Fernando Bastarrachea

ColE1 hybrid plasmids containing Escherichia coli genes involved in the biosynthesis of glutamate and glutamine

Abstract The Clarke-Carbon bank of Escherichia coli strains carrying ColE1 hybrid plasmids was screened for complementation of gdh, gltB, and glnA mutations affecting nitrogen metabolism in E. coli. Plasmids which complemented each one of these mutations were isolated. In every case, the plasmids conferred to otherwise mutant cells the capacity to synthesize the corresponding wild-type enzymes: glutamate dehydrogenase, glutamate synthase, and glutamine synthetase (GS), respectively. For three representative plasmids, endonuclease restriction maps were constructed. One of the plasmids, pACR1, which complemented glnA mutations, including the glnA21::Tn5 insertion, was deemed to carry the glnA+ allele. GS synthesis by pACR1 glnA + glnA20 heterozygous merodiploids was subjected to repression by growth on 15 m m NH4+ and had a twofold high derepressed level than wild-type (glnA+) haploid cells when grown on 0.5 m m NH4+ or on glutamate as only nitrogen sources. The presence of glutamine as sole nitrogen source promoted repressed GS synthesis in the glnA + glnA20 merodiploids. By contrast, glutamine allowed almost fully derepressed synthesis of GS in glnA+ haploid cells.

Nitrogen Regulation of Synthesis of the High Affinity Methylammonium Transport System of Escherichia coli

Uptake of 14CH3NH+3 (methylammonium) was measured as a probe of NH+4 transport in intact Escherichia coli cells and derivatives impaired in the Ntr regulatory system. The results suggest that expression of the high affinity 14CH3NH+3 transport system (a) requires de novo polypeptide synthesis, (b) is activated by the glnG and glnF regulatory products under nitrogen limitation, and (c) is repressed under nitrogen excess by the glnL product. Cells deficient in glutamate synthase activity by virtue of their harbouring the gltB31 mutation were unable to activate synthesis of 14CH3NH+3 transport. This could explain the inability of cells carrying gltB mutations to grow on low concentrations of NH+4.

Adhesiveness and root hair deformation capacity of Azospirillum strains for wheat seedlings

Abstract A binding assay for Azospirillum cells adsorbed to wheat roots was developed. It consisted of incubation exposing germinating seedlings to bacterial cells in liquid Fahreaus solution at 30°C for 2 h. The seedlings were removed and washed four times in Fahreaus solution followed by centrifugation for 15 min at 300 rev min −1 after every washing. The number of Azospirilla that remained attached to the roots was estimated by plating serial dilutions on agar media of root homogenates to enable colony counts. Optimal assay conditions for inoculum size, pH, age of the cultures and exposure time were determined. Binding kinetic data best fitted a Langmuir adsorption isotherm thus indicating a saturable, specific number of available sites on the roots for the adsorption of Azospirillum brasilense Sp245. Competition assays with different Rhizobium species were carried out showing a preferential adsorption for Azospirillum .

Nitrogen regulation in an Escherichia coli strain with a temperature sensitive glutamyl-tRNA synthetase

Escherichia coli cells carrying the gltX351 allele are unable to grow at 42° C (Ts phenotype) due to an altered glutamyl-tRNA synthetase. We found that gltX351 cells display a new phenotype termed Gsd−, i.e. an inability to raise glutamine synthetase activity above low constitutive levels in minimal medium with 6.8 mM glutamine as sole nitrogen source. When 0.5 mM NH4+or 12 mM glutamate replaced glutamine, the glutamine synthetase activities of gltX351 cells were raised to wildtype levels. Northern experiments showed that the Gsd− phenotype is the result of an impairment in transcription initiation from the Ntr-regulated promoter, glnAp2. Intragenic and extragenic secondary mutations appeared frequently in gltX351 cells, which suppressed their Gsd− but not their TS phenotype. Moreover, in heterozygous gltX+/gltX351 partial diploids, gltX351 was dominant for the Gsd− phenotype and recessive for the Tr phenotype. A slight increase in the glutamine pool and in the intracellular glutamine: 2-oxoglutarate ratio was also observed but this could not account for the Gsd− phenotype of gltX351 cells. In cells carrying gltX351 and a suppressor of the Gsd− phenotype, sup-1, tightly linked to gltX351, the glutamine pool and glutamine: 2-oxoglutarate intracellular ratio were even higher than in the gltX351 single mutant. These results indicate that the gltX351 mutant polypeptide may be the direct cause of the Gsd− phenotype. The possibility that it interacts with one or more components that trigger the Ntr response is discussed.

Nucleotide sequence of the glnA control region of Escherichia coli

SummaryThe RNA polymerase binding sites present along a DNA segment encompasing the glnA, glnL, and glnG genes have been identified in a hybrid plasmid carrying this chromosomal region of Escherichia coli. The DNA sequence was determined of an 817 base pair segment that contains the region coding for the first 42 amino acids of the NH2-terminal and of the glnA structural gene, as well as its regulatory region. Analysis of this nucleotide sequence revealed three probable RNA polymerase recognition sites, imperfect palindromes, inverted repeats, and direct repeated sequences.

Genetic characterization of streptomycin-resistant and -dependent mutants of Escherichia coli K12.

SummaryStreptomycin high resistant and dependent mutants of E. coli K12 were isolated and characterized with regard to a) permissiveness for phenotypic suppression by streptomycin of the conditional streptomycin-dependent mutation thr-1, and b) restriction of the activity of the sup-59 amber suppressor. The majority of the streptomycin-resistant the strA mutation. Streptomycin-dependent (SmHD) mutants were grouped into three main classes, SmD Et− PmS, SmD EtD PmS and SmD EtD PmD or DrugD, according to their ability to allow paromomycin or ethanol to act as substitutes for streptomycin for growth. In addition, streptomycin-dependent mutants were classified as either permissive (Per+) or nonpermissive (Per−) with regard to the streptomycin-mediated phenotypic suppression of the thr-1 mutation and of two T4 amber mutations. This behavior was shown to be due to different allelic states of another gene, strM. Mutations at strM were responsible for the Per− phenotype in SmHD strains. In streptomycin-sensitive or-resistant bacteria, strM remained largely unexpressed. strM was located at approximately min 68 of the standard E. coli map. In strM heterozygous merodiploids, the wild-type Per+ phenotype was dominant over Per−. The nature of the strM product remains unknown. Restriction of sup-59 activity by streptomycin resistant and dependent mutations was infrequent, and was not correlated with the mutants behavior in phenotypic suppression. Although strM-mediated phenotypic suppression counteracted the restriction imposed by strA mutations, apparently it did not act by restoring the activity of genetic suppressors.