Utilization Of Nitrogen Sources Research Articles

Metabolic reconstruction of the human pathogen Candida auris: using a cross-species approach for drug target prediction.

Candida auris is an emerging human pathogen, associated with antifungal drug resistance and hospital candidiasis outbreaks. In this work, we present iRV973, the first reconstructed Genome-scale metabolic model (GSMM) for C. auris. The model was manually curated and experimentally validated, being able to accurately predict the specific growth rate of C. auris and the utilization of several sole carbon and nitrogen sources. The model was compared to GSMMs available for other pathogenic Candida species and exploited as a platform for cross-species comparison, aiming the analysis of their metabolic features and the identification of potential new antifungal targets common to the most prevalent pathogenic Candida species. From a metabolic point of view, we were able to identify unique enzymes in C. auris in comparison with other Candida species, which may represent unique metabolic features. Additionally, 50 enzymes were identified as potential drug targets, given their essentiality in conditions mimicking human serum, common to all four different Candida models analysed. These enzymes represent interesting drug targets for antifungal therapy, some are already known targets of antifungal agents used in clinical practice, but other new potential drug targets also stand out without any human homolog or drug association in Candida species.

P323 Virulence not required? Albumin promotes pathogenicity of (non)-damaging Candida strains

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMThe pathogenicity of the dimorphic yeast Candida albicans is associated with filamentation, adhesion, invasion, and production of the toxin Candidalysin. However, there are certain clinical isolates and other Candida spp., that cause infection independent of filamentation or the production of Candidalysin. Consequently, these strains and species are often non-damaging in vitro, this does not correlate with their potential to cause infection in patients. We hypothesize that specific host factors, which trigger pathogenicity, are absent in in vitro models, and thereby not reflecting the situation in the host. To determine the impact of albumin, the most abundant protein in the human body, vaginal epithelial cells were infected with different C. albicans strains and Candida species. Interestingly, after prolonged infection (45 h) albumin increased the damage potential, even in otherwise non-damaging and non-filamentous strains. This included deletion mutants deficient in filamentation, als3 adhesin/invasin, thigmotropism, or Candidalysin production. Yet, the increased damage was likely not solely an effect of increased growth and nutrient competition between the fungus and epithelial cells. Reduced damage in presence of protease inhibitors and albumin hint toward the role of proteases in the utilization of albumin. Albumin enhanced C. albicans metabolism, by stimulating the utilization of various nitrogen sources. This metabolic adaption could explain the advantage and enhanced growth as a strain and species-independent feature. Our data suggest that common host factors can impact C. albicans to cause damage independent of adhesion, invasion, filamentation, and toxin production. Possibly, also other host-derived factors can drive the pathogenic potential of fungi through unresolved mechanisms.

Transcription Factor Mavib-1 Negatively Regulates Conidiation by Affecting Utilization of Carbon and Nitrogen Source in Metarhizium acridum.

Conidium is the main infection unit and reproductive unit of pathogenic fungi. Exploring the mechanism of conidiation and its regulation contributes to understanding the pathogenicity of pathogenic fungi. Vib-1, a transcription factor, was reported to participate in the conidiation process. However, the regulation mechanism of Vib-1 in conidiation is still unclear. In this study, we analyzed the function of Vib-1 and its regulation mechanism in conidiation through knocking out and overexpression of Vib-1 in entomopathogenic fungus Metarhizium acridum. Results showed that the colonial growth of Mavib-1 disruption mutant (ΔMavib-1) was significantly decreased, and conidiation was earlier compared to wild type (WT), while overexpression of Mavib-1 led to a delayed conidiation especially when carbon or nitrogen sources were insufficient. Overexpression of Mavib-1 resulted in a conidiation pattern shift from microcycle conidiation to normal conidiation on nutrient-limited medium. These results indicated that Mavib-1 acted as a positive regulator in vegetative growth and a negative regulator in conidiation by affecting utilization of carbon and nitrogen sources in M. acridum. Transcription profile analysis demonstrated that many genes related to carbon and nitrogen source metabolisms were differentially expressed in ΔMavib-1 and OE strains compared to WT. Moreover, Mavib-1 affects the conidial germination, tolerance to UV-B and heat stresses, cell wall integrity, conidial surface morphology and conidial hydrophobicity in M. acridum. These findings unravel the regulatory mechanism of Mavib-1 in fungal growth and conidiation, and enrich the knowledge to conidiation pattern shift of filamentous fungi.

Chromatin Regulators Ahc1p and Eaf3p Positively Influence Nitrogen Metabolism in Saccharomyces cerevisiae.

There is a complex regulatory network of nitrogen metabolism in Saccharomyces cerevisiae, and many details of this regulatory network have not been revealed. This study explored the global regulation of nitrogen metabolism in S. cerevisiae from an epigenetic perspective. Comparative transcriptome analysis of S. cerevisiae S288C treated with 30 nitrogen sources identified nine chromatin regulators (CRs) that responded significantly to different nitrogen sources. Functional analysis showed that among the CRs identified, Ahc1p and Eaf3p promoted the utilization of non-preferred nitrogen sources through global regulation of nitrogen metabolism. Ahc1p regulated nitrogen metabolism through amino acid transport, nitrogen catabolism repression (NCR), and the Ssy1p-Ptr3p-Ssy5p signaling sensor system. Eaf3p regulated nitrogen metabolism via amino acid transport and NCR. The regulatory mechanisms of the effects of Ahc1p and Eaf3p on nitrogen metabolism depended on the function of their histone acetyltransferase complex ADA and NuA4. These epigenetic findings provided new insights for a deeper understanding of the nitrogen metabolism regulatory network in S. cerevisiae.

A systems approach discovers the role and characteristics of seven LysR type transcription factors in Escherichia coli

Although Escherichia coli K-12 strains represent perhaps the best known model bacteria, we do not know the identity or functions of all of their transcription factors (TFs). It is now possible to systematically discover the physiological function of TFs in E. coli BW25113 using a set of synergistic methods; including ChIP-exo, growth phenotyping, conserved gene clustering, and transcriptome analysis. Among 47 LysR-type TFs (LTFs) found on the E. coli K-12 genome, many regulate nitrogen source utilization or amino acid metabolism. However, 19 LTFs remain unknown. In this study, we elucidated the regulation of seven of these 19 LTFs: YbdO, YbeF, YcaN, YbhD, YgfI, YiaU, YneJ. We show that: (1) YbdO (tentatively re-named CitR) regulation has an effect on bacterial growth at low pH with citrate supplementation. CitR is a repressor of the ybdNM operon and is implicated in the regulation of citrate lyase genes (citCDEFG); (2) YgfI (tentatively re-named DhfA) activates the dhaKLM operon that encodes the phosphotransferase system, DhfA is involved in formate, glycerol and dihydroxyacetone utilization; (3) YiaU (tentatively re-named LpsR) regulates the yiaT gene encoding an outer membrane protein, and waaPSBOJYZU operon is also important in determining cell density at the stationary phase and resistance to oxacillin microaerobically; (4) YneJ, re-named here as PtrR, directly regulates the expression of the succinate-semialdehyde dehydrogenase, Sad (also known as YneI), and is a predicted regulator of fnrS (a small RNA molecule). PtrR is important for bacterial growth in the presence of l-glutamate and putrescine as nitrogen/energy sources; and (5) YbhD and YcaN regulate adjacent y-genes on the genome. We have thus established the functions for four LTFs and identified the target genes for three LTFs.

Reconstruction and analysis of genome-scale metabolic model for thermophilic fungus Myceliophthora thermophila.

Myceliophthora thermophila, a thermophilic fungus that can degrade and utilize all major polysaccharides in plant biomass, has great potential in biotechnological industries. Here, the first manually curated genome-scale metabolic model iDL1450 for M. thermophila was reconstructed using an autogenerating pipeline with thorough manual curation. The model contains 1450 genes, 2592 reactions, and 1784 unique metabolites. High accuracy was shown in predictions related to carbon and nitrogen source utilization based on data obtained from Biolog experiments. Besides, metabolism profiles were analyzed using iDL1450 integrated with transcriptomics data of M. thermophila at various growth temperatures. The refined model provides new insights into thermophilic fungi metabolism and sheds light on model-driven strain design to improve biotechnological applications of this thermophilic lignocellulosic fungus.

A DasA family sugar binding protein Ste2 links nutrient and oxidative stress to exopolysaccharides production in Streptomyces sp. 139

BackgroundEbosin is an exopolysaccharide produced by Streptomyces sp. 139, and its biosynthetic gene cluster (ste) has been previously described. Ste234 has high homology to the well-known ATP-binding cassette transport system DasABC, which has been linked to the regulation of morphological differentiation, antibiotics biosynthesis and aminosugars utilization in Streptomycetes. This study was conducted to evaluate the effect of the DasA family sugar binding protein Ste2 on Streptomyces sp. 139.ResultsThe disruption of ste2 results in the upregulation of transcription of genes within Ebosin biosynthetic gene cluster and a two-fold increase in Ebosin production. RNA sequencing data suggests that the disruption of ste2 results in the decreased utilization of carbon and nitrogen sources, increased sensitivity to oxidative stress, as well as differed strain morphology, all of which have been experimentally proven.ConclusionsTaken together, Ste2 controls Ebosin yields, aminosugars uptake, sensitivity to oxidative stress, and morphological differentiation of Streptomyces sp. 139.

Gene co-expression network analysis reveals the positive impact of endocytosis and mitochondria-related genes over nitrogen metabolism in Saccharomyces cerevisiae

Nitrogen metabolism is essential for most cellular activities. Therefore, a deep understanding of its regulatory mechanisms is necessary for the efficient utilization of nitrogen sources for Saccharomyces cerevisiae. In this study, a gene co-expression network was constructed for S. cerevisiae S288C with different nitrogen sources. From this, a key gene co-expression module related to nitrogen source preference utilization was obtained, and 10 hub genes centrally located in the co-expression network were identified. Functional studies verified that the endocytosis-related genes CAP1 and END3 significantly increased the utilization of multiple non-preferred amino acids and reduced the accumulation of the harmful nitrogen metabolite precursor urea by regulating amino acid transporters and TOR pathway. The mitochondria-related gene ATP12, MRPL22, MRP1 and NAM9 significantly increased the utilization of multiple non-preferred amino acids and reduced accumulation of the urea by coordinately regulating nitrogen catabolism repression, Ssy1p-Ptr3p-Ssy5p signaling sensor system, amino acid transporters, TOR pathway and urea metabolism-related pathways. Furthermore, these data revealed the potential positive effects of endocytosis and mitochondrial ribosomes protein translation on nitrogen source preference. This study provides new analytical perspectives for complex regulatory networks involving nitrogen metabolism in S. cerevisiae.

Highly Stretchable Bacterial Cellulose Produced by Komagataeibacter hansenii SI1.

A new strain of bacteria producing cellulose was isolated from Kombucha and identified as Komagataeibacter hansenii, named SI1. In static conditions, the strain synthesises bacterial nanocellulose with an improved ability to stretch. In this study, utilisation of various carbon and nitrogen sources and the impact of initial pH was assessed in terms of bacterial nanocellulose yield and properties. K. hansenii SI1 produces cellulose efficiently in glycerol medium at pH 5.0–6.0 with a yield of 3.20–3.60 g/L. Glucose medium led to the synthesis of membrane characterised by a strain of 77%, which is a higher value than in the case of another Komagataeibacter species. Supplementation of medium with vitamin C results in an enhanced porosity and improves the ability of bacterial nanocellulose to stretch (up to 123%). The properties of modified membranes were studied by scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction and mechanical tests. The results show that bacterial nanocellulose produced in SH medium and vitamin C-supplemented medium has unique properties (porosity, tensile strength and strain) without changing the chemical composition of cellulose. The method of production BNC with altered properties was the issue of Polish patent application no. P.431265.

What makes Komagataella phaffii non-conventional?

ABSTRACTThe important industrial protein production host Komagataella phaffii (syn Pichia pastoris) is classified as a non-conventional yeast. But what exactly makes K. phaffii non-conventional? In this review, we set out to address the main differences to the ‘conventional’ yeast Saccharomyces cerevisiae, but also pinpoint differences to other non-conventional yeasts used in biotechnology. Apart from its methylotrophic lifestyle, K. phaffii is a Crabtree-negative yeast species. But even within the methylotrophs, K. phaffii possesses distinct regulatory features such as glycerol-repression of the methanol-utilization pathway or the lack of nitrate assimilation. Rewiring of the transcriptional networks regulating carbon (and nitrogen) source utilization clearly contributes to our understanding of genetic events occurring during evolution of yeast species. The mechanisms of mating-type switching and the triggers of morphogenic phenotypes represent further examples for how K. phaffii is distinguished from the model yeast S. cerevisiae. With respect to heterologous protein production, K. phaffii features high secretory capacity but secretes only low amounts of endogenous proteins. Different to S. cerevisiae, the Golgi apparatus of K. phaffii is stacked like in mammals. While it is tempting to speculate that Golgi architecture is correlated to the high secretion levels or the different N-glycan structures observed in K. phaffii, there is recent evidence against this. We conclude that K. phaffii is a yeast with unique features that has a lot of potential to explore both fundamental research questions and industrial applications.

MaNmrA, a Negative Transcription Regulator in Nitrogen Catabolite Repression Pathway, Contributes to Nutrient Utilization, Stress Resistance, and Virulence in Entomopathogenic Fungus Metarhizium acridum.

Simple SummaryNutrient metabolism is closely related to the growth, development, and pathogenicity of pathogenic fungi. The nitrogen catabolite repression (NCR) pathway is a well-known fungal nitrogen source regulation path, in which NmrA plays an important regulatory role. Here, we reported a negative regulatory protein MaNmrA, the NmrA homologous protein, in the entomopathogenic fungus Metarhizium acridum, and found that it played important roles in carbon and nitrogen metabolism, growth, stress tolerance, and virulence of M. acridum. Our work will provide a theoretical basis for further exploring the functions of NCR pathway related genes in entomopathogenic fungi.The NCR pathway plays an important regulatory role in the nitrogen metabolism of filamentous fungi. NmrA, a central negative regulatory protein in the NCR pathway and a key factor in sensing to the carbon metabolism, plays important roles in pathogenic fungal nutrition metabolism. In this study, we characterized the functions of MaNmrA in the insect pathogenic fungus M. acridum. Multiple sequence alignments found that the conserved domain (NAD/NADP binding domain) of MaNmrA was highly conservative with its homologues proteins. Deletion of MaNmrA improved the utilization of multiple carbon sources (such as glucose, mannose, sucrose, and trehalose) and non-preferred nitrogen sources (such as NaNO3 and urea), significantly delayed the conidial germination rate and reduced the conidial yield. The MaNmrA-disruption strain (ΔMaNmrA) significantly decreased tolerances to UV-B and heat-shock, and it also increased the sensitivity to the hypertonic substance sorbitol, oxygen stress substance H2O2, and cell wall destroyer calcofluor white, indicating that loss of MaNmrA affected cell wall integrity, tolerances to hypertonic and oxidative stress. Bioassays demonstrated that disruption of MaNmrA decreased the virulence in both topical inoculation and intrahemocoel injection tests. Further studies revealed that the appressorium formation, turgor pressure, and colonization in hemolymph were significantly reduced in the absence of MaNmrA. Our work will deepen the functional cognition of MaNmrA and make a contribution to the study of its homologous proteins.

Novel Glutamate-Putrescine Ligase Activity in Haloferax mediterranei: A New Function for glnA-2 Gene.

The genome of the halophilic archaea Haloferax mediterranei contains three ORFs that show homology with glutamine synthetase (GS) (glnA-1, glnA-2, and glnA-3). Previous studies have focused on the role of GlnA-1, suggesting that proteins GlnA-2 and GlnA-3 could play a different role to that of GS. Glutamine synthetase (EC 6.3.1.2) belongs to the class of ligases, including 20 subclasses of other different enzymes, such as aspartate–ammonia ligase (EC 6.3.1.1), glutamate–ethylamine ligase (EC 6.3.1.6), and glutamate–putrescine ligase (EC 6.3.1.11). The reaction catalyzed by glutamate–putrescine ligase is comparable to the reaction catalyzed by glutamine synthetase (GS). Both enzymes can bind a glutamate molecule to an amino group: ammonium (GS) or putrescine (glutamate–putrescine ligase). In addition, they present the characteristic catalytic domain of GS, showing significant similarities in their structure. Although these proteins are annotated as GS, the bioinformatics and experimental results obtained in this work indicate that the GlnA-2 protein (HFX_1688) is a glutamate–putrescine ligase, involved in polyamine catabolism. The most significant results are those related to glutamate–putrescine ligase’s activity and the analysis of the transcriptional and translational expression of the glnA-2 gene in the presence of different nitrogen sources. This work confirms a new metabolic pathway in the Archaea domain which extends the knowledge regarding the utilization of alternative nitrogen sources in this domain.

MaAreB, a GATA Transcription Factor, Is Involved in Nitrogen Source Utilization, Stress Tolerances and Virulence in Metarhizium acridum.

The nitrogen catabolite repression (NCR) pathway is involved in nitrogen utilization, in which the global GATA transcription factor AreA plays an indispensable role and has been reported in many fungi. However, relatively few studies are focused on AreB, another GATA transcription factor in the NCR pathway and the functions of AreB are largely unknown in entomopathogenic fungi. Here, we characterized MaAreB in the model entomopathogenic fungus Metarhizium acridum. Sequence arrangement found that MaAreB had a conserved GATA zinc finger DNA binding domain and a leucine zipper domain. Disruption of MaAreB affected the nitrogen utilization and led to decelerated conidial germination and hyphal growth, decreased conidial yield, and lower tolerances to UV-B irradiation and heat-shock. Furthermore, the MaAreB mutant (ΔMaAreB) exhibited increased sensitivity to CFW (Calcofluor white), decreased cell wall contents (chitin and β-1,3-glucan) and reduced expression levels of some genes related to cell wall integrity, indicating that disruption of MaAreB affected the cell wall integrity. Bioassays showed that the virulence of the ΔMaAreB strain was decreased in topical inoculation but not in intra-hemocoel injection. Consistently, deletion of MaAreB severely impaired the appressorium formation and reduced the turgor pressure of appressorium. These results revealed that MaAreB regulated fungal nitrogen utilization, cell wall integrity and biological control potential, which would contribute to the functional characterization of AreB homologous proteins in other insect fungal pathogens, and even filamentous fungi.

Biochemical Characterizations of Selected Indigenous Endophytic Bacteria Potential as Growth Promoters and Biocontrol Agents on Tomato

Nine indigenous endophytic bacteria strains showing the best ability to promote growth and control tomato pathogens had been screened in our previous research. The strains’ biochemical properties, such as nutrition and other traits, must be characterized to design the best formulations for the strains’ biochemical properties. This study aimed to describe the biochemical characteristics of the selected indigenous endophytic bacteria. The variables observed were utilization of carbon sources (glucose, fructose, sucrose, lactose, glycerol, and olive oil), nitrogen sources (peptone, yeast extract, urea, NH4Cl, NH4SO4, and NH4NO3), and citrate, hydrolytic activities (urease, triple sugar iron, starch hydrolysis, gelatin hydrolysis, chitinase, cellulase, protease, lipase, and catalase), oxidative/fermentative assay, salt tolerance, and growth ability at 4° and 44°C. This study showed that all the endophytic bacteria strains characterized had various biochemical characteristics. All strains showed the different ability to utilize nitrogen and carbon source. Some strains survived to grow at 4°C except Bacillus cereus AGBE 1.2 TL. All strains tolerate growth in 4% NaCl concentrations, while some strains can tolerate up to 6%. This result can be used for further studies to develop the most suitable formulations for each strain to get the best results of the growth-promoting and biocontrol activities of the indigenous endophytic bacteria strains.

Detection of Capsular Polysaccharide Virulence Genes rmpA and magA of Klebsiella Pneumonia Isolate from Diabetic Foot Ulcer Patient in Najaf Governorate in Iraq

Klebsiella Pneumoniae is an opportunistic pathogen, which mostly cause nosocomial infections. K.Pneumoniae utilizes a variety of virulence factors, especially capsule polysaccharide, lipopolysaccharide,fimbriae, outer membrane proteins and determinants for iron acquisition and nitrogen source utilization,for survival and immune evasion during infection. A total of (36) clinical swab specimens isolated frompatient with diabetic foot ulcer in Al-Sdder Medical City (Central Diabetic Foot) in Najaf Governorate,Iraq from November 2019 to February 2020 , for detection some virulence capsular polysaccharide genessuch as rmpA and magA that related with K1 serotype of K . pneumonia and associated with antibioticresistance pattern in patient of diabetic foot ulcer. Antibiotic susceptibility test of K. Pneumoniae isolatesagainst (20) of commonly used antibiotic showed there 25 (69%) of K. pneumonia isolate from diabeticfoot patient were Multi-drug resistance (MDR),while11(31%) were extensive-drug resistance (XDR). Theresults of amplified product demonstrated that K1 serotype of K . Pneumonia was positive in 23 (64%)of totally clinical specimen than other types of serotype ( Non-K1 serotype). (magA) gene that associatedwith hypermucoid viscosity was positive in 21 (58%) of total specimens while (rmpA) gene that regulate ofcapsular polysaccharide of K .pneumonia was positive in 22 (62%) of totally samples

DNA affinity purification sequencing and transcriptional profiling reveal new aspects of nitrogen regulation in a filamentous fungus

Sensing available nutrients and efficiently utilizing them is a challenge common to all organisms. The model filamentous fungus Neurospora crassa is capable of utilizing a variety of inorganic and organic nitrogen sources. Nitrogen utilization in N. crassa is regulated by a network of pathway-specific transcription factors that activate genes necessary to utilize specific nitrogen sources in combination with nitrogen catabolite repression regulatory proteins. We identified an uncharacterized pathway-specific transcription factor, amn-1, that is required for utilization of the nonpreferred nitrogen sources proline, branched-chain amino acids, and aromatic amino acids. AMN-1 also plays a role in regulating genes involved in responding to the simple sugar mannose, suggesting an integration of nitrogen and carbon metabolism. The utilization of nonpreferred nitrogen sources, which require metabolic processing before being used as a nitrogen source, is also regulated by the nitrogen catabolite regulator NIT-2. Using RNA sequencing combined with DNA affinity purification sequencing, we performed a survey of the role of NIT-2 and the pathway-specific transcription factors NIT-4 and AMN-1 in directly regulating genes involved in nitrogen utilization. Although previous studies suggested promoter binding by both a pathway-specific transcription factor and NIT-2 may be necessary for activation of nitrogen-responsive genes, our data show that pathway-specific transcription factors regulate genes involved in the catabolism of specific nitrogen sources, while NIT-2 regulates genes involved in utilization of all nonpreferred nitrogen sources, such as nitrogen transporters. Together, these transcription factors form a nutrient sensing network that allows N. crassa cells to regulate nitrogen utilization.

Transcriptome analyses reveal the utilization of nitrogen sources and related metabolic mechanisms of Sporosarcina pasteurii.

In recent years, Sporosarcina pasteurii (S. pasteurii) has become one of the most popular bacteria in microbially induced calcium carbonate precipitation (MICP). Various applications have been developed based on the efficient urease that can induce the precipitation of calcium carbonate. However, the metabolic mechanism related to biomineralization of S. pasteurii has not been clearly elucidated. The process of bacterial culture and biomineralization consumes a large amount of urea or ammonium salts, which are usually used as agricultural fertilizers, not to mention probable environmental pollutions caused by the excessive use of these raw materials. Therefore, it is urgent to reveal the mechanism of nitrogen utilization and metabolism of S. pasteurii. In this paper, we compared the growth and gene expression of S. pasteurii under three different culture conditions through transcriptome analyses. GO and KEGG analyses revealed that both ammonium and urea were direct nitrogen sources of S. pasteurii, and the bacteria could not grow normally in the absence of ammonium or urea. To the best of our knowledge, this paper is the first one to reveal the nitrogen utilization mechanism of S. pasteurii through transcriptome methods. Furthermore, the presence of ammonium might promote the synthesis of intracellular ATP and enhance the motility of the bacteria. There should be an ATP synthesis mechanism associated with urea hydrolysis catalyzed by urease in S. pasteurii.

A highly efficient protein degradation system in Bacillus sp. CN2: a functional-degradomics study.

A novel protease-producing Bacillus sp. CN2 isolated from chicken manure composts exhibited a relatively high proteolytic specific activity. The strain CN2 degradome consisted of at least 149 proteases and homolog candidates, which were distributed into 4 aspartic, 30 cysteine, 55 metallo, 56 serine, and 4 threonine proteases. Extracellular proteolytic activity was almost completely inhibited by PMSF (phenylmethylsulfonyl fluoride) rather than o-P, E-64, or pepstatin A, suggesting that strain CN2 primarily secreted serine protease. More importantly, analysis of the extracellular proteome of strain CN2 revealed the presence of a highly efficient protein degradation system. Three serine proteases of the S8 family with different active site architectures firstly fragmented protein substrates which were then degraded to smaller peptides by a M4 metalloendopeptidase that prefers to degrade hydrophobic peptides and by a S13 carboxypeptidase. Those enzymes acted synergistically to degrade intact substrate proteins outside the cell. Furthermore, highly expressed sequence-specific intracellular aminopeptidases from multiple families (M20, M29, and M42) accurately degraded peptides into oligopeptides or amino acids, thus realizing the rapid acquisition and utilization of nitrogen sources. In this paper, a systematic study of the functional-degradome provided a new perspective for understanding the complexity of the protease hydrolysis system of Bacillus, and laid a solid foundation for further studying the precise degradation of proteins with the cooperative action of different family proteases. KEY POINTS: • Bacillus sp. CN2 has relatively high proteolytic specific activity. • Bacillus sp. CN2 harbors a highly efficient protein degradation system. • The site-specific endopeptidases were secreted extracellular, while the sequence-specific aminopeptidases played a role in the cell.

Identification and bio-control activity of Streptomyces strain (Koyanogensis) against Magnaporthe grisea

Rice blast is a major threat to the rice crop in rice-growing areas around the globe. However, synthetic fungicides are injurious for the ecosystem and the existing bio-control agents facing the resistance from the pathogens. In this study, an isolated novel Streptomyces strain (TA-47), from the rice field was identified by culturing on different media, physiological tests, biochemical tests, chemo-taxonomy and molecular biological approaches. In-vitro and in-vivo bio-control activity of the isolated strain was evaluated. Growth on the media, spore production, utilization of sole carbon and nitrogen sources, presence of diaminopimelic acid (DAP) in the cell wall of the strain and fatty acid production were observed.16S rRNA (gene sequence) and a neighbour-joining hood tree indicated that the strain TA-47 is Streptomyces koyanogensis. In-vitro inhibition zone of 29 mm against the M. grisea showed the antagonistic potential of the novel strain TA-47. On the other hand, in the in-vivo studies, strain TA-47, showed the strong bio-control effect, as disease severity decreased to 19.66 ± 1.6%, while in the non-treated sample the disease severity was recorded as 44.66 ± 5.249%. Moreover, after the treatment of the strain, effects on the growth, such as increased height and root length by 72.22 and 16.54 cm, respectively, were also recorded. In conclusion, rhizosphere Streptomyces TA-47, was a novel Streptomyces koyanogensis strain. The study found that the strain had strong bio-control and growth-promoting abilities.

Metabolic network of ammonium in cereal vinegar solid-state fermentation and its response to acid stress

Shanxi aged vinegar (SAV), a Chinese traditional vinegar, is produced by various microorganisms. Ammonium is an important nitrogen source for microorganisms and a key intermediate for the utilization of non-ammonium nitrogen sources. In this work, an ammonium metabolic network during SAV fermentation was constructed through the meta-transcriptomic analysis of in situ samples, and the potential mechanism of acid affecting ammonium metabolism was revealed. The results showed that ammonium was enriched as the acidity increased. Meta-transcriptomic analysis showed that the conversion of glutamine to ammonia is the key pathway of ammonium metabolism in vinegar and that Lactobacillus and Acetobacter are the dominant genera. The construction and analysis of the metabolic network showed that amino acid metabolism, nucleic acid metabolism, pentose phosphate pathway and energy metabolism were enhanced to resist acid damage to the intracellular environment and cell structures. The enhancement of nitrogen assimilation provides nitrogen for metabolic pathways that resist acid cytotoxicity. In addition, the concentration gradient allows ammonium to diffuse outside the cell, which causes ammonium to accumulate during fermentation.