems. This study provided the NO reduction kinetics of various bacteria under aerobic and anaerobic conditions and classified the bacteria into oxygen-tolerant, -sensitive, and -intolerant NO reducers. Oxygen-tolerant NO reducers rapidly consumed O, which could help maintain the low O concentration in the cells and keep their NO reductase active. These findings are important and useful when selecting NO reducers for bioremediation applications.
PubMed: 36926990
DOI: 10.1128/spectrum.04709-22 -
FEBS Open Bio Jun 2022Helicobacter pylori infection is a prevalent infectious disease, associated with many gastric diseases, including gastritis, gastric ulcer, and gastric cancer. To...
Helicobacter pylori infection is a prevalent infectious disease, associated with many gastric diseases, including gastritis, gastric ulcer, and gastric cancer. To reveal the characteristics of the gastric microbiome in patients infected with H. pylori, we performed metagenomic shotgun sequencing of stomach swab samples from 96 patients and then conducted metagenomic association analyses between alterations in the gastric microbiome and H. pylori infection status. The overall composition of the gastric microbiota in H. pylori-infected individuals was distinctly different from the negative controls; H. pylori became the dominant species after colonizing the human stomach and significantly decreased the α-diversity of the gastric community (P < 0.05, Wilcoxon rank-sum test). We also identified 6 HPI-associated microbial species (FDR < 0.05, Wilcoxon rank-sum test): Stenotrophomonas maltophilia, Stenotrophomonas unclassified, Chryseobacterium unclassified, Pedobacter unclassified, Variovorax unclassified, and Pseudomonas stutzeri. Furthermore, 55 gastric microbial pathways were enriched in the H. pylori-positive group, whereas only 2 pathways were more abundant in the H. pylori-negative group: dTDP-L-rhamnose biosynthesis and tetrapyrrole biosynthesis (FDR < 0.05, Wilcoxon rank-sum test). Gastritis was not associated with non-H. pylori species in the stomach (P > 0.05, Wilcoxon rank-sum test). This study revealed alterations in gastric microbial taxa and function associated with HPI in the Chinese population, which provides an insight into gastric microbial interactions and their potential role in the pathological process of gastric diseases.
Topics: Gastritis; Gastrointestinal Microbiome; Helicobacter Infections; Helicobacter pylori; Humans; Microbiota
PubMed: 35243810
DOI: 10.1002/2211-5463.13390 -
World Journal of Microbiology &... Sep 2021Pseudomonas stutzeri A1501 is a model strain used to study associative nitrogen fixation, and it possesses the nitrogen regulatory NtrC protein in the core genome....
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.
Topics: Bacterial Proteins; Gene Expression Regulation, Bacterial; Mutation; Nitrogen; Nitrogen Fixation; Pseudomonas stutzeri; RNA-Seq; Rhizosphere; Transcription Factors
PubMed: 34524580
DOI: 10.1007/s11274-021-03144-w -
Scientific Reports Jun 2022In this report, we systematically characterize 32 response regulators (RRs) from a metal tolerant groundwater isolate, Pseudomonas stutzeri RCH2 to assess the impact of...
In this report, we systematically characterize 32 response regulators (RRs) from a metal tolerant groundwater isolate, Pseudomonas stutzeri RCH2 to assess the impact of host-derived post-translational phosphorylation. As observed by distinct shifted bands in a phos-tag gel, 12 of the 24 detected RRs show homogenous mixtures of phosphorylated proteins or heterogenous mixtures of unphosphorylated and phosphorylated proteins. By evaluating the phosphorylation state of CzcR and CopR II under varying assay parameters, we found that changes to pH and exogenous addition of phospho-donors (e.g. acetyl phosphate) have little to no effect on phosphorylation state. By applying protein production conditions that decrease the pool of intracellular acetyl-phosphate in E. coli, we found a reduction in the phosphorylated population of CopR II when magnesium was added to the medium, but observed no change in phosphorylated population when CopR II is expressed in E. coli BL21 (DE3) ∆pta, a mutant with a metabolic disruption to the acetyl-phosphate pathway. Therefore, the specific mechanism of post-translational phosphorylation of RRs in E. coli remains obscure. These findings show the importance of characterizing the phosphorylation state of proteins when heterologously expressed, since their biochemical and physiological properties can be dependent on post-translational modification.
Topics: Escherichia coli; Phosphates; Phosphorylation; Protein Processing, Post-Translational; Proteins; Pseudomonas stutzeri
PubMed: 35725867
DOI: 10.1038/s41598-022-13525-2 -
Applied and Environmental Microbiology Apr 2020Biodegradation of alkanes by microbial communities is ubiquitous in nature. Interestingly, the microbial communities with high hydrocarbon-degrading performances are...
Biodegradation of alkanes by microbial communities is ubiquitous in nature. Interestingly, the microbial communities with high hydrocarbon-degrading performances are sometimes composed of not only hydrocarbon degraders but also nonconsumers, but the synergistic mechanisms remain unknown. Here, we found that two bacterial strains isolated from Chinese oil fields, sp. strain DQ12-45-1b and SLG510A3-8, had a synergistic effect on hexadecane (C compound) biodegradation, even though could not utilize C individually. To gain a better understanding of the roles of the alkane nonconsumer in the C-degrading consortium, we reconstructed a two-species stoichiometric metabolic model, BH1908, and integrated prediction with the following validation, a comparative proteomics analysis, and extracellular metabolomic detection. Metabolic interactions between and sp. were successfully revealed to have importance in efficient C degradation. In the process, survived on C metabolic intermediates from sp., including hexadecanoate, 3-hydroxybutanoate, and α-ketoglutarate. In return, reorganized its metabolic flux distribution to fed back acetate and glutamate to sp. to enhance its C degradation efficiency by improving cell accumulation and by regulating the expression of succinate dehydrogenase. By using the synergistic microbial consortium of sp. and with the addition of the -predicted key exchanged metabolites, diesel oil was effectively disposed of in 15 days with a removal fraction of 85.54% ± 6.42%, leaving small amounts of C to C isomers. Our finding provides a novel microbial assembling mode for efficient bioremediation or chemical production in the future. Many natural and synthetic microbial communities are composed of not only species whose biological properties are consistent with their corresponding communities but also ones whose chemophysical characteristics do not directly contribute to the performance of their communities. Even though the latter species are often essential to the microbial communities, their roles are unclear. Here, by investigation of an artificial two-member microbial consortium in -alkane biodegradation, we showed that the microbial member without the -alkane-degrading capability had a cross-feeding interaction with and metabolic regulation to the leading member for the synergistic -alkane biodegradation. Our study improves the current understanding of microbial interactions. Because "assistant" microbes showed importance in communities in addition to the functional microbes, our findings also suggest a useful "assistant-microbe" principle in the design of microbial communities for either bioremediation or chemical production.
Topics: Actinomycetales; China; Microbial Consortia; Oil and Gas Fields; Pseudomonas stutzeri
PubMed: 32033953
DOI: 10.1128/AEM.02931-19 -
Journal of Environmental Health Science... Dec 2022A consortium of bacteria capable of decomposing oily hydrocarbons was isolated from tarballs on the beaches of Terengganu, Malaysia, and classified as , , and . The...
UNLABELLED
A consortium of bacteria capable of decomposing oily hydrocarbons was isolated from tarballs on the beaches of Terengganu, Malaysia, and classified as , , and . The Taguchi design was used to optimize the biodegradation of diesel using these bacteria as a consortium. The highest biodegradation of diesel-oil in the experimental tests was 93.6%, and the individual n-alkanes decomposed 87.6-97.6% over 30 days. Optimal settings were inoculum size of 2.5 mL (1.248 OD); 12% (v/v) the initial diesel-oil in a minimal salt medium of pH 7.0, 30.0 gL NaCl and 2.0 gL NHNO concentration, incubated at 42 °C temperature and 150 rpm agitation speed. Parameters significantly improved diesel-oil removal by consortium as shown by the model determination coefficient (R = 90.89%; < 0.001) with a synergistic effect of agitation speed significantly contributing 81.03%. Taguchi design determined the optimal settings for the parameters under study, which significantly improved diesel-oil removal by consortium. This can be used to design a novel bioremediation strategy that can achieve optimal decontamination of oil pollution in a shorter time.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s40201-022-00812-3.
PubMed: 36406595
DOI: 10.1007/s40201-022-00812-3