Use of 5-fluorouracil for the isolation of auxotrophic mutants of Bacillus megaterium

Wachsman, J. T. (University of Illinois, Urbana), and L. Hogg. Use of 5-fluorouracil for the isolation of auxotrophic mutants of Bacillus megaterium. J. Bacteriol. 87:1137-1139. 1964.-The combination of 5-fluorouracil (FU) and uridine was used to selectively kill wild-type cells of Bacillus megaterium KM, thereby providing surviving populations greatly enriched in auxotrophic mutants. Exponentially growing cells were irradiated with ultraviolet light, incubated in a basal medium containing sucrose and, in most experiments, a complete amino acid mixture. Exponentially growing cells were then washed and incubated in the basal medium containing only sucrose, to deplete intracellular reserves. FU and uridine were added, and incubation was continued. After 5 hr, auxotrophs may account for up to 50% of the survivors. Organisms requiring each of the following compounds were identified: alanine, arginine, asparagine, cysteine, histidine, phenylalanine, serine, threonine, tyrosine, adenine, and guanine.     

The inhibition of staphylococcal beta-lactamase by clavulanic acid.

Clavulanic acid inhibited both the extracellular and cell-extract beta-lactamases of the four Staphylococcus aureus strains tested. The inhibition of S. aureus Russell cell-extract enzyme appeared to be active-site-directed and proceeded in a first-order fashion consistent with the formation of a covalent intermediate. Inhibited enzyme free of excess clavulanic acid was shown to regenerate enzyme activity slowly at pH 7.0, but the rate of reactivation increased at acid pH. When the enzyme was incubated with excess clavulanic acid complete inhibition was rapidly obtained, during further incubation clavulanic acid was shown to disappear slowly and complete loss of clavulanic acid from the reaction mixture coincided with the onset of the return of enzyme activity. A reactive enamine resulting from enzymic hydrolysis of the beta-lactam ring of clavulanic acid has been proposed as a possible intermediate in the inhibitory mechanism.     

Use of thymineless death to enrich for doubly auxotrophic mutants of Bacillus megaterium

Wachsman, J. T. (University of Illinois, Urbana), and L. Hogg. Use of thymineless death to enrich for doubly auxotrophic mutants of Bacillus megaterium. J. Bacteriol. 87:1118-1122. 1964.-When strain KM:T(-), a thymine auxotroph of Bacillus megaterium strain KM, is allowed to undergo thymineless death on a minimal medium, the survivors are greatly enriched in polyauxotrophic mutants. Cells were irradiated with ultraviolet light, grown in the presence of thymidine and a complete amino acid mixture, and then starved for thymidine in the absence of amino acids. Doubly auxotrophic mutants (thymine(-) amino acid(-)) may account for more than 90% of the survivors. The most reproducible results were obtained when sucrose (0.4 m) was added to both growth and starvation media. Although the percentage of mutants among the survivors increases with the time of thymine starvation, the absolute number of double auxotrophs per milliliter decreases. It is probable that the extent of cross-feeding determines both the mutant yield and the mutants types. Substrains of KM:T(-) having additional requirements for each of the following amino acids have been isolated: histidine, threonine, tyrosine, tryptophan, arginine, isoleucine, methionine, serine, and cysteine.     

UV-mutagenesis in Bacillus subtilis. VII. Induction of auxotrophic mutants]

An experimental testing of material from thin-layered, transparent in passing light, colonies which appear with some frequency after plating Bacillus subtilis cells on agar medium with limited enrichment, has shown that such colonies are formed by auxotrophic mutants. The growth requirements for many of them has been identified. The most of mutants can be reversed to original phenotype by UV-irradiation. The frequency of auxotrophs increases after UV-irradiation of suspension of original cells. The sensitivity of auxotrophic mutants to inactivating action of UV-light is near to that of original cells, hence the increase of the frequency of mutants with dose is a result of induction, but not of selection of preexisting spontaneous auxotrophic mutants. The frequency of induced auxotrophs, in contrast to that of suppressor revertants, badly give way to declining in the time of temporary inhibition of postradiation growth. In the case of Bac, subtilis, the system of induced auxotrophic mutants on the medium with limited enrichment is rather comfortable in use and can be recommended for studying UV-induced mutagenesis in structural genes as well as for testing mutagenic activities.     

Export and folding of signal-sequenceless Bacillus licheniformis beta-lactamase in Escherichia coli.

Two genetically engineered variants of the Bacillus licheniformis beta-lactamase gene were expressed in Escherichia coli. One variant coded for the exo-small mature enzyme without the signal peptide. The other coded for the exo-large mature enzyme preceded by 10, mostly polar, residues from an incomplete heterologous signal. As observed following the extraction by a lysozyme-EDTA treatment, the signal-less variant was exported to the periplasm with nearly 20% efficiency, whereas the variant with the N-terminal extension was translocated to a lesser degree; interestingly, nearly all of the former and half of the latter were extracted by osmotic shock, which may be of importance for our understanding of cellular compartments. The fact that a signal-less protein is translocated with substantial yields raises questions about the essential role of signal peptides for protein export. As folding and export are related processes, we investigated the folding in vitro of the two variants. No differences were found between them. In the absence of denaturant, they are completely folded, fully active and have a large DeltaG of unfolding. Under partially denaturing conditions they populate several partially folded states. The absence of significant amounts of a non-native state under native conditions makes a thermodynamic partitioning between folding and export less likely. In addition, kinetic measurements indicated that these B. licheniformis lactamases fold much faster than E. coli beta-lactamase. This behavior suggests that they are exported by a kinetically controlled process, mediated by one or more still unidentified interactions that slow folding and allow a folding intermediate to enter the export pathway.     

L-Lysine production by mutants of Bacillus licheniformis

Summary A bacterium able to grow at 46°C was isolated from soil and identified asBacillus licheniformis. L-Lysine producing mutants were derived from the bacterium by the introduction of resistance to S-(2-amino)-ethylcysteine (thialysine) and auxotrophy.One of the mutants, HBR-2 (Thialysine^r, Leu^–, Homoserine^–), produced L-lysine at a concentration of 30 mg/ml in a molasses medium containing 10% reducing sugar.     

Kinetics of beta-lactamase inhibition by clavulanic acid]

The mechanisms of action of 3 R-factors on beta-lactamases (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) (TEM-1 pI = 5.4, TEM-2 pI = 5.6 and Pitton's type 2 pI = 7.7) have been kinetically analyzed for clavulanic acid inactivation. Clavulanic acid appears as a competitive and irreversible inhibitor (Kcat inhibitor) reacting in two steps: a, formation of a reversible enzyme . inhibitor complex (characterized by a Ki); b, evolution of the reversible complex into a new derivative (covalent, stable and inactive) by monomolecular kinetics characterized by a k6 (or Kcat) related to half-life. The kinetic constants are: TEM-1: Ki = 0.8 micrometer, k6 = 0.027 s-1; TEM-2: Ki = 0.7 micrometer, k6 = 0.03 s-1; type 2: Ki = 0.6 micrometer, k6 = 0.046 s-1. These results justify the 'progressive irreversible' character of the inhibition generally described.     

Molecular cloning and expression of Bacillus licheniformis beta-lactamase gene in Escherichia coli and Bacillus subtilis.

The chromosomal beta-lactamase (penicillinase, penP) gene from Bacillus licheniformis 749/C has been cloned in Escherichia coli. The locations of the target sites for various restriction enzymes on the 4.2-kilobase EcoRI fragment were determined. By matching the restriction mapping data with the potential nucleotide sequences of the penP gene deduced from known protein sequence, we established the exact position of the penP gene on the fragment. A bifunctional plasmid vector carrying the penP gene, plasmid pOG2165, was constructed which directs the synthesis of the heterologous beta-lactamase in both E. coli and Bacillus subtilis hosts. The protein synthesized in E. coli and B. subtilis is similar in size to the processed beta-lactamase made in B. licheniformis. Furthermore, the beta-lactamase made in B. subtilis is efficiently secreted by the host into the culture medium, indicating that B. subtilis is capable of carrying out the post-translational proteolytic cleavage(s) to convert the membrane-bound precursor enzyme into the soluble extracellular form.     

Clavulanic acid dehydrogenase: structural and biochemical analysis of the final step in the biosynthesis of the beta-lactamase inhibitor clavulanic acid

The ultimate step in the biosynthesis of the medicinally important beta-lactamase inhibitor clavulanic acid is catalyzed by clavulanic acid dehydrogenase (CAD). CAD is responsible for the NAPDH-dependent reduction of the unstable intermediate clavulanate-9-aldehyde to yield clavulanic acid. Here, we report biochemical and structural studies on CAD. Biophysical analyses demonstrate that CAD exists as dimeric and tetrameric species in solution. The reaction performed by CAD was shown to be reversible, allowing the use of clavulanic acid for activity analyses. The crystal structure of CAD was solved using single-wavelength anomalous diffraction with a seleno-methionine derivative. The structure reveals that the individual monomers comprise a single domain possessing the Rossmann fold, characteristic of dinucleotide-binding enzymes. The monomers are arranged as tetramers, similar to other tetrameric members of the short-chain dehydrogenase/reductase family. The structure of the unreactive complex of CAD with clavulanic acid and NADPH suggests how CAD is able to catalyze the reduction of clavulanate-9-aldehyde without fragmentation of the bicyclic beta-lactam ring structure. The relative positions of NADPH and clavulanic acid, in the active site, together with the presence of the latter in an eclipsed conformation, rationalizes previous labeling studies demonstrating that the incorporation of the C5 pro-R, but not pro-S, hydrogen of ornithine/arginine into the C9 position of clavulanic acid occurs with overall inversion of configuration.     

A note on the isolation of auxotrophic mutants of Rhizobium japonicum

Z lotnikov K.M. C hatuev B.M. khmelnitsky , M.I. 1984. A note on the isolation of auxotrophic mutants of Rhizobium japonicum. Journal of Applied Bacteriology 56 , 173–174.
Several different stable auxotrophic mutants of Rhizobium japonicum were isolated following NG mutagenesis. It was found that NG mutagenesis of the rhizobia was most efficient at pH 70.     

Two oligopeptide-permease-encoding genes in the clavulanic acid cluster of Streptomyces clavuligerus are essential for production of the beta-lactamase inhibitor

orf7 (oppA1) and orf15 (oppA2) are located 8 kb apart in the clavulanic acid gene cluster of Streptomyces clavuligerus and encode proteins which are 48.0% identical. These proteins show sequence similarity to periplasmic oligopeptide-binding proteins. Mutant S. clavuligerus oppA1::acc, disrupted in oppA1, lacks clavulanic acid production. Clavulanic acid production is restored by transformation with plasmid pIJ699-oppA1, which carries oppA1, but not with the multicopy plasmid pIJ699-oppA2, which carries oppA2. The mutant S. clavuligerus oppA2::aph also lacks clavulanic acid production, shows a bald phenotype, and overproduces holomycin (5). Clavulanic acid production at low levels is restored in the oppA2-disrupted mutants by transformation with plasmid pIJ699-oppA2, but it is not complemented by the multicopy plasmid pIJ699-oppA1. Both genes encode oligopeptide permeases with different substrate specificities. The disrupted S. clavuligerus oppA2::aph is not able to grow on RPPGFSPFR (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; bradykinin), but both mutants grow on VAPG (Val-Ala-Pro-Gly) as the only nitrogen source, indicating differences in the peptide bound by the proteins encoded by both genes. The null S. clavuligerus oppA1::acc and S. clavuligerus oppA2::aph mutants are more resistant to the toxic tripeptide phosphinothricyl-alanyl-alanine (also named bialaphos) than the wild-type strain, suggesting that this peptide might be transported by these peptide-binding proteins.     

Removal of the fermentation by-product succinyl L-tyrosine from the beta-lactamase inhibitor clavulanic acid using a molecularly imprinted polymer

Clavulanic acid is a beta-lactamase inhibitor used in therapeutic combinations with the penicillin-type antibiotics. During the fermentation leading to clavulanic acid, a succinyl L-tyrosine by-product is unavoidably formed. Occasionally, the amount of this by-product is found to be as high as 2% of the product even after standard purification operations. To further remove this impurity, we prepared a highly specific adsorbent for succinyl L-tyrosine with the molecular imprinting technique. This was performed by simultaneously using vinylbenzyl trimethylammonium chloride and methacrylic acid as the functional monomers. The imprinted polymer selectively bound succinyl L-tyrosine, and could be successfully used to remove this impurity at concentrations of less than 2% in the presence of clavulanic acid.     

Analysis of mutants of Bacillus subtilis, auxotrophic for lysine

A number of B. subtilis mutants auxotrophic for lysine has been isolated and mapped in relation to the flanking ser2 marker. One of the mutants has been found to have a mutation in lys A gene coding for DAP-decarboxylase activity. The expression of DAP-decarboxylase is dependent on the product of lys R gene that is not linked with lysine genes cluster.     

Chemical studies on the inactivation of Escherichia coli RTEM beta-lactamase by clavulanic acid.

Incubation of clavulanic acid with the beta-lactamase from Escherichia coli RTEM leads to enzyme-catalyzed depletion of clavulanic acid, to transient inhibition, and to irreversible inactivation of the enzyme. Both the transiently inhibited and the irreversibly inactivated species show a marked increase in the absorbance at 281 nm that is proportional to the decrease in enzyme activity. Hydroxylamine treatment of irreversibly inactivated enzyme restores about one-third of the catalytic activity, with a concomitant decrease in absorbance at 281 nm. Polyacrylamide isoelectric focusing of the irreversibly inactivated enzyme shows three bands of approximately equal intensity, different from native enzyme. Upon hydroxylamine treatment, one of the three bands disappears and now focuses identically with native enzyme. It is evident that the irreversible inactivation of enzyme by an excess of clavulanic acid generates three products, one of which can be reactivated by hydroxylamine.     

The use of vancomycin for the isolation of auxotrophic mutants inMycobacterium smegmatis

A method using vancomycin for the accumulation of auxotrophic mutants ofMycobacterium smegmatis M54/81 induced by N-methyl-N′-nitro-N-nitrosoguanidine was developed. As compared with the simple replication technique the yield of auxotrophic mutants was twenty-fold.