tyrB Gene Research Articles

Molecular study on Efflux pumps of Klebsiella pneumonia Isolated from patients with Cystitis

Background: Klebsiella pneumoniae can be defined as one of the clinically relevant pathogens that is a common cause of community-acquired and hospital-acquired urinary tract infections (UTI). Objective: The current study was conducted to investigate most common members of 5 classes of efflux pumps among K. Pneumonia isolates. Methodology: K. Pneumonia isolates was diagnosed on EMB and confirmed by tyrB gene. Antibiotic susceptibility test has been done based on the CLSI-2019. Efflux pumps genes were examined via PCR. Results: All isolates were high resist to ceftazidime, Amoxicillin, cefotaxime, ceftriaxone, Cefixime, cefepime, streptomycin and trimethoprime. Moderate resistance were showed to nitrofurantion, Aztreonam, Kanamycin, Cefoxitin, Gentamycine and Tobramycine. Low resistance was exhibited to Ciprofloxacine, Azithromycin, Doxycycline, piperacillin, Nalidixic acid, Imipenem and Amikacine. High sensitivity were exploited to Levofloxacine, ofloxacine, Meropenem and Netilamicin Concern antibiotic resistance patterns PDR, XDR and MDR), the results revealed that (10%) isolates were non-MDR while MDR compile (90%). Results of molecular investigation of efflux pumps in K pneumonia revealed that, AcrAB-TolC, AcrAD-TolC and AcrFE-TolC genes, EmrAB-TolC, EmrD and MdfA, EmrE, YnfA and TehA, MacAB-TolC and MdlAB-TolC, MdtK and DinF) genes were investigated for K. pneumoniae. Results of biofilm formation revealed that 100% were biofilm former (40% weak biofilm, 44% moderate and 16% strong biofilm former). Conclusion: The study concludes that, all efflux pumps may be highly associated with resistance to penicillins and cephalosporins and moderately with streptomycin, trimethoprime, nitrofuraniton, Aztreonam, Kanamycin. Additionally, biofilm formation was highly related to presence of studied pumps.

Metabolic Engineering of Escherichia coli for Production of 2-phenylethanol from Renewable Glucose

2-Phenylethanol (2-PE) is an important aromatic alcohol with a rose-like odor and has wide applications. The present work aims to construct a synthetic pathway for 2-PE synthesis from glucose in Escherichia coli. First, the genes adh1 (encoding alcohol dehydrogenase) and kdc (encoding phenylpyruvate decarboxylase) from Saccharomyces cerevisiae S288c and Pichia pastoris GS115 were investigated in E. coli, respectively, and single overexpression of adh1 or kdc significantly increased 2-PE accumulation. When co-overexpressing adh1 and kdc, 2-PE was increased up to 130 from 57 mg/L. Furthermore, by optimizing coordinated expression of the four committed genes aroF, pheA, adh1 and kdc, 2-PE was improved to 285 mg/L which was the highest production of 2-PE by the recombinant E. coli system. In addition, our results also demonstrated that the tyrB gene, which encodes aromatic-amino-acid transaminase, plays an important role on 2-PE synthesis.

Rapid Identification of <i>Klebsiella pneumoniae</i>, <i>Corynebacterium kutscheri</i>, and <i>Streptococcus pneumoniae</i> Using Triplex Polymerase Chain Reaction in Rodents

Klebsiella pneumoniae, Corynebacterium kutscheri, and Streptococcus pneumoniae are important pathogens that cause respiratory infections in laboratory rodents. In this study, we used species-specific triplex PCR analysis to directly detect three common bacterial pathogens associated with respiratory diseases. Specific targets were amplified with conventional PCR using the tyrB gene from K. pneumoniae, gyrB gene from C. kutscheri, and ply gene from S. pneumoniae. Our primers were tested against purified DNA from another eleven murine bacteria to determine primer specificity. Under optimal PCR conditions, the triplex assay simultaneously yielded a 931 bp product from K. pneumoniae, a 540 bp product from C. kutscheri, and a 354 bp product from S. pneumoniae. The triplex assay detection thresholds for pure cultures were 10 pg for K. pneumoniae and S. pneumoniae, and 100 pg for C. kutscheri. All three bacteria were successfully identified in the trachea and lung of experimentally infected mice at the same time. Our triplex PCR method can be used as a useful method for detecting pathogenic bacterial infections in laboratory rodents.

Co-expression of five genes inE colifor L-phenylalanine inBrevibacterium flavum

To study the effect of co-expression of ppsA, pckA, aroG, pheA and tyrB genes on the production of L-phenylalanine, and to construct a genetic engineering strain for L-phenylalanine. ppsA and pckA genes were amplified from genomic DNA of E. coli by polymerase chain reaction, and then introduced into shuttle vectors between E coli and Brevibacterium flavum to generate constructs pJN2 and pJN5. pJN2 was generated by inserting ppsA and pckA genes into vector pCZ; whereas pJN5 was obtained by introducing ppsA and pckA genes into pCZ-GAB, which was originally constructed for co-expression of aroG, pheA and tyrB genes. The recombinant plasmids were then introduced into B. flavum by electroporation and the transformants were used for L-phenylalanine fermentation. Compared with the original B. flavum cells, all the transformants were showed to have increased five enzyme activities specifically, and have enhanced L-phenylalanine biosynthesis ability variably. pJN5 transformant was observed to have the highest elevation of L-phenylalanine production by a 3.4-fold. Co-expression of ppsA and pckA increased activity of DAHP synthetase significantly. Co-expression of ppsA and pckA genes in B. flavum could remarkably increase the expression of DAHP synthetase; Co-expression of ppsA, pckA, aroG, pheA and tyrB of E. coli in B. flavum was a feasible approach to construct a strain for phenylalanine production.

Novel biosynthetic routes to non-proteinogenic amino acids as chiral pharmaceutical intermediates

Transaminases catalyse the reversible transfer of amino and keto groups between an amino acid and keto acid substrate pair. Many bacterial transaminases accept a wide array of keto acids as amino acceptors and are useful as commercial biocatalysts in the preparation of amino acids. Since the reaction equilibrium typically lies close to unity, several approaches have been described to improve upon the 50% product yield, using additional enzymes. The present work describes an efficient means to significantly increase product yield in transamination using the aromatic transaminase of Escherichia coli encoded by the tyrB gene, with l-aspartate as the amino donor. This is achieved by the introduction of the alsS gene encoding the acetolactate synthase of Bacillus subtilis, which eliminates pyruvate and alanine produced as a by-product of aspartate transamination. The biosynthesis of the non-proteinogenic amino acid l-2-aminobutyrate is described using a recombinant strain of E. coli containing the cloned tyrB and alsS genes. The strain additionally carries the cloned ilvA gene of E. coli encoding threonine deaminase to produce the substrate 2-ketobutyrate from l-threonine. An alternate coupled process uses lysine ε-aminotransferase in concert with a transaminase using l-glutamate as the amino donor.

Use of lac Fusion to Approach the High TyrB Production with a Runaway-Replication Vector

An easy and efficient method was proposed to achieve high production of the tyrB gene product on a runaway-replication vector. The DNA containing the tyrB promoter fused transcriptionally with the lacZ gene was cloned into a runaway-replication plasmid, pRA90. With the aid of this gene fusion, the expression level of tyrB in response to temperature alter-ations, the induction cell density, the temperature upshift rate, and various culture medium was investigated by measuring LacZ activities. The optimal condition thus set up for LacZ production was adopted to yield TyrB by a similar clone harboring an intact tyrE gene. As a result, TyrB was over-amplified 135-fold in tenns of specific activity in comparison with the wild-type level. This work demonstrates the potential use of lacZ fusion to probe the sound condition for high TryB production with uncontrolled-replication plasmids.

Genetic rearrangements in the tyrB-uvrA region of the enterobacterial chromosome: a potential cause for different class B acid phosphatase regulation in Salmonella enterica and Escherichia coli.

Unlike in Escherichia coli, in Salmonella enterica production of class B acid phosphatase (AphA) was detectable also in cells growing in the presence of glucose. Characterization of the aphA locus from a S. enterica ser. typhi strain showed that the aphA determinant is very similar to the E. coli homolog, and that its chromosomal location between the highly conserved tyrB and uvrA genes is retained. However, the aphA flanking regions were found to be markedly different in the two species, either between tyrB and aphA or between aphA and uvrA. The differences in the aphA 5'-flanking region, which in S. enterica is considerably shorter than in E. coli (183 vs. 1121 bp) and includes potential promoter sequences not present in E. coli, could be responsible for the different regulation of class B acid phosphatase observed in the two species.

Cloning and characterization of the tyrB gene from Salmonella typhimurium

We found a gene homologous to tyrB which encodes aromatic amino acid aminotransferase (ArAT, EC2.6.1.57) in Escherichia coli, in the genome of Salmonella typhimurium IFO 13245. The S. typhymurium tyrB product consists of 397 amino acid residues. The amino acid sequence shows 87.9% identity with that of E. coli ArAT, but shows lower identity (42.3%) with that of E. coli aspartate aminotransferase (AspAT, EC2.6.1.1). When the S. typhimurium tyrB gene was expressed in an E. coli mutant whose intrinsic tyrB gene had been inactivated, the activity of transaminating tyrosine and phenylalanine could be recovered, indicating that the S. tphimurium tyrB gene product possesses transamination activities similar to those of the E. coli ArAT. Elucidation of the molecular features of a new ArAT may be helpful for structural and functional analyses of ArAT and AspAT with regard to the different but overlapping substrate specificity of the two enzymes.

Molecular analysis of the regulatory region of the Escherichia coli K-12 tyrB gene.

The tyrB gene from Escherichia coli K-12 was cloned and sequenced, and the transcriptional start point of tyrB was determined by primer extension. By using a fusion plasmid in which the lacZ structural gene is transcribed from the tyrB promoter, it was shown that the expression of tyrB is controlled at the transcriptional level by the TyrR protein, with tyrosine as corepressor. The fusion plasmid was used to isolate mutants in which the repression of tyrB had been abolished. The tyrB promoter-operator region of these mutants was sequenced, and the tyrB operator was identified. A comparison between the tyrB operator and those of the other genes belonging to the tyrR regulon is presented.

The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K12. Comparison of the primary structures of the aspartate aminotransferase and aromatic aminotransferase of E. coli with those of the pig aspartate aminotransferase isoenzymes.

In this paper we describe the cloning and sequence analysis of the tyrB and aspC genes from Escherichia coli K12, which encode the aromatic aminotransferase and aspartate aminotransferase respectively. The tyrB gene was isolated from a cosmid carrying the nearby dnaB gene, identified by its ability to complement a dnaB lesion. Deletion and linker insertion analysis located the tyrB gene to a 1.7-kilobase NruI-HindIII-digest fragment. Sequence analysis revealed a gene encoding a 43 000 Da polypeptide. The gene starts with a GTG codon and is closely followed by a structure resembling a rho independent terminator. The aspC gene was cloned by screening gene banks, prepared from a prototrophic E. coli K12 strain, for plasmids able to complement the aspC tyrB lesions in the aminotransferase-deficient strain HW225. Sub-cloning and deletion analysis located the aspC gene on a 1.8-kilobase HincII-StuI-digest fragment. Sequence analysis revealed the presence of a gene encoding a 43 000 Da protein, the sequence of which is identical with that previously obtained for the aspartate aminotransferase from E. coli B. Considerable overproduction of the two enzymes was demonstrated. We compared the deduced protein sequences with those of the pig mitochondrial and cytoplasmic aspartate aminotransferases. From the extensive homology observed we are able to propose that the two E. coli enzymes possess subunit structures, subunit interactions and coenzyme-binding and substrate-binding sites that are very similar both to each other and to those of the mammalian enzymes and therefore must also have very similar catalytic mechanisms. Comparison of the aspC and tyrB gene sequences reveals that they appear to have diverged as much as is possible within the constraints of functionality and codon usage.

Aromatic amino acid aminotransferase of [formula omitted]: Nucleotide sequence of the [formula omitted] gene

The tyrB gene of E. coli K-12, which encodes aromatic amino acid aminotransferase (EC 2.6.1.57) was cloned. The nucleotide sequence of about 2 kilobase pairs containing the gene was determined. The coding region of the tyrB gene and the deduced amino acid sequence revealed that the aromatic amino acid aminotransferase of E. coli is homologous with the aspartate aminotransferase.

Role of nuclear genes in expression of a mitochondrial tRNA gene in Saccharomyces cerevisiae.

In yeast mitochondria, most of the isoaccepting species of tyrosyl tRNA are coded by a mitochondrial gene, tyrA. A particular isoaccepting species is coded by a second mitochondrial gene, tyrB. This gene is not expressed in certain strains of yeast which show no deficient phenotype. Genetic crosses between strains expressing or not expressing the tyrB gene demonstrate that expression is controlled by specific nuclear genes and that a mutation of the tyrA gene can be bypassed when the tyrB gene is operative.

Role of nuclear genes in expression of a mitochondrial tRNA gene in Saccharomyces cerevisiae.

In yeast mitochondria, most of the isoaccepting species of tyrosyl tRNA are coded by a mitochondrial gene, tyrA. A particular isoaccepting species is coded by a second mitochondrial gene, tyrB. This gene is not expressed in certain strains of yeast which show no deficient phenotype. Genetic crosses between strains expressing or not expressing the tyrB gene demonstrate that expression is controlled by specific nuclear genes and that a mutation of the tyrA gene can be bypassed when the tyrB gene is operative.

Role of the Escherichia coli aromatic amino acid aminotransferase in leucine biosynthesis

Strains of Escherichia coli that lack the branched-chain amino acid amino-transferase because of mutations in the ilvE gene had no growth requirement for leucine when the cells contained the aromatic amino acid aminotransferase that is the product of the tyrB gene. The presence of leucine increased the generation time of these cells and decreased the specific activity of the aromatic amino acid aminotransferase. It is concluded that this enzyme functions efficiently in leucine biosynthesis and can be repressed by leucine as well as by tyrosine.