glpD Gene Research Articles

Molecular Surveillance of Yersinia pestis from Stray Dogs and Cats and their Fleas in Algiers.

In recent years, plague has re-emerged in several countries around the world and remains endemic in some regions. In a natural environment and in contact with rodents and their fleas, stray carnivores are most at risk of catching the disease and maintaining the spread of the bacillus. The present study aimed to demonstrate the presence or absence of Yersinia pestis in stray dogs and cats in the Algiers region by molecular methods and thus determine their role in epidemiology of this disease. Molecular research of Yersinia pestis has also been conducted on fleas from these carnivores. Preliminary identification of ectoparasites to genus and species level was performed. Real-time polymerase chain reaction targeting Yersinia pestis pla gene was used to survey the plague agent in fleas and carnivores captured as stray animals in Algiers (Algeria). Positive qPCR results were tested by PCR sequencing using glpD gene. Among 327 fleas captured from 107 dogs and 365 fleas from 140 cats, prevalence of Ctenocephalides felis was higher in cats (86,96%), whereas that of Ctenocephalides canis and Xenopsylla cheopis were higher in dogs (90,57% and 92,63%, respectively). While internal and external PCR positive controls were positive, none of the 107 dogs spleens and 140 cats spleens and none of the 256 analyzed fleas were positive for Y. pestis. These results suggested that stray cats and dogs are unlikely sources of plague in Algeria, contrary to what has been reported in other plague-endemic countries. This observation illustrates that the plague epidemiological chain varies from one region to another.

Development of a plasmid stabilization system in Vibrio natriegens for the high production of 1,3-propanediol and 3-hydroxypropionate

Vibrio natriegens is a promising industrial chassis with a super-fast growth rate and high substrate uptake rates. V. natriegens was previously engineered to produce 1,3-propanediol (1,3-PDO) from glycerol by overexpressing the corresponding genes in a plasmid. However, antibiotic selection pressure for plasmid stability was not satisfactory and plasmid loss resulted in reduced productivity of the bioprocess. In this study, we developed an antibiotic-free plasmid stabilization system for V. natriegens. The system was achieved by shifting the glpD gene, one of the essential genes for glycerol degradation, from the chromosome to plasmid. With this system, engineered V. natriegens can stably maintain a large expression plasmid during the whole fed-batch fermentation and accumulated 69.5 g/L 1,3-PDO in 24 h, which was 23% higher than that based on antibiotic selection system. This system was also applied to engineering V. natriegens for the production of 3-hydroxypropionate (3-HP), enabling the engineered strain to accumulate 64.5 g/L 3-HP in 24 h, which was 30% higher than that based on antibiotic system. Overall, the developed strategy could be useful for engineering V. natriegens as a platform for the production of value-added chemicals from glycerol.Graphic

Identification of the key functional genes in salt-stress tolerance of Cyanobacterium Phormidium tenue using in silico analysis.

The online version contains supplementary material available at 10.1007/s13205-021-03050-w.

Cofactor Regeneration Using Permeabilized Escherichia coli Expressing NAD(P)+-Dependent Glycerol-3-Phosphate Dehydrogenase.

Oxidoreductases are effective biocatalysts, but their practical use is limited by the need for large quantities of NAD(P)H. In this study, a whole-cell biocatalyst for NAD(P)H cofactor regeneration was developed using the economical substrate glycerol. This cofactor regeneration system employs permeabilized Escherichia coli cells in which the glpD and gldA genes were deleted and the gpsA gene, which encodes NAD(P)+-dependent glycerol-3-phosphate dehydrogenase, was overexpressed. These manipulations were applied to block a side reaction (i.e., the conversion of glycerol to dihydroxyacetone) and to switch the glpD-encoding enzyme reaction to a gpsA-encoding enzyme reaction that generates both NADH and NADPH. We demonstrated the performance of the cofactor regeneration system using a lactate dehydrogenase reaction as a coupling reaction model. The developed biocatalyst involves an economical substrate, bifunctional regeneration of NAD(P)H, and simple reaction conditions as well as a stable environment for enzymes, and is thus applicable to a variety of oxidoreductase reactions requiring NAD(P)H regeneration.

Molecular-Genetic and Phenotypic Peculiarities of Plague Agent Strains Isolated in Vietnam

Objective of the study is to investigate phenotypic and molecular-genetic features and perform whole genome sequencing of Y. pestis strains isolated in Vietnam. Materials and methods. Studied were phenotypic and genotypic peculiarities of 20 plague agent strains isolated in different prefectures of Vietnam. Carried out was SNP-analysis of the strains, sequenced were genomes of 8 Y. pestis strains. Results and conclusions. Based on the results of studies of differential biochemical characteristics all the investigated strains were attributed to oriental biovar of the main subspecies of plague agent, which was confirmed by the presence of marker indel mutation – deletion of 93 bps in glpD gene. Investigated was also the plasmid composition of the strains. On the basis of the conducted genome sequencing and SNP-analysis appurtenance of 19 out of 20 strains under examination was determined. They belong to 1.ORI2v phylogenetic branch, relative to the strains isolated in Yunnan Province, China, which points to their common origin. Identified was a marker SNP and developed the method of SNP-typing for 1.ORI2v strains from Vietnam.

Improving β-carotene production in Escherichia coli by metabolic engineering of glycerol utilization pathway

Glycerol is a byproduct during biodiesel production. It is an important feedstock for fermentation due to its low price and high reduced status. Multiple genes of the glycerol utilization pathway were modulated in a previously engineered high β-carotene producing Escherichia coli strain CAR015 to enhance glycerol utilization capability for improving isoprenoids production. The glpR gene, encoding glycerol 3-phosphate repressor, was firstly deleted. The glpFK, glpD and tpiA genes were then modulated by three artificial regulatory parts, M1-37, M1-46 and M1-93, respectively. β-carotene titer reached 64.82 mg/L after modulating glpD with M1-46, which was 4.86 times higher than that of CAR015, and glycerol consumption rate also increased 100%. Modulating tpiA led to a little increase of β-carotene titer, whereas modulating glpFK led to a little decrease of β-carotene titer. This demonstrated that GlpD was a rate-limiting step in glycerol utilization pathway. Q-PCR of glpF, glpK, glpD and tpiA results showed that decrease the transcription level of glpF, glpK, glpD, or decrease the transcription level of tpiA could increase the cell growth and β-carotene production, probably for the decrease of methylglyoxal toxicity. Modulating glpD and tpiA genes in combination resulted in the best strain Gly003, which produced 72.45 mg/L β-carotene with a yield of 18.65 mg/g dry cell weight. The titer was 5.23 and yield 1.99 times of that of the parent strain CAR015. Our work suggested that appropriate activation of glpD and tpiA genes in glycerol utilization pathway could effectively improve β-carotene production. This strategy can be used for production of other terpenoids in E. coli.

The glpD gene is a novel reporter gene for E. coli that is superior to established reporter genes like lacZ and gusA

Reporter genes facilitate the characterization of promoter activities, transcript stabilities, translational efficiencies, or intracellular localization. Various reporter genes for Escherichia coli have been established, however, most of them have drawbacks like transcript instability or the inability to be used in genetic selections. Therefore, the glpD gene encoding glycerol-3-phosphate dehydrogenase was introduced as a novel reporter gene for E. coli. The enzymatic assay was optimized, and it was verified that growth on glycerol strictly depends on the presence of GlpD. The 5′-UTRs of three E. coli genes were chosen and cloned upstream of the new reporter gene glpD as well as the established reporter genes lacZ and gusA. Protein and transcript levels were quantified and translational efficiencies were calculated. The lacZ transcript was very unstable and its level highly depended on its translation, compromising its use as a reporter. The results obtained with gusA and glpD were similar, however, only glpD can be used for genetic selections. Therefore, glpD was found to be a superior novel reporter gene compared to the established reporter genes lacZ and gusA.

Enzootic plague foci, Algeria.

In Algeria, PCR sequencing of pla, glpD and rpoB genes found Yersinia pestis in 18/237 (8%) rodents of five species, including Apodemus sylvaticus, previously undescribed as pestiferous; and disclosed three new plague foci. Multiple spacer typing confirmed a new Orientalis variant. Rodent survey should be reinforced in this country hosting reemerging plague.

Recombinant expression of glpK and glpD genes improves the accumulation of shikimic acid in E. coli grown on glycerol.

Shikimic acid (SA) is an industrially important chiral compound used in diverse commercial applications, and the insufficient supply by isolation from plants and expensive chemical synthesis of SA has increased the importance of developing strategies for SA synthesis. In our previous studies, glycerol was observed to be an effective carbon source for SA accumulation in E. coli DHPYAAS-T7, where the PTS operon (ptsHIcrr) and aroL and aroK genes were inactivated, and the tktA, glk, aroE, aroF (fbr) , and aroB genes were overexpressed. For further investigation of the effects of glycerol aerobic fermentation on SA accumulation in E. coli BL21(DE3), the glpD, glpK genes and tktA, glk, aroE, aroF (fbr) , aroB genes were overexpressed simultaneously. The results indicated that SA production was increased 5.6-fold, while the yield was increased 5.3-fold over that of parental strain in shake flasks. It is demonstrated that the aerobic fermentation of glycerol associated with glpD and glpK gene overexpression increased glycerol flux, resulting in higher SA accumulation in E. coli BL21(DE3)-P-DK.

Functional characterization of Yersinia pestis aerobic glycerol metabolism

Yersinia pestis biovar Orientalis isolates have lost the capacity to ferment glycerol. Herein we provide experimental validation that a 93 bp in-frame deletion within the glpD gene encoding the glycerol-3-phosphate dehydrogenase present in all biovar Orientalis strains is sufficient to disrupt aerobic glycerol fermentation. Furthermore, the inability to ferment glycerol is often insured by a variety of additional mutations within the glpFKX operon which prevents glycerol internalization and conversion to glycerol-3-phosphate. The physiological impact of functional glpFKX in the presence of dysfunctional glpD was assessed. Results demonstrate no change in growth kinetics at 26 °C and 37 °C. Mutants deficient in glpD displayed decreased intracellular accumulation of glycerol-3-phosphate, a characterized inhibitor of cAMP receptor protein (CRP) activation. Since CRP is rigorously involved in global regulation Y. pestis virulence, we tested a possible influence of a single glpD mutation on virulence. Nonetheless, subcutaneous and intranasal murine challenge was not impacted by glycerol metabolism. As quantified by crystal violet assay, biofilm formation of the glpD-deficient KIM6+ mutant was mildly repressed; whereas, chromosomal restoration of glpD in CO92 resulted in a significant increase in biofilm formation.

Phenotypic and Molecular-Genetic Peculiarities of Yersinia pestis Strains from Trans-Baikal Steppe Plague Focus

The paper contains the data on the complex phenotypic and genotypic characteristics of Yersinia pestis strains isolated in Trans-Baikal steppe plague focus before and after the succession of the main carrier which took place in the 1960s. Based on the PCR-analysis and multilocus sequence typing of glpD, melB, napA, rhaA , and iclR genes, encoding differentially significant properties such as glycerin, melibiose, rhamnose fermentation, isocitrate lyase production, and denitrification activity, developed are the proofs of affiliation of the studied strains to the biovariant antiqua of the plague agent main subspecies. Multilocus VNTR-analysis by seven locuses of variable tandem repeats – ms01, ms04, ms06, ms07, ms46, ms62 – has been used to establish genetic relations between the strains. Thus on the basis of the results obtained, it is established that the strains of the plague agent, isolated in the Trans-Baikal steppe focus in various periods of its existence, form one common branch on the phylogenetic tree of Y. pestis evolution.

High Throughput, Multiplexed Pathogen Detection Authenticates Plague Waves in Medieval Venice, Italy

BackgroundHistorical records suggest that multiple burial sites from the 14th–16th centuries in Venice, Italy, were used during the Black Death and subsequent plague epidemics.Methodology/Principal FindingsHigh throughput, multiplexed real-time PCR detected DNA of seven highly transmissible pathogens in 173 dental pulp specimens collected from 46 graves. Bartonella quintana DNA was identified in five (2.9%) samples, including three from the 16th century and two from the 15th century, and Yersinia pestis DNA was detected in three (1.7%) samples, including two from the 14th century and one from the 16th century. Partial glpD gene sequencing indicated that the detected Y. pestis was the Orientalis biotype.ConclusionsThese data document for the first time successive plague epidemics in the medieval European city where quarantine was first instituted in the 14th century.

Distinct clones of Yersinia pestis caused the black death.

From AD 1347 to AD 1353, the Black Death killed tens of millions of people in Europe, leaving misery and devastation in its wake, with successive epidemics ravaging the continent until the 18th century. The etiology of this disease has remained highly controversial, ranging from claims based on genetics and the historical descriptions of symptoms that it was caused by Yersinia pestis to conclusions that it must have been caused by other pathogens. It has also been disputed whether plague had the same etiology in northern and southern Europe. Here we identified DNA and protein signatures specific for Y. pestis in human skeletons from mass graves in northern, central and southern Europe that were associated archaeologically with the Black Death and subsequent resurgences. We confirm that Y. pestis caused the Black Death and later epidemics on the entire European continent over the course of four centuries. Furthermore, on the basis of 17 single nucleotide polymorphisms plus the absence of a deletion in glpD gene, our aDNA results identified two previously unknown but related clades of Y. pestis associated with distinct medieval mass graves. These findings suggest that plague was imported to Europe on two or more occasions, each following a distinct route. These two clades are ancestral to modern isolates of Y. pestis biovars Orientalis and Medievalis. Our results clarify the etiology of the Black Death and provide a paradigm for a detailed historical reconstruction of the infection routes followed by this disease.

New Rural Focus of Plague, Algeria

New Rural Focus of Plague, Algeria

Atypical Transcriptional Regulation and Role of a New Toxin-Antitoxin-Like Module and Its Effect on the Lipid Composition of Bradyrhizobium japonicum

A toxin-antitoxin (TA)-like system (designated as bat/bto genes) was identified in Bradyrhizobium japonicum, based on sequence homology and similarities in organization and size to known TA systems. Deletion of the bat/bto module resulted in pleiotropic alterations in cell morphology and metabolism. The generation time of the mutant was considerably decreased in rich media. Atomic force microscopy revealed the modified shape (shorter and wider) and softness of mutant cells. The synthesis of phosphatidylcholine was completely blocked in the mutant bacteria, and vaccenic acid, the predominant fatty acid of membranes of the wild-type cell, was replaced by palmitic acid in the mutant membranes. The mutant bacteria synthesized incomplete lipopolysaccharide molecules. Remarkable changes in the membrane lipid composition may explain the observed morphological alterations and growth properties of the mutant bacteria. The overlapping promoter region of bat/bto and glpD (coding for the aerobic sn-glycerol-3-phosphate dehydrogenase) genes suggests a complex regulation and the involvement of bat/bto in the control of main metabolic pathways and an important role in the maintenance of a normal physiological state of B. japonicum. These data reveal new aspects of the role of TA systems in bacteria.

Yersinia pestisOrientalis in Remains of Ancient Plague Patients

Yersinia pestis DNA was recently detected in human remains from 2 ancient plague pandemics in France and Germany. We have now sequenced Y. pestis glpD gene in such remains, showing a 93-bp deletion specific for biotype Orientalis. These data show that only Orientalis type caused the 3 plague pandemics.

The Bacillus subtilis glpD leader and antiterminator protein GlpP provide a target for glucose repression in Escherichia coli

Expression of the Bacillus subtilis glpD gene which encodes glycerol-3-phosphate (G3P) dehydrogenase is regulated by the GlpP protein which, in the presence of G3P, causes antitermination of transcription of glpD. The glpD gene leader fused to lacZ was integrated into the chromosome of Escherichia coli deleted for the lac operon and carrying the B. subtilis glpP gene on a plasmid. β-Galactosidase activity of this strain was increased by the addition of G3P. When G3P and glucose, glucose-6-phosphate or fructose-6-phosphate were added, β-galactosidase activity was reduced showing that GlpP mediates catabolite repression of transcription from the glpD leader in the absence of any other B. subtilis protein.

The Bacillus subtilis glpD leader and antiterminator protein GlpP provide a target for glucose repression in Escherichia coli.

Expression of the Bacillus subtilis glpD gene which encodes glycerol-3-phosphate (G3P) dehydrogenase is regulated by the GlpP protein which, in the presence of G3P, causes antitermination of transcription of glpD. The glpD gene leader fused to lacZ was integrated into the chromosome of Escherichia coli deleted for the lac operon and carrying the B. subtilis glpP gene on a plasmid. beta-Galactosidase activity of this strain was increased by the addition of G3P. When G3P and glucose, glucose-6-phosphate or fructose-6-phosphate were added, beta-galactosidase activity was reduced showing that GlpP mediates catabolite repression of transcription from the glpD leader in the absence of any other B. subtilis protein.

Antiterminator protein GlpP of Bacillus subtilis binds to glpD leader mRNA.

The Bacillus subtilis glpD gene encodes glycerol-3-phosphate (G3P) dehydrogenase. Expression of glpD is mainly controlled by termination/antitermination of transcription at an inverted repeat in the glpD leader. Antitermination is mediated by the antiterminator protein GlpP in the presence of G3P. In this paper, interaction between GlpP and glpD leader mRNA in vivo and in vitro is reported. In vivo, the antiterminating effect of GlpP can be titrated in a strain carrying the glpD leader on a plasmid. GlpP has been purified and gel shift experiments have shown that it binds to glpD leader mRNA in vitro. GlpP is not similar to other known antiterminator proteins, but database searches have revealed an Escherichia coli ORF which has a high degree of similarity to GlpP.

Action at a distance for negative control of transcription of the glpD gene encoding sn-glycerol 3-phosphate dehydrogenase of Escherichia coli K-12.

Aerobic sn-glycerol 3-phosphate dehydrogenase is a cytoplasmic membrane-associated respiratory enzyme encoded by the glpD gene of Escherichia coli. The glpD operon is tightly controlled by cooperative binding of the glp repressor to tandem operators (O(D)1 and O(D)2) that cover the -10 promoter element and 30 bp downstream of the transcription start site. In this work, two additional operators were identified within the glpD structural gene at positions 568 to 587 (0(D)3) and 609 to 628 (0(D)4). The two internal operators bound the glp repressor in the presence or absence of the tandem operators (O(D)1 and O(D)2) in vitro, as shown by DNase I footprinting. To assess a potential regulatory role for the two internal operators in vivo, a glpD-lacZ transcriptional fusion containing all four operators was constructed. The response of this fusion to the glp repressor was compared with those of fusion constructs in which O(D)3 and O(D)4 were inactivated by either deletion or site-directed mutagenesis. It was found that the repression conferred by binding of the glp repressor to O(D)1 and O(D)2 was increased five- to sevenfold upon introduction of the internal operators. A regulatory role for HU was suggested when it was found that repressor-mediated control of glpD transcription was increased fourfold in strains containing HU compared with that of strains deficient in HU. The effect of HU was apparent only in the presence of all four glpD operators. The results suggest that glpD is controlled by formation of a repression loop between the tandem and internal operators. HU may assist repression by bending the DNA to facilitate loop formation.