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At least 19 recordsLinked to original sources
PubMed1974

Induction by mutagens of tandem gene duplications in the glyS region of the Escherichia coli chromosome.

Four mutagens (ultraviolet light, nitrous acid, the acridine half mustard ICR 372, and niridazole) have been found to increase the frequency of tandem gene duplications in the glyS region of the Escherichia coli chromosome. This result was obtained by quantitating the spontaneous and mutagen-induced reversion frequency of a glycyl-tRNA synthetase (glyS) mutant. Following mutagenesis, as many as 0.2% of the survivors were observed to contain duplications in the glyS region. In addition, several classes of stable revertants of the glyS mutant have been identified.

Acridines↗
PubMed2025

Gene model for the ortholog of Glys in Drosophila ananassae.

Gene model for the ortholog of Glycogen synthase (Glys) in the May 2011 (Agencourt dana_caf1/DanaCAF1) Genome Assembly (GenBank Accession: GCA_000005115.1) of Drosophila ananassae. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Bioinformatics↗
PubMed1995

Role of protein kinase C alpha in the induction of carcinoembryonic antigen by transforming growth factor beta 1.

Previous studies showed that transforming growth factor beta 1 (TGF beta 1) regulates the expression of carcinoembryonic antigen (CEA) and CEA-cross-reactive glycoproteins (CEA-GLYs) in human colon carcinoma cells through a signal-transducing pathway associated with protein kinase C (PKC) (Chakrabarty, J. Cell. Physiol., 1992, 152:494-499). In this study we determined the role of the PKC alpha isoform in the regulation of CEA and CEA-GLYs expression by TGF beta 1. Expression of PKC alpha antisense RNA, through transfection experiments with an antisense PKC alpha expression vector, resulted in down-modulation of PKC alpha RNA and protein expression. TGF beta 1 was unable to stimulate the expression and secretion of CEA in cells in which the expression of PKC alpha protein was substantially reduced. The ability of TGF beta 1 to stimulate the expression of the 95- and 55-kDa CEA-GLYs, however, was not affected. We therefore conclude that TGF beta 1 regulates the secretion and expression of CEA through a signal-transducing pathway associated with PKC alpha. TGF beta 1 may also regulate the expression of CEA-GLYs through signal-transducing pathways associated with other PKC isoforms.

Carcinoembryonic Antigen↗
PubMed1982

A thioamide substrate of carboxypeptidase A.

Carbobenzoxythioglycyl-L-phenylalanine [CbzNHCH2C(==S)Phe, Z-Glys-Phe] was synthesized as thioamide analogue of Z-Gly-Phe, a known substrate of carboxypeptidase A (CPA). By use of a ninhydrin-based assay and Z-Gly-Gly-Phe as the substrate, Z-Glys-Phe was shown to be a weak competitive inhibitor of CPA (Ki = 1.4 mM). The L isomer (but not the D) of Z-Glys-Phe proved to be a substrate for CPA (Km = 1.1 mM and kcat = 5.3 s-1 at pH 7.5), binding with comparable affinity to, but hydrolyzing at 10% the rate of, the oxo analogue Z-Gly-Phe. The CPA-catalyzed hydrolysis of Z-Glys-Phe was shown to involve only C-N bond cleavage, to give carbobenzoxythioglycine and phenylalanine.

Amides↗
PubMed1970

Isolation and partial characterization of Escherichia coli mutants with altered glycyl transfer ribonucleic acid synthetases.

Isolates with mutations in glyS, the structural gene for glycyl-transfer ribonucleic acid (tRNA) synthetase (GRS) in Escherichia coli, are frequently found among glycine auxotrophs. Extracts of glyS mutants have altered GRS activities. The mutants grow with normal growth rates in minimal media when high levels of glycine are provided. No other metabolite of a variety tested is capable of restoring normal growth. The glyS mutants fail to make ribonucleic acid (RNA) when depleted of exogenous glycine in strains which are RC(str) but do so when the cells are RC(rel). In contrast, biosynthetic mutants which are unable to synthesize glycine (glyA mutants) do not make RNA when deprived of glycine even if they are RC(rel); in this case, RNA is synthesized upon glycine deprivation only when the nucleic acid precursors made from glycine are provided in the medium. The level of serine transhydroxymethylase is unaltered in extracts of any of the glyS mutants, even though the level of charged tRNA(Gly) is at least 20-fold lower than that found in a prototrophic parent; this indicates that, if there is control over the synthesis of serine transhydroxymethylase, it is not modified by reduced levels of charging of the major species of tRNA(Gly).

Carbon Isotopes↗
PubMed1970

Characterization of altered forms of glycyl transfer ribonucleic acid synthetase and the effects of such alterations on aminoacyl transfer ribonucleic acid synthesis in vivo.

The glycyl transfer ribonucleic acid (tRNA) synthetase (GRS) activities of several Escherichia coli glyS mutants have been partially characterized; the K(m) for glycine and the apparent V(max) of several of the altered GRS differ significantly from the parental GRS. Paradoxically, some of the altered forms exhibit more activity in vitro than the GRS from a prototrophic strain (GRS(L)); several parameters of these activities have been studied in an attempt to resolve this problem. The amount of acylated tRNA(Gly) in vivo was examined to assess the GRS activities inside the cells. During exponential growth in media containing glycine, moderate amounts of acylated tRNA(Gly) occur in the glyS mutants; glycine deprivation leads to a dramatic drop in the amount of acylated tRNA(Gly). An alternative measure of the in vivo activities of the altered enzymes is the efficiency of suppression of the trpA36 locus by su(36) (+); glyS mutants grown with added glycine exhibit one-third to one-fourth the suppression efficiency of the prototrophic glyS(H) parent, presumably because they are less efficient, even in the presence of high levels of glycine, in charging the tRNA(Gly) species which functions as the translational suppressor.

Amino Acids↗
PubMed1982

A Col E1 hybrid plasmid containing Escherichia coli genes complementing d-xylose negative mutants of Escherichia coli and Salmonella typhimurium.

The Clarke and Carbon bank of Col El - Escherichia coli DNa hybrid plasmids was screened for complementation of d-xylose negative mutants of E. coli. Of several obtained, the smallest, pRM10, was chosen for detailed study. Its size was 16 kilobases (kb) and that of the insert was 9.7 kg. By transformation or F'-mediated conjugation this plasmid complemented mutants of E. coli defective in either D-xylose isomerase or D-xylulose kinase activity, or both. The activity of D-xylulose kinase in E. coli transformants which bear an intact chromosomal gene for this enzyme was greater than that for the host, due to a gene dosage effect. The plasmid also complemented D-xylose negative mutants of Salmonella typhimurium by F'-mediated conjugation between E. coli and S. typhimurium. Salmonella typhimurium mutants complemented were those for D-xylose isomerase and for D-xylulose kinase in addition to pleiotropic D-xylose mutants which were defective in a regulatory gene of the D-xylose operon. In addition, the plasmid complemented the glyS mutation in E. coli and S. typhimurium. The glyS mutant of E. coli was temperature sensitive, indicating that the plasmid carried the structural gene for glycine synthetase. The glyS mutation in E. coli maps at 79 min, as do the xyl genes. The behaviour of the plasmid is consistent with the existence of a d-xylose operon in E. coli. The data also suggest that the plasmid carries three of the genes of this operon, specifically those for D-xylose isomerase, D-xylulose kinase, and a regulatory gene.

Cloning, Molecular↗
PubMed1984

A repetitive DNA sequence, rhs, responsible for duplications within the Escherichia coli K-12 chromosome.

A novel family of large, imperfectly repeated DNA sequences has been found in Escherichia coli. Two members of this family, rhsA and rhsB, occur as direct repeats, flanking the pit glyS xyl segment of the chromosome. Unequal sister-chromatid crossing over between rhsA and rhsB accounts for the frequent tandem duplication of the glyS locus that has been observed by various workers. This unequal recombination is recA-dependent. The rhsA locus is operationally defined as the segment between xyl and mtl that is repeated at other chromosomal locations. Using this definition, rhsA extends minimally 5500 base-pairs; 3800 base-pairs of rhsA are sufficiently homologous to rhsB to form an S1 nuclease-resistant heteroduplex with it. The rhsA sequence also exhibits internal repetition. At least one additional rhs sequence occurs in the E. coli chromosome unlinked to either rhsA or rhsB. Southern analysis of restriction digests of genomic DNA from E. coli strains C and B/5 showed that both of these strains have rhs hybridizable patterns similar to strain K-12, but the rhs sequence is absent in Salmonella typhimurium. The function of the rhs sequences has not been discovered. In the course of this work we developed a technique, termed "transductional walking", by which chromosomal DNA adjacent to a previously cloned DNA segment can be cloned through genetic procedures.

Chromosome Mapping↗
PubMed1983

Synthesis of two polypeptide subunits of an aminoacyl tRNA synthetase as a single polypeptide chain.

E. coli aminoacyl tRNA synthetases are typically comprised of a single type of polypeptide chain. Glycine tRNA synthetase is an exception, and is comprised of two different subunits. Previous work showed that glyS encodes both subunits in a tandem arrangement of coding regions which are in the same reading frame. Nine nucleotides separate the TAA stop of the first coding segment (alpha-subunit) from the ATG start of the second one (beta-subunit). A plasmid containing glyS was put into four different ochre suppressor strains. In three of them, significant quantities of an alpha-beta fusion protein were synthesized in maxicells, in genetic backgrounds which retained cellular proteases. This shows that the fusion protein is stable in vivo and suggests that Gly-tRNA synthetase is operationally a single polypeptide which is the ancestor of the two subunits.

Amino Acid Sequence↗
PubMed1996

Mapping of 61 genes on the refined physical map of the chromosome of Thermus thermophilus HB27 and comparison of genome organization with that of T. thermophilus HB8.

We have constructed refined physical maps of the chromosome (1 center dot 82 Mb) and the large plasmid pTT27 (250 kb) of Thermus thermophilus HB27. A total of 49 cleavage sites with five restriction enzymes, EcoRI, SspI, MunI, EcoRV and ClaI, were determined on the maps. The location of 61 genes was determined by using as probes 64 genes cloned from T. thermophilus or other Thermus strains. Comparison of the genomic organization of the chromosomes of T. thermophilus HB27 and HB8 revealed that they were basically identical, but some genes were located in different regions. Among 32 genes whose locations were determined on both the HB27 and the HB8 chromosomes, the copy number of rpsL-rpsG-fus-tufA, the locations of glyS, pol, and one copy of nusG-rplK-rplA were different. The IS1000 sequence was located only in one region on the HB27 chromosome. In contrast, IS1000 sequences were scattered over four regions on the chromosome of HB8. As each region in which glyS, pol, or one copy of nusG-rplK-rplA are present also contained IS1000 in HB8, it is suggested that IS1000 may play an important role in genomic rearrangements in Thermus strains.

Chromosome Mapping↗
PubMed1990

A mutation in the small (alpha) subunit of glycyl-tRNA synthetase affects amino acid activation and subunit association parameters.

A strategy was designed to isolate mutants of glycyl-tRNA synthetase that are altered at the amino acid binding site, including a class with altered amino acid specificity. For this purpose, the plasmid pBR322 was mutated so that the codon (AGC) of the active site Ser-68 in the beta-lactamase gene was changed to the glycine codon GGC to inactivate the encoded enzyme. Suppressors that increase the amount of beta-lactamase activity of the Gly-68 allele of beta-lactamase were isolated and some mapped to the gene encoding glycyl-tRNA synthetase (glyS). While in vitro misaminoacylation of tRNA(Gly) with serine was not detected for any of the mutants, glycyl-tRNA synthetase activity was altered. One severely affected glyS mutant (N302) was studied in more detail. For this mutant, a single Pro-61----Leu substitution in the alpha chain confers an elevation of the Km values for glycine (25-fold) and for ATP (45-fold) in the aminoacylation reaction, but only a minor perturbation of the Km for tRNA. There also was a severely reduced adenylate synthesis activity (greater than 100-fold). In addition, a nonlinear dependence between aminoacylation activity and enzyme concentration was observed which implies that the alpha chain Pro-61----Leu mutation has disrupted the functionally essential subunit interactions of the holoenzyme. The results of the preceding paper have shown that the alpha chain and parts of the beta chain are required for aminoacylation and adenylate synthesis activity. The results of this study suggest that the alpha chain specifically contributes to amino acid and to ATP binding in a way that is affected by proper subunit interactions.

Alleles↗
PubMed2023

Teicoplanin associated gene tcaA inactivation increases persister cell formation in Staphylococcus aureus.

Staphylococcus aureus is part of normal human flora and is widely associated with hospital-acquired bacteremia. S. aureus has shown a diverse array of resistance to environmental stresses and antibiotics. Methicillin-resistant S. aureus (MRSA) is on the high priority list of new antibiotics discovery and glycopeptides are considered the last drug of choice against MRSA. S. aureus has developed resistance against glycopeptides and the emergence of vancomycin-intermediate-resistant, vancomycin-resistant, and teicoplanin-resistant strains is globally reported. Teicoplanin-associated genes tcaR-tcaA-tcaB (tcaRAB) is known as the S. aureus glycopeptide resistance operon that is associated with glycopeptide resistance. Here, for the first time, the role of tcaRAB in S. aureus persister cells formation, and ΔtcaA dependent persisters' ability to resuscitate the bacterial population was explored. We recovered a clinical strain of MRSA from a COVID-19 patient which showed a high level of resistance to teicoplanin, vancomycin, and methicillin. Whole genome RNA sequencing revealed that the tcaRAB operon expression was altered followed by high expression of glyS and sgtB. The RNA-seq data revealed a significant decrease in tcaA (p = 0.008) and tcaB (p = 0.04) expression while tcaR was not significantly altered. We knocked down tcaA, tcaB, and tcaR using CRISPR-dCas9 and the results showed that when tcaA was suppressed by dCas9, a significant increase was witnessed in persister cells while tcaB suppression did not induce persistence. The results were further evaluated by creating a tcaA mutant that showed ΔtcaA formed a significant increase in persisters in comparison to the wild type. Based on our findings, we concluded that tcaA is the gene that increases persister cells and glycopeptide resistance and could be a potential therapeutic target in S. aureus.

MRSA↗
PubMed1997

Diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) controls the timing of cell division in Escherichia coli.

BACKGROUND: The timing of the cell division in Escherichia coli is highly regulated, but its mechanism has not been identified. Previously we have found that the cfcA1 mutation uncouples DNA replication and cell division and elevates the frequency of cell division. We further analysed the structure and the role of the cfc genes of cfcA11, a derivative of cfcA1, and another cfc mutant, cfcB1. RESULTS: The cfc mutants divided prior to the ordinary stage of cell division and produced many small cells with nucleoid. However, the cells grew exponentially, and the length of a cell cycle and the initiation mass for chromosome replication were not altered by the cfc mutations. These properties resulted from a reduction of the period between the nucleoid division and the cell division in a cell cycle, and a compensatory lengthening of the period between the cell division and the initiation of the next round of DNA replication. CfcA11 has a mutation in glySa which encodes the alpha-subunit of glycyl-tRNA synthetase, and cfcB1 has an IS2 insertion in apaH which encodes Ap4A hydrolase. The cfc properties of both cfc mutants were suppressed by a multicopy plasmid carrying apaH+, and the intracellular level of Ap4A in cfcA1 was 15-fold higher, and cfcB1 was 100-fold higher than their parent. Experiments using wild-type cells showed that a high level of Ap4A caused early cell division, and that a low level of Ap4A caused delayed cell division. CONCLUSION: Ap4A is a signal for the induction of cell division. High levels of Ap4A are responsible for the initiation of cell division. The glyS mutation permits an efficient synthesis of Ap4A.

Cell Cycle↗
PubMed1997

Role of the glycine triad in the ATP-binding site of cAMP-dependent protein kinase.

A glycine-rich loop in the ATP-binding site is one of the most highly conserved sequence motifs in protein kinases. Each conserved glycine (Gly-50, Gly-52, and Gly-55) in the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) was replaced with Ser and/or Ala. Active mutant proteins were expressed in Escherichia coli, purified to apparent homogeneity, separated into phosphoisoforms, and characterized. Replacing Gly-55 had minimal effects on steady-state kinetic parameters, whereas replacement of either Gly-50 or Gly-52 had major effects on both Km and kcat values consistent with the prediction of the importance of the tip of the glycine-rich loop for catalysis. Substitution of Gly-50 caused a 5-8-fold reduction in Km (ATP), an 8-12-fold increase in Km (peptide), and a 3-5-fold drop in kcat. The Km (ATP) and Km (peptide) values of C(G52S) were increased 8- and 18-fold, respectively, and the kcat was decreased 6-fold. In contrast to catalytic efficiency, the ATPase rates of C(G50S) and C(G52S) were increased by more than an order of magnitude. The thermostability of each mutant was slightly increased. Unphosphorylated C(G52S) was characterized as well as several isoforms phosphorylated at a single site, Ser-338. All of these phosphorylation-defective mutants displayed a substantial decrease in both enzymatic activity and thermal stability that correlated with the missing phosphate at Thr-197. These results are correlated with the crystal structure, models of the respective mutant proteins, and conservation of the Glys within the protein kinase family.

Adenosine Triphosphatases↗
PubMed2025

The toxin-antitoxin system SavRS contributes to vancomycin resistance in vancomycin-intermediate Staphylococcus aureus by mediating cell wall thickening.

BACKGROUND: The emergence of vancomycin-intermediate Staphylococcus aureus (VISA) has significantly challenged the treatment of S. aureus infection. Toxin-antitoxin (TA) systems have been reported to mediate bacterial stress adaptation and virulence, but their role in vancomycin resistance remains elusive. This study investigated the vancomycin resistance mechanism regulated by the TA system SavRS in VISA. METHODS: savRS mutants in Mu50 and XN108 were generated via homologous recombination. To investigate the regulatory mechanism of vancomycin resistance mediated by savRS in VISA, phenotypic analyses including MICs, growth kinetics and cell wall thickness measurements were performed. Expression of cell wall synthesis-related genes was analysed using quantitative RT-PCR (RT-qPCR) and promoter-lacZ reporter assay. Electrophoretic mobility shift assay (EMSA) was performed to assess the binding of SavRS to the promoters of the cell wall synthesis-related genes. Pull-down assay identified an upstream regulatory element of savRS associated with vancomycin resistance. Quantitative assessment of bacterial burden in murine organ systems following vancomycin administration revealed the critical regulatory role of savRS in mediating vancomycin resistance in vivo. RESULTS: Compared with the WT, the savRS mutant exhibited enhanced vancomycin sensitivity, accelerated growth and reduced cell wall thickness. Correspondingly, RT-qPCR revealed marked down-regulation of the cell wall synthesis-related genes (glyS, dltA, scdA, pbp2, ddl). EMSA and promoter-lacZ reporter assay confirmed direct binding of SavRS to a conserved promoter motif, MGHYYTCCTCA. Pull-down assay identified UspA as an upstream regulator of SavRS, demonstrating that UspA directly controls savRS transcription and modulates VISA resistance. Mouse infection experiments showed that savRS promotes VISA to vancomycin resistance in vivo. CONCLUSIONS: SavRS critically regulates vancomycin resistance in VISA.

Cell Wall↗
PubMed1979

Genetics and regulation of D-xylose utilization in Salmonella typhimurium LT2.

Twenty-one Xyl- mutants of Salmonella typhimurium were selected: all had lost one or more of the activities for D-xylose isomerase, C-xylulokinase, or D-xylose transport. The mutants were classified into five functional groups: xylR, pleiotropic negative (12 mutants); xylA, D-xylose isomerase defective (3 mutants); xylB, D-xylulokinase defective (2 mutants); xylT, D-xylose transport defective (1 mutant); and 3 mutants with defective D-xylose isomerase and D-xylulokinase. Some nonsense mutations were identified among the xylR mutants. Two F'xyl plasmids were isolated by selection for early transfer of xyl+ by an Hfr which transfers xyl as a terminal gene; a plasmid with a mutation in the xyl genes, F'xylR1, was also isolated. Complementation tests using F'xyl plasmids indicate that expression of the xylA, xylB, and xylT genes is under the positive control of the xylR regulatory gene. Conjugation crosses and P22-mediated transduction data indicate that all the xyl mutations tested are in a cluster of genes at 78 units on the linkage map, and that the gene order is xylT--xylR--xylB--xylA--glyS--mtlA,D.

Biological Transport, Active↗
PubMed1975

Effect of Nepsilon-alkylation of the substrate on the hydrolysis of benzoylglycyl-L-lysine by carboxypeptidase B1.

The action of carboxypeptidase B (EC 3.4.12.3) on the benzoylglycyl dipeptides Bz-Gly-Lys(X) where X = methyl, ethyl, propyl, formyl, dimethyl, isopropyl, trimethyl, and benzyl has been investigated. All were hydrolyzed, at a rate decreasing in the order indicated, except where X = trimethyl and benzyl. Compounds where X = dimethyl, formyl, and isopropyl were hydrolyzed very slowly, and did not inhibit the hydrolysis of Bz-Glys-Lys by the enzyme. The kinetic parameters kcat and Km were determined for compounds with X = methyl and ethyl. The observed decrease in rate of hydrolysis of substrates with increasing size of X is consistent with increasing steric hindrance effects arising from the interaction of the Nepsilon-alkyl group with residues of the protein in the cleft which accommodates the substrate side-chain, and resulting in weaker binding of the substrate. Bz-Gly-Lys(Me2) was prepared by reductive methylation of Bz-Gly-Lys. Bz-Gly-Lys(Me3) was prepared by the reaction of Bz-Gly-Lys(Me2) with diazomethane in aqueous solution. Bz-Gly-Lys(Me) and Bz-Gly-Lys(Et) were synthesized by classical coupling procedures from the appropriately protected lysine derivatives.

Alcohols↗