Nitrogen regulation in Corynebacterium glutamicum: isolation of genes involved and biochemical characterization of corresponding proteins

The regulation of nitrogen assimilation was investigated in the Gram-positive actinomycete Corynebacterium glutamicum. Biochemical studies and site-directed mutagenesis revealed that glutamine synthetase activity is regulated via adenylylation in this organism. The genes encoding the central signal transduction protein PH (glnB) and the primary nitrogen sensor uridylyltransferase (glnD) were isolated and sequenced. Additionally, genes putatively involved in the degradation of ornithine (ocd) and sarcosine (soxA), ammonium uptake (amtP) and protein secretion (ftsY, srp) were identified in C. glutamicum. Based on these observations, the mechanism of N regulation in C. glutamicum is similar to that of the Gram-negative Escherichia coli. As deduced from data base searches, the described regulation may also hold true for the important pathogen Mycobacterium glutamicum.     

Effect of lysine succinylation on the regulation of 2-oxoglutarate dehydrogenase inhibitor,OdhI, involved in glutamate production in Corynebacterium glutamicum

In Corynebacterium glutamicum, the activity of the 2-oxoglutarate dehydrogenase (ODH) complex is negatively regulated by the unphosphorylated form of OdhI protein, which is critical for L-glutamate overproduction. We examined the potential impact of protein acylation at lysine (K)-132 of OdhI in C. glutamicum ATCC13032. The K132E succinylation-mimic mutation reduced the ability of OdhI to bind OdhA, the catalytic subunit of the ODH complex, which reduced the inhibition of ODH activity. In vitro succinylation of OdhI protein also reduced the ability to inhibit ODH, and the K132R mutation blocked the effect. These results suggest that succinylation at K132 may attenuate the OdhI function. Consistent with these results, the C. glutamicum mutant strain with OdhI-K132E showed decreased L-glutamate production. Our results indicated that not only phosphorylation but also succinylation of OdhI protein may regulate L-glutamate production in C. glutamicum.     

Aspartokinase genes lysC alpha and lysC beta overlap and are adjacent to the aspartate beta-semialdehyde dehydrogenase gene asd in Corynebacterium glutamicum

Summary A 2.1 kb DNA fragment of the recombinant plasmid pCS2, isolated from an aminoethyl cysteine (AEC)-resistant and lysine-producing Corynebacterium glutamicum mutant strain, and which confers AEC resistance and lysine production on the wild-type G. glutamicum ATCC 13032 was analysed. DNA sequence analysis of this fragment revealed three large open reading frames (ORFs). The incomplete ORF1 does not contain the 5 end of the coding region. ORF2, which uses the same reading frame as ORF1, is identical to the 3 end of ORF1 and encodes a putative protein of 172 amino acids (aa) and of M_r 18 584. ORF3 encodes a putative protein of 344 as and of M_r 36275. The amino acid sequences deduced from ORF1 and ORF2 display strong homologies to those of the - and -subunits of the Bacillus subtilis aspartokinase II. It is therefore proposed that the incomplete ORF1, termed lysC, encodes part of the -subunit of the C. glutamicum aspartokinase whereas the complete ORF2, termed lysC, encodes the -subunit of the same enzyme. ORF2 is responsible for AEC resistance and lysine production due to a feedback-resistant aspartokinase. The amino acid sequence deduced from ORF3, termed asd, is highly homologous to that of the Streptococcus mutans aspartate -semialdehyde dehydrogenase (ASD). Plasmids carrying the C. glutamicum asd gene complemented Escherichia coli asd mutants. Increase in ASD activity by a factor of 30–60 was measured for C. glutamicum cells harbouring high copy-number plasmids with the C. glutamicum asd gene.     

The tetAB genes of the Corynebacterium striatum R-plasmid pTP10 encode an ABC transporter and confer tetracycline, oxytetracycline and oxacillin resistance in Corynebacterium glutamicum

The tetracycline resistance region of the 50-kb R-plasmid pTP10 from the clinical isolate Corynebacterium striatum M82B was analyzed in Corynebacterium glutamicum ATCC 13032 and confined to a 4.4-kb SphI-Sa/I DNA fragment. Nucleotide sequence analysis revealed two open reading frames, termed tetA and tetB, specifying proteins of 513 and 528 amino acids, respectively. The deduced amino acid sequences of tetAB displayed similarity to ATP-binding cassette transporters including StrV and StrW of Streptomyces glaucescens which are proposed to play a role in the export of streptomycin-like aminoglycosides. An antibiotic susceptibility screening in C. glutamicum showed that the tetAB genes confer resistance to tetracycline, oxytetracycline and to the structurally and functionally unrelated beta-lactam antibiotic oxacillin.     

Inactivation of the phosphoglucomutase gene pgm in Corynebacterium glutamicum affects cell shape and glycogen metabolism

In Corynebacterium glutamicum formation of glc-1-P (α-glucose-1-phosphate) from glc-6-P (glucose-6-phosphate) by α-Pgm (phosphoglucomutase) is supposed to be crucial for synthesis of glycogen and the cell wall precursors trehalose and rhamnose. Furthermore, Pgm is probably necessary for glycogen degradation and maltose utilization as glucan phosphorylases of both pathways form glc-1-P. We here show that C. glutamicum possesses at least two Pgm isoenzymes, the cg2800 (pgm) encoded enzyme contributing most to total Pgm activity. By inactivation of pgm we created C. glutamicum IMpgm showing only about 12% Pgm activity when compared to the parental strain. We characterized both strains during cultivation with either glucose or maltose as substrate and observed that (i) the glc-1-P content in the WT (wild-type) and the mutant remained constant independent of the carbon source used, (ii) the glycogen levels in the pgm mutant were lower during growth on glucose and higher during growth on maltose, and (iii) the morphology of the mutant was altered with maltose as a substrate. We conclude that C. glutamicum employs glycogen as carbon capacitor to perform glc-1-P homeostasis in the exponential growth phase and is therefore able to counteract limited Pgm activity for both anabolic and catabolic metabolic pathways.     

The cytosolic, cell surface and extracellular proteomes of the biotechnologically important soil bacterium Corynebacterium efficiens YS-314 in comparison to those of Corynebacterium glutamicum ATCC 13032

Reference maps of the cytosolic, cell surface and extracellular proteome fractions of the amino acid-producing soil bacterium Corynebacterium efficiens YS-314 were established. The analysis window covers a pI range from 3 to 7 along with a molecular mass range from 10 to 130 kDa. After second-dimensional separation on SDS-PAGE and Coomassie staining, computational analysis detected 635 protein spots in the cytosolic proteome fraction, whereas 76 and 102 spots were detected in the cell surface and extracellular proteomes, respectively. By means of MALDI-TOF-MS and tryptic peptide mass fingerprinting, 164 cytosolic proteins, 49 proteins of the cell surface and 89 extracellular protein spots were identified, representing in total 177 different proteins. Additionally, reference maps of the three cellular proteome fractions of the close phylogenetic relative Corynebacterium glutamicum ATCC 13032 were generated and used for comparative proteomics. Classification according to the Clusters of Orthologous Groups of proteins scheme and abundance analysis of the identified proteins revealed species-specific differences. The high abundance of molecular chaperones and amino acid biosynthesis enzymes in C. efficiens points to environmental adaptations of this recently discovered amino acid-producing bacterium.     

Investigation of central carbon metabolism and the 2-methylcitrate cycle in Corynebacterium glutamicum by metabolic profiling using gas chromatography-mass spectrometry

The 2-methylcitrate cycle as the primary way to metabolize propionate was investigated using metabolic profiling. For this purpose, a fast harvesting procedure was applied in which cells growing in liquid minimal medium were harvested by a short centrifugation and freeze-dried. Subsequently, gas chromatography–mass spectrometry of polar extracts derivatized by MSTFA was employed for metabolite characterization. Routinely more than 300 different peaks were obtained in the chromatograms, and 74 substances were identified unequivocally by using pure standards. The procedure provided reliable data which closely relate to prior knowledge on flux distributions during growth on glucose and acetate as carbon sources.

Propionate degradation via the 2-methylcitrate cycle was demonstrated on the metabolite level by the detection of the intermediates 2-methylcitrate and 2-methylisocitrate. Further characterization of the 2-methylcitrate cycle was carried out by comparing different mutant strains of this pathway. The growth deficit of a prpD2-mutant strain observed when propionate is added to a culture growing on acetate indicates that the toxic effect of propionate is based on the accumulation of 2-methylcitrate. It could also be shown that the 2-methylcitrate cycle is active in the absence of propionate and might fulfill house-keeping functions in the degradation of fatty acids or branched-chain amino acids.     

Development of food-grade expression system for d-allulose 3-epimerase preparation with tandem isoenzyme genes in Corynebacterium glutamicum and its application in conversion of cane molasses to D-allulose

D -Allulose 3-epimerase (DAE) has been applied to produce D -allulose, a low-calorie and functional sweetener. In this study, a new DAE from Paenibacillus senegalensis was characterized in Escherichia coli. Furthermore, we presented a tandem isoenzyme gene expression strategy to express multiple DAEs in one cell and construct food-grade expression systems based on Corynebacterium glutamicum. Seventeen expression cassettes based on three DAE genes from different organisms were constructed. Among all recombinant strains, DAE16 harboring three DAE genes in an expression vector exhibited the highest enzyme activity with 22.7 U/mg. Whole-cell transformation of DAE16 produced 225 g/L D -allulose with a volumetric productivity of 353 g·g ⁻¹·hr ⁻¹. The catalytic efficiency of strain C-DAE9 integrating total 11 DAE genes in chromosome was 16.4-fold higher than strains carrying one DAE. Fed-batch culture of C-DAE9 gave enzyme activity of 44,700 U/L. We also expressed a thermostable invertase in C. glutamicum and obtained enzyme activity of 29 U/mg. Immobilized cells expressing DAE or invertase exhibited 80% of retained activity after 30 cycles of catalytic reactions. Those immobilized cells were coupled to produce 61.2 g/L D -allulose from cane molasses in a two-step reaction process. This study provided an efficient approach for enzyme preparation and allowed access to produce D -allulose from other abundant and low-cost feedstock enriched with sucrose.     

The Corynebacterium glutamicum gene pmt encoding a glycosyltransferase related to eukaryotic protein-O-mannosyltransferases is essential for glycosylation of the resuscitation promoting factor (Rpf2) and other secreted proteins

Two-dimensional gel electrophoresis and immunoassays revealed several proteins of the secretory subproteome of Corynebacterium glutamicum to be glycosylated. By genome-wide searches for genes involved in glycosylation, the C. glutamicum gene cg1014 was found to exhibit significant similarity to eukaryotic protein-O-mannosyltransferases (PMTs) and to a recently identified orthologue of Mycobacterium tuberculosis, Rv1002c, which is responsible for protein-O-mannosylation. The putative membrane protein Cg1014 showed the same predicted transmembrane topology as Saccharomyces cerevisiae PMT1 and M. tuberculosis Rv1002c along with conserved amino acid residues responsible for catalytic activity. Deletion of the C. glutamicum pmt gene (cg1014) caused a complete loss of glycosylation of secreted proteins including the resuscitation promoting factor 2 (Rpf2), which is involved in intercellular communication and growth stimulation of C. glutamicum. Because the gene pmt as well as rpf genes are present in the genomes of all actinobacteria sequenced so far, this work provides new insights into bacterial protein glycosylation and new opportunities to elucidate the molecular mechanisms of Rpf activity in pathogenic growth and infection.     

The anaerobic fungusPiromyces sp. strain E2: nitrogen requirement and enzymes involved in primary nitrogen metabolism

The anaerobic fungusPiromyces sp. strain E2 appeared restricted in nitrogen utilization. Growth was only supported by ammonium as source of nitrogen. Glutamine also resulted in growth, but this was due to release of ammonia rather than to uptake and utilization of the amino acid. The fungus was not able to grow on other amino acids, albumin, urea, allantoin, or nitrate. Assimilation of ammonium is very likely to be mediated by NADP-linked glutamate dehydrogenase (NADP-GDH) and glutamine synthetase (GS). One transaminating activity, glutamate-oxaloacetate transaminase (GOT), was demonstrated. Glutamate synthase (GOGAT), NAD-dependent glutamate dehydrogenase (NAD-GDH), and the transaminating activity glutamate-pyruvate transaminase (GPT) were not detected in cell-free extracts ofPiromyces sp. strain E2. Specific enzyme activities of both NADP-GDH and GS increased four-to sixfold under nitrogen-limiting conditions.Abbreviations GDH Glutamate dehydrogenase - GOGAT Glutamate synthase - GOT Glutamate-oxaloacetate transaminase - GPT Glutamate-pyruvate transaminase - GS Glutamine synthetase     

Bioinformatics analysis of the genes involved in the extension of prostate cancer to adjacent lymph nodes by supervised and unsupervised machine learning methods: The role of SPAG1 and PLEKHF2

The present study aimed to identify the genes associated with the involvement of adjunct lymph nodes of patients with prostate cancer (PCa) and to provide valuable information for the identification of potential diagnostic biomarkers and pathological genes in PCa metastasis. The most important candidate genes were identified through several machine learning approaches including K-means clustering, neural network, Naïve Bayesian classifications and PCA with or without downsampling.In total, 21 genes associated with lymph nodes involvement were identified. Among them, nine genes have been identified in metastatic prostate cancer, six have been found in the other metastatic cancers and four in other local cancers. The amplification of the candidate genes was evaluated in the other PCa datasets. Besides, we identified a validated set of genes involved in the PCa metastasis. The amplification of SPAG1 and PLEKHF2 genes were associated with decreased survival in patients with PCa.     

The OSBP-related proteins: a novel protein family involved in vesicle transport,cellular lipid metabolism,and cell signalling

Proteins/genes showing high sequence homology to the mammalian oxysterol binding protein (OSBP) have been identified in a variety of eukaryotic organisms from yeast to man. The unifying feature of the gene products denoted as OSBP-related proteins (ORPs) is the presence of an OSBP-type ligand binding (LB) domain. The LB domains of OSBP and its closest homologue bind oxysterols, while data on certain other family members suggest interaction with phospholipids. Many ORPs also have a pleckstrin homology (PH) domain in the amino-terminal region. The PH domains of the family members studied in detail are known to interact with membrane phosphoinositides and play an important role in the intracellular targeting of the proteins. It is plausible that the ORPs constitute a regulatory apparatus that senses the status of specific lipid ligands in membranes, using the PH and/or LB domains, and mediates information to yet poorly known downstream machineries. Functional studies carried out on the ORP proteins in different organisms indicate roles of the gene family in diverse cellular processes including control of lipid metabolism, regulation of vesicle transport, and cell signalling events.     

The cps gene cluster of Salmonella strain LT2 includes a second mannose pathway: sequence of two genes and relationship to genes in the rfb gene cluster

Summary We report the presence in Salmonella enterica strain LT2 (serovar thyphimurium) of duplicate genes for two steps in the synthesis of GDP-mannose. The previously known genes, rfbK (phosphomannomutase) and rfbM (mannose-l-phosphate guanyltransferase), are part of the gene cluster for the O antigen. The two new genes, cpsB and cpsG, respectively, are thought to be part of the gene cluster for the M antigen capsular polysaccharide, present in many Enterobacteriaceae. The two genes have been sequenced and have a GC content of 0.61, suggesting an origin outside of Salmonella. Comparison of the inferred protein sequences for cpsB and rfbM shows 57% identity of amino acids whereas for cpsG and rfbK there is only 19% identity. It is suggested that the greater divergence between cpsG and rfbK may be due to a period of accelerated evolution, perhaps precipitated by transfer of the genes from another species.     

Use of vitamin D4 analogs to investigate differences in hepatic and target cell metabolism of vitamins D2 and D3

In this study, we used molecules with either of the structural differences in the side chains of vitamin D₂ and vitamin D₃ to investigate which feature is responsible for the significant differences in their respective metabolism, pharmacokinetics and toxicity. We used two cell model systems—HepG2 and HPK1A-ras—to study hepatic and target cell metabolism, respectively. Studies with HepG2 revealed that the pattern of 24- and 26-hydroxylation of the side chain reported for 1α-hydroxyvitamin D₂ (1α-OH-D₂) but not for 1α-OH-D₃ is also observed in both 1α-OH-D₄ and Δ²²-1α-OH-D₃ metabolism. This suggests that the structural feature responsible for targeting the enzyme to the C24 or C26 site could be either the C24 methyl group or the 22–23 double bond. In HPK1A-ras cells, the pattern of metabolism observed for the 24-methylated derivative, 1α,25-(OH)₂D₄, was the same pattern of multiple hydroxylations at C24, C26 and C28 seen for vitamin D₂ compounds without evidence of side chain cleavage observed for vitamin D₃ derivatives, suggesting that the C24 methyl group plays a major role in this difference in target cell metabolism of D₂ and D₃ compounds. Novel vitamin D₄ compounds were tested and found to be active in a variety of in vitro biological assays. We conclude that vitamin D₄ analogs and their metabolites offer valuable insights into vitamin D analog design, metabolic enzymes and maybe useful clinically.