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1.
Rostirolla DC Breda A Rosado LA Palma MS Basso LA Santos DS 《Archives of biochemistry and biophysics》2011,(2):202-212
The pyrH-encoded uridine 5′-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP–Mg2+ as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents. 相似文献
2.
Martín Hugo Koen Van Laer Aníbal M. Reyes Didier Vertommen Joris Messens Rafael Radi Madia Trujillo 《The Journal of biological chemistry》2014,289(8):5228-5239
Mycobacterium tuberculosis (M. tuberculosis), the pathogen responsible for tuberculosis, detoxifies cytotoxic peroxides produced by activated macrophages. M. tuberculosis expresses alkyl hydroxyperoxide reductase E (AhpE), among other peroxiredoxins. So far the system that reduces AhpE was not known. We identified M. tuberculosis mycoredoxin-1 (MtMrx1) acting in combination with mycothiol and mycothiol disulfide reductase (MR), as a biologically relevant reducing system for MtAhpE. MtMrx1, a glutaredoxin-like, mycothiol-dependent oxidoreductase, directly reduces the oxidized form of MtAhpE, through a protein mixed disulfide with the N-terminal cysteine of MtMrx1 and the sulfenic acid derivative of the peroxidatic cysteine of MtAhpE. This disulfide is then reduced by the C-terminal cysteine in MtMrx1. Accordingly, MtAhpE catalyzes the oxidation of wt MtMrx1 by hydrogen peroxide but not of MtMrx1 lacking the C-terminal cysteine, confirming a dithiolic mechanism. Alternatively, oxidized MtAhpE forms a mixed disulfide with mycothiol, which in turn is reduced by MtMrx1 using a monothiolic mechanism. We demonstrated the H2O2-dependent NADPH oxidation catalyzed by MtAhpE in the presence of MR, Mrx1, and mycothiol. Disulfide formation involving mycothiol probably competes with the direct reduction by MtMrx1 in aqueous intracellular media, where mycothiol is present at millimolar concentrations. However, MtAhpE was found to be associated with the membrane fraction, and since mycothiol is hydrophilic, direct reduction by MtMrx1 might be favored. The results reported herein allow the rationalization of peroxide detoxification actions inferred for mycothiol, and more recently, for Mrx1 in cellular systems. We report the first molecular link between a thiol-dependent peroxidase and the mycothiol/Mrx1 pathway in Mycobacteria. 相似文献
3.
Thomas Garbe Spiros Servos Alastair Hawkins George Dimitriadis Douglas Young Gordon Dougan Ian Charles 《Molecular & general genetics : MGG》1991,228(3):385-392
Summary The Mycobacterium tuberculosis shikimate pathway genes designated aroB and aroQ encoding 3-dehydroquinate synthase and 3-dehydroquinase, respectively were isolated by molecular cloning and their nucleotide sequences determined. The deduced dehydroquinate synthase amino acid sequence from M. tuberculosis showed high similarity to those of equivalent enzymes from prokaryotes and filamentous fungi. Surprisingly, the deduced M. tuberculosis 3-dehydroquinase amino acid sequence showed no similarity to other characterised prokaryotic biosynthetic 3-dehydroquinases (bDHQases). A high degree of similarity was observed, however, to the fungal catabolic 3-dehydroquinases (cDHQases) which are active in the quinic acid utilisation pathway and are isozymes of the fungal bDHQases. This finding indicates a common ancestral origin for genes encoding the catabolic dehydroquinases of fungi and the biosynthetic dehydroquinases present in some prokaryotes. Deletion of genes encoding shikimate pathway enzymes represents a possible approach to generation of rationally attenuated strains of M. tuberculosis for use as live vaccines. 相似文献
4.
Ragunathan Priya Goran Biuković Malathy Sony Subramanian Manimekalai Jackwee Lim Srinivasa P. S. Rao Gerhard Grüber 《Journal of bioenergetics and biomembranes》2013,45(1-2):121-129
Tuberculosis, caused by the strain Mycobacterium tuberculosis, is in focus of interest due to the emergence of multi- and extensive drug-resistant TB strains. The F1FO ATP synthase is one of the essential enzymes in energy requirement of both proliferating aerobic and hypoxic dormant stage of mycobacterium life cycle, and therefore a potential TB drug target. Subunit γ of F-ATP synthases plays an important role in coupling and catalysis via conformational transitions of its N- and C-termini as well as the bottom segment of the globular domain of γ, which is in close proximity to the rotating and ion-pumping c-ring. Here we describe the first production, purification and low resolution solution structure of subunit γ (γ1–204, Mtγ1–204) of the M. tuberculosis F-ATP synthase. Mtγ1–204 is a pear-like shaped protein with a molecular weight of 23?±?2 kDa. Protein sequence analysis of Mtγ revealed differences in the amino acid composition to γ subunits from other sources, in particular the presence of a unique stretch of 13 amino acid residues (Mtγ165–178). NMR studies showed that Mtγ165–178 forms a loop of polar residues. Mtγ165–178 has been aligned at the bottom of the globular domain of the Escherichia coli subunit γ, being in close vicinity to the polar residues R41, Q42, E44 and Q46 (M. tuberculosis nomenclature) of the c-ring. The putative role(s) of Mtγ165–178 in coupling and as a potential drug target are discussed. 相似文献
5.
Riccardo Miggiano Valentina Casazza Silvia Garavaglia Maria Ciaramella Giuseppe Perugino Menico Rizzi Franca Rossi 《Journal of bacteriology》2013,195(12):2728-2736
Mycobacterium tuberculosis displays remarkable genetic stability despite continuous exposure to the hostile environment represented by the host''s infected macrophages. Similarly to other organisms, M. tuberculosis possesses multiple systems to counteract the harmful potential of DNA alkylation. In particular, the suicidal enzyme O6-methylguanine-DNA methyltransferase (OGT) is responsible for the direct repair of O6-alkylguanine in double-stranded DNA and is therefore supposed to play a central role in protecting the mycobacterial genome from the risk of G·C-to-A·T transition mutations. Notably, a number of geographically widely distributed M. tuberculosis strains shows nonsynonymous single-nucleotide polymorphisms in their OGT-encoding gene, leading to amino acid substitutions at position 15 (T15S) or position 37 (R37L) of the N-terminal domain of the corresponding protein. However, the role of these mutations in M. tuberculosis pathogenesis is unknown. We describe here the in vitro characterization of M. tuberculosis OGT (MtOGT) and of two point-mutated versions of the protein mimicking the naturally occurring ones, revealing that both mutated proteins are impaired in their activity as a consequence of their lower affinity for alkylated DNA than the wild-type protein. The analysis of the crystal structures of MtOGT and MtOGT-R37L confirms the high level of structural conservation of members of this protein family and provides clues to an understanding of the molecular bases for the reduced affinity for the natural substrate displayed by mutated MtOGT. Our in vitro results could contribute to validate the inferred participation of mutated OGTs in M. tuberculosis phylogeny and biology. 相似文献
6.
Francesca De Luca Mauro Di Vito Elena Fanelli Aurelio Reyes Nicola Greco Carla De Giorgi 《Gene》2009,440(1-2):16-22
The full-length cDNA and the corresponding gene of the heat shock protein 90, Mt-Hsp90, were isolated and characterized in the plant parasitic nematode Meloidogyne artiellia. The full-length Mt-Hsp90 cDNA contained a 5′ untranslated region (UTR) of 45 bp with the 22 bp trans-spliced leader SL1, an ORF of 2172 bp encoding a polypeptide of 723 amino acids and a 3′ UTR of 191 bp. The deduced amino acid sequence of Mt-hsp90 showed high similarity with other known Hsp90s. Five conserved amino acid signatures indicated that Mt-hsp90 is a cytosolic member of the Hsp90 family. The gene consists of 10 exons and 9 introns, a more expanded gene structure compared to the corresponding Caenorhabditis elegans gene, daf-21. Mt-hsp90 gene was constitutively expressed at high levels in all developmental stages of M. artiellia. Egg masses and second stage juveniles (J2s) were exposed at 5° and 30 °C for different periods of times in order to explore the impact of adverse temperature on Mt-hsp90 gene expression. Expression levels of Mt-hsp90 were examined by fluorescent real-time PCR. At 30 °C a burst of expression for Mt-hsp90 was observed in J2s after 2 h of heat shock treatment, then expression dropped with longer exposing times, although remaining still relatively high after 24 h. This temperature did not affect Mt-hsp90 gene expression in the egg masses. However, egg masses exposed at 5 °C showed a little but gradual increase in the mRNA level with time. By contrast, no significant changes in the Mt-hsp90 level were observed in J2s exposed to cold. These data show that egg masses and J2s exposed to cold and heat stresses have different expression profiles suggesting that Mt-Hsp90 may provide a link between environmental conditions and the life cycle of the nematode. 相似文献
7.
Anne Drumond Villela Rodrigo Gay Ducati Leonardo Astolfi Rosado Carlos Junior Bloch Maura Vianna Prates Danieli Cristina Gon?alves Carlos Henrique Inacio Ramos Luiz Augusto Basso Diogenes Santiago Santos 《PloS one》2013,8(2)
Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PPi). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PPi product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis. 相似文献
8.
Biling Huang Jinglin Fu Chenyun Guo Xueji Wu Donghai Lin Xinli Liao 《Biomolecular NMR assignments》2016,10(2):321-324
Ribosomal protein S1 of Mycobacterium tuberculosis (MtRpsA) binds to ribosome and mRNA, and plays significant role in the regulation of translation initiation, conventional protein synthesis and transfer-messenger RNA (tmRNA) mediated trans-translation. It has been identified as the target of pyrazinoic acid (POA), a bactericidal moiety from hydrolysis of pyrazinamide, which is a mainstay of combination therapy for tuberculosis. POA prevented the interactions between the C-terminal S1 domain of MtRpsA (residues 280–368, MtRpsACTD_S1) and tmRNA; so that POA can inhibit the trans-translation, which is a key component of multiple quality control pathways in bacteria. However, the details of molecular mechanism and dynamic characteristics for MtRpsACTD_S1 interactions with POA, tmRNA or mRNA are still unclear. Here we present the 1H, 15N, 13C resonance assignments of MtRpsACTD_S1 as well as the secondary structure information based on backbone chemical shifts, which lay foundation for further solution structure determination, dynamic properties characterization and interactions investigation between MtRpsACTD_S1 and tmRNA, RNA or POA. 相似文献
9.
Satya Tapas Girijesh Kumar Patel Sonali Dhindwal Shailly Tomar 《Journal of molecular modeling》2011,17(4):621-631
The shikimate pathway is involved in production of aromatic amino acids in microorganisms and plants. The enzymes of this
biosynthetic pathway are a potential target for the design of antimicrobial compounds and herbicides. 3-deoxy-D-arabinoheptulosonate-7-phosphate
synthase (DAHPS) catalyzes the first step of the pathway. The gene encoding DAHPS was cloned and sequenced from Pseudomonas fragi, the bacterium responsible for spoilage of milk, dairy products and meat. Amino acid sequence deduced from the nucleotide
sequence revealed that P. fragi DAHPS (Pf-DAHPS) consists of 448 amino acids with calculated molecular weight of ∼50 kDa and isoelectric point of 5.81. Primary sequence
analysis of Pf-DAHPS shows that it has more than 84% identity with DAHPS of other Pseudomonas species, 46% identity with Mycobacterium tuberculosis DAHPS (Mt-DAHPS), the type II DAHPS and less than 11% sequence identity with the type I DAHPS. The three-dimensional structure of Pf-DAHPS was predicted by homology modeling based on the crystal structure of Mt-DAHPS. Pf-DAHPS model contains a (β/α)8 TIM barrel structure. Sequence alignment, phylogenetic analysis and 3D structure model classifies Pf-DAHPS as a type II DAHPS. Sequence analysis revealed the presence of DAHPS signature motif DxxHxN in Pf-DAHPS. Highly conserved sequence motif RxxxxxxKPRT(S/T) and xGxR present in type II DAHPS were also identified in Pf-DAHPS sequence. High sequence homology of DAHPS within Pseudomonas species points to the option of designing a broad spectrum drug for the genus. Pf-DAHPS 3D model provides molecular insights that may be beneficial in rationale inhibitor design for developing effective
food preservative against P. fragi. 相似文献
10.
《Biochimica et Biophysica Acta (BBA)/General Subjects》2017,1861(9):2354-2366
Mycobacteria employ a versatile machinery of the mycothiol-dependent system, containing the proteins mycothiol disulfide reductase (Mtr), the oxido-reductase Mycoredoxin-1 (Mrx-1) and the alkyl-hydroperoxide subunit E (AhpE). The mycothiol-dependent protein ensemble regulates the balance of oxidized-reduced mycothiol, to ensure a reductive intracellular environment for optimal functioning of its proteins even upon exposure to oxidative stress. Here, we determined the first low-resolution solution structure of Mycobacterium tuberculosis Mtr (MtMtr) derived from small-angle X-ray scattering data, which provides insight into its dimeric state. The solution shape reveals the two NADPH-binding domains inside the dimeric MtMtr in different conformations. NMR-titration shows that the MtMtr-MtMrx-1 interaction is characterized by a fast exchange regime and critical residues involved in the protein-protein interaction were identified. Using NMR spectroscopy and docking studies, the epitopes of MtMrx-1 and MtAhpE interaction are described, shedding new light into the interaction interface and mechanism of action. Finally, the essential residue of MtMrx-1 identified in the interaction with MtMtr and MtAhpE form a platform for structure-guided drug design against the versatile enzyme machinery of the mycothiol-dependent system inside M. tuberculosis. 相似文献
11.
Fatty acid synthesis is essential for cell growth and viability. The 3-oxoacyl-acyl carrier protein synthase II (KAS II) from
Mycobacterium tuberculosis catalyses initiation of the fatty acid synthesis pathway by condensation of acyl CoA and mycolic acid during the elongation
phase. KAS II is a key regulator of bacterial fatty acid synthesis, and a promising target in the search for potent antibacterial
drugs. Homology modelling was used to generate the 3-D protein structure using the known crystal structure, and the stereochemical
quality of KAS II was validated. Effective drugs were selected that target the active amino acid residues of KAS II. The drugs
thiolactomycin, thiophenone and the multidrug cerulenin isoniazed were found to be more potent for inhibition of M. tuberculosis due to the robust binding affinity of their protein–drug interactions. KAS II enzymes of M. tuberculosis and other species of Mycobacterium are conserved, as revealed by their close phylogenetic relationships. This study may provide new insights towards understanding
the 3-D structural conformation and active amino acids of KAS II, thus providing rationale for the design of novel antibacterial
drugs. 相似文献
12.
《Bioorganic & medicinal chemistry》2020,28(15):115600
The enzyme dihydrofolate reductase from M. tuberculosis (MtDHFR) has a high unexploited potential to be a target for new drugs against tuberculosis (TB), due to its importance for pathogen survival. Preliminary studies have obtained fragment-like molecules with low affinity to MtDHFR which can potentially become lead compounds. Taking this into account, the fragment MB872 was used as a prototype for analogue development by bioisosterism/retro-bioisosterism, which resulted in 20 new substituted 3-benzoic acid derivatives. Compounds were active against MtDHFR, with IC50 values ranging from 7 to 40 μM, where compound 4e not only had the best inhibitory activity (IC50 = 7 μM), but also was 71-fold more active than the original fragment MB872. The 4e inhibition kinetics indicated an uncompetitive mechanism, which was supported by molecular modeling which suggested that the compounds can access an independent backpocket from the substrate and competitive inhibitors. Thus, based on these results, substituted 3-benzoic acid derivatives have strong potential to be developed as novel MtDHFR inhibitors and also anti-TB agents. 相似文献
13.
An important cause of bacterial resistance to aminoglycoside antibiotics is the enzymatic acetylation of their amino groups by acetyltransferases, which abolishes their binding to and inhibition of the bacterial ribosome. Enhanced intracellular survival (Eis) protein from Mycobacterium tuberculosis (Mt) is one of such acetyltransferases, whose upregulation was recently established as a cause of resistance to aminoglycosides in clinical cases of drug-resistant tuberculosis. The mechanism of aminoglycoside acetylation by MtEis is not completely understood. A systematic analysis of steady-state kinetics of acetylation of kanamycin A and neomycin B by Eis as a function of concentrations of these aminoglycosides and the acetyl donor, acetyl coenzyme A, reveals that MtEis employs a random-sequential bisubstrate mechanism of acetylation and yields the values of the kinetic parameters of this mechanism. The implications of these mechanistic properties for the design of inhibitors of Eis and other aminoglycoside acetyltransferases are discussed. 相似文献
14.
Lorenzo Chiariotti Pietro Alifano M. Stella Carlomagno Carmelo B. Bruni 《Molecular & general genetics : MGG》1986,203(3):382-388
Summary In this paper we report the nucleotide sequence of the hisD gene of Escherichia coli and of the hisIE region of both E. coli and Salmonella typhimurium. The hisD gene codes for a bifunctional enzyme, l-histidinol: NAD+ oxidoreductase, of 434 amino acids with a molecular mass of 46,199 daltons. We established that the hisIE region of both S. typhimurium and E. coli is composed of a single gene and not, as previously believed, of two separate genes. The derived amino acid sequence indicates that the hisIE gene codes for a bifunctional protein of 203 amino acids with an approximate molecular mass of 22,700 daltons. We also determined the nucleotide sequence of a deletion mutant in S. typhimurium which abolishes the hisF and hisI functions but retains the hisE function. We deduced that the mutant produces a chimeric protein fusing the aminoterminal region of the upstream hisF gene to the carboxylterminal domain of the hisIE gene which encodes for the hisE function. In view of these results the structural and functional organization of the histidine operon in enteric bacteria needs to be revised. The operon is composed of only 8 genes and the pathway leading to the biosynthesis of the amino acid requires 11 enzymatic steps. 相似文献
15.
Irina A. Rodionova Harmon J. Zuccola Leonardo Sorci Alexander E. Aleshin Marat D. Kazanov Chen-Ting Ma Eduard Sergienko Eric J. Rubin Christopher P. Locher Andrei L. Osterman 《The Journal of biological chemistry》2015,290(12):7693-7706
Nicotinate mononucleotide adenylyltransferase NadD is an essential enzyme in the
biosynthesis of the NAD cofactor, which has been implicated as a target for
developing new antimycobacterial therapies. Here we report the crystal structure
of Mycobacterium tuberculosis NadD (MtNadD) at
a resolution of 2.4 Å. A remarkable new feature of the
MtNadD structure, compared with other members of this
enzyme family, is a 310 helix that locks the active site in an
over-closed conformation. As a result, MtNadD is rendered
inactive as it is topologically incompatible with substrate binding and
catalysis. Directed mutagenesis was also used to further dissect the structural
elements that contribute to the interactions of the two MtNadD
substrates, i.e. ATP and nicotinic acid mononucleotide (NaMN).
For inhibitory profiling of partially active mutants and wild type
MtNadD, we used a small molecule inhibitor of
MtNadD with moderate affinity
(Ki ∼ 25 μm) and
antimycobacterial activity (MIC80) ∼ 40–80
μm). This analysis revealed interferences with some of the
residues in the NaMN binding subsite consistent with the competitive inhibition
observed for the NaMN substrate (but not ATP). A detailed steady-state kinetic
analysis of MtNadD suggests that ATP must first bind to allow
efficient NaMN binding and catalysis. This sequential mechanism is consistent
with the requirement of transition to catalytically competent (open)
conformation hypothesized from structural modeling. A possible physiological
significance of this mechanism is to enable the down-regulation of NAD synthesis
under ATP-limiting dormancy conditions. These findings point to a possible new
strategy for designing inhibitors that lock the enzyme in the inactive
over-closed conformation. 相似文献
16.
17.
Gisele Biazus Cristopher Z. Schneider Mario S. Palma Luiz A. Basso Digenes S. Santos 《Protein expression and purification》2009,66(2):185-190
Human tuberculosis (TB) is a major cause of morbidity and mortality worldwide, especially in poor and developing countries. Moreover, the emergence of Mycobacterium tuberculosis strains resistant to first- and second-line anti-TB drugs raises the prospect of virtually incurable TB. Enzymes of the purine phosphoribosyltransferase (PRTase) family are components of purine salvage pathway and have been proposed as drug targets for the development of chemotherapeutic agents against infective and parasitic diseases. The PRTase-catalyzed chemical reaction involves the ribophosphorylation in one step of purine bases (adenine, guanine, hypoxanthine, or xanthine) and their analogues to the respective nucleoside 5′-monophosphate and pyrophosphate. Hypoxanthine–guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8) is a purine salvage pathway enzyme that specifically recycles hypoxanthine and guanine from the medium, which are in turn converted to, respectively, IMP and GMP. Here we report cloning, DNA sequencing, expression in Escherichia coli BL21 (DE3) cells, purification to homogeneity, N-terminal amino acid sequencing, mass spectrometry analysis, and determination of apparent steady-state kinetic parameters for an in silico predicted M. tuberculosis HGPRT enzyme. These data represent an initial step towards future functional and structural studies, and provide a solid foundation on which to base M. tuberculosis HGPRT-encoding gene manipulation experiments to demonstrate its role in the biology of the bacillus. 相似文献
18.
19.
Carmelo B. Bruni M. Stella Carlomagno Silvestro Formisano Giovanni Paolella 《Molecular & general genetics : MGG》1986,203(3):389-396
Summary A detailed comparative analysis of the Escherichia coli and Salmonella typhimurium hisIE and hisD gene products and the functionally equivalent, single, HIS4 gene product of Saccharomyces cerevisiae permitted several insights concerning the relationship between these genes. Our analysis supports the idea that HIS4 results from the fusion of hisIE and hisD. The comparison permitted a more precise definition of the functional domains of hisI/HIS4A and hisE/HIS4B as well as the two functional domains of hisD/HIS4C. The homologies between the bacterial and yeast sequences suggest a region of the hisD/HIS4C protein that may constitute one of the active centres. A large fragment at the amino terminal region of the yeast protein is missing from the bacterial hisIE gene product and is probably not needed for catalytic activity. Another region of non-homology in the yeast protein is probably a peptide bridge connecting the HIS4AB domain to HIS4C. Although the overall homology at the level of amino acid sequence is modest (about 38%) there is a striking similarity when the hydropathic patterns and predicted secondary structural configurations of these proteins are compared. 相似文献
20.
Manuel Megias Miguel A. Caviedes Mercedes Andres Carolina Sousa Francisco Ruiz-Berraquero Antonio J. Palomares 《Molecular & general genetics : MGG》1988,211(2):369-372
Summary We have studied N-methyl-N-nitro-N-nitrosoguanidine (NTG)- and Tn5-induced histidine auxotrophic mutants in Rhizobium trifolii. HisGE, hisD and hisH mutants have been characterized. Using the Kemper's equation we have located them on the R. trifolii linkage map. The hisGE and hisD genes are clustered in the same region and are closely linked to the spectinomycin marker. The hisH gene is located in another region equidistant from the streptomycin and rifampicin markers. The two regions carrying his genes are separated by a distance approximately one-third of the length of the chromosome. 相似文献