Single amino acid replacements affecting the thermostability of kanamycin nucleotidyltransferase

Summary Amino acid residues of the carboxyl-terminal region of kanamycin nucleotidyltransferase were modified using segment-directed mutagenesis. Six different mutant enzymes with single amino acid replacements were selected out of 59 clones by DNA sequence analyses. The mutant enzymes were purified and it was found that the thermostability of one mutant enzyme was identical to the wild type, whereas the other five were less thermostable at varying degrees. The data suggested that changes in the enzyme thermostability depend not only on the position but also on the species of amino acid residue replaced.     

The isolation and characterization of ngm2, a mutation that affects nitrosoguanidine mutagenesis in yeast

Summary We have isolated and characterized a new mutant of Saccharomyces cerevisiae, carrying a single mutant allele that we designate ngm2-1, which is defective with respect to induced mutagenesis. This mutant was isolated by screening mutagenized clones for reduced frequencies of reversion of the his1-7 allele, induced by N-methyl-N-nitro-N-nitrosoguanidine. As judged by the reversion of his1-7 and ilv1-92, ngm2-1 mutant strains are also deficient with respect to mutability induced by methyl methane sulfonate, ethyl methane sulfonate and, at least partially, by UV. UV-induced reversion of the ochre mutation arg4-17 and the frameshift mutation his4-38 was not much affected by ngm2-1, however. Like rev3 and rev7 mutations, ngm2-1 also has little influence on the reversion of the proline missense allele, cyc1-115. Ngm2-1 mutants are only at best very slightly more sensitive to the toxicity of the four mutagens used, and homozygous diploids sporulate normally.     

Complementation of an Erwinia carotovora subsp. carotovora protease mutant with a protease-encoding cosmid

Summary We report the complementation of a genetic lesion in the genome of Erwinia carotovora subsp. carotovora (Ecc), a pathogenic bacterium that incites soft rot of plants. A Sau3AI genomic library of Ecc was constructed using the conjugal cosmid pLAFR-3 as a vector. Sixteen cosmid clones encoding various plant tissue-degrading enzymes were identified, including a proteolytic clone, five cellulolytic clones, and ten pectolytic clones. We detected a mutant of Ecc with no proteolytic activity following transposon mutagenesis with an unstable Tn5-carrying plasmid. Conjugal transfer of the protease-encoding cosmid to this mutant restored near-wildtype extracellular protease production. Further manipulation and study of genes encoding pathogenic determinants in Ecc will be possible using this system.     

Mutational specificity of a proof-reading defective Escherichia coli dnaQ49 mutator

Summary The dnaQ (mutD) gene product which encodes the -subunit of the DNA polymerase III holoenzyme has a central role in controlling the fidelity of DNA replication because both mutD5 and dnaQ49 mutations severely decrease the 3–5 exonucleolytic editing capacity.It is shown in this paper that more than 95% of all anaQ49-induced base pair substitutions are transversions of the types G:C-T:A and A:T-T:A. Not only is this unusual mutational specificity precisely that observed recently for a number of potent carcinogens such as benzo(a) pyrene diolepoxide (BPDE) and aflatoxin B1 (AFB1), which are dependent on the SOS system to mutagenize bacteria, but it is also seen for the constitutively expressed SOS mutator activity in E. coli tif-1 strains as well as for the SOS mutator activity mediated gap filling of apurinic sites. Because the G:C-T:A and A:T-T:A transversions can either result from the insertion of an adenine across from apurinic sites or arise due to the incorporation of syn-adenine opposite a purine base, we postulate that the DNA polymerase III holoenzyme also has a reduced discrimination ability in a dnaQ49 background.The introduction of a lexA (Ind^-) allele, which prevents the expression of SOS functions, led to a significant reduction in the dnaQ49-caused mutator effect.Both, the mutational specificity observed and the partial lexA ⁺ dependence of the mutator effect provoke a reanalysis of the hypothesis that the DNA polymerase III holoenzyme can be converted into the postulated but until now unidentified SOS polymerase.     

Symbiotically defective histidine auxotrophs of Bradyrhizobium japonicum

Four histidine auxotrophs of Bradyrhizobium japonicum strain USDA 122 were isolated by random transposon Tn5 mutagenesis. These mutants arose from different, single transposition events as shown by the comparison of EcoRI and XhoI-generated Tn5 flanking sequences of genomic DNA. The mutants grew on minimal medium supplemented with l-histidine or l-histidinol but failed to grow with l-histidinol phosphate. While two of the muants were symbiotically defective and did not form nodules on Glycine max cvs. Lee and Peking and on Glycine soja, the other two mutants were symbiotically competent. Reversion to prototrophy occurred at a frequency of about 10^-7 on growth medium without added antibiotics, but prototrophs could not be isolated from growth medium containing 200 g/ml kanamycin and streptomycin. The prototrophic revertants formed nodules on all the soybean cultivars examined. When histidine was supplied to the plant growth medium, both nodulation deficient mutants formed effective symbioses. On histidine unamended plants, nodules were observed infrequently. Three classes of bacterial colonies were isolated from such infrequent nodules: class 1 were kanamycin resistant-auxotrophs; class 2 were kanamycin sensitive-prototrophs; and class 3 were kanamycin-sensitive auxotrophs. Our results suggest that two Tn5 insertion mutations in B. japonicum leading to histidine auxotrophy, affect nodulation in some way. These mutations are in regions that show no homology to the Rhizobium meliloti common nodulation genes.     

Substrate specificities in class A beta-lactamases: preference for penams vs. cephems. The role of residue 237

Site saturation mutagenesis has been carried out at Ala-237 in RTEM-1 beta-lactamase to assess the role of this site in modulating differences in specificity of beta-lactamases for penams vs. cephems as substrates. (An Ala-237 Thr mutation had previously been shown to increase activity on cephems by about 30-80%.) Screening of all 19 possible mutants on penams and cephems revealed the even more active Ala-237 Asn mutant. Detailed kinetic analysis shows that this mutant has about four times the activity toward cephalothin and cephalosporin C as the wild-type enzyme. Both mutations reduce the activity toward penams to about 10% that of RTEM-1 beta-lactamase and lower by about 5 degrees C the temperature at which the enzyme denatures. Functional properties of the other mutants have also been surveyed. The most interesting aspect of these results is that two quite disparate amino acids, threonine and asparagine, when introduced for Ala-237, cause such similar changes in enzyme specificity while more similar residues do not alter the catalytic properties of the enzyme to such a significant degree.     

Genetic approaches for studying rhizosphere colonization

Most bacterial traits involved in colonization of plant roots are yet to be defined. Studies were initiated to identify genes in Pseudomonas which play significant roles in this process. The general approach is to use transposons to construct collections of insertion mutants, each of which is then screened for alterations in its interactions with the host plant. In one study a Tn5 derivative containing a constitutively expressed -galactosidase (lacZ) gene was used to generate a collection of insertion mutants which could be distinguished from the wild-type parent on X-gal plates. Each mutant was examined for its ability to colonize wheat seedlings in the presence of the wild-type parent. Mutants which gave wild-type:mutant ratio of 20:1 or greater were obtained. In a second study a Tn5 derivative which carries a promoterless lacZ gene located near one end of the transposon was constructed. Expression of the lacZ gene depends on the presence of an active promoter outside of the transposon in the correct orientation. Insertion mutants generated with this transposon were examined for changes in -galactosidase expression in the presence and absence of plant root exudate. A number of mutants which showed differential lacZ expression have been identified.     

The induction of lincomycin resistance in Lycopersicon peruvianum and Lycopersicon esculentum

Lincomycin-resistant calli were induced from both Lycopersicon esculentum and Lycopersicon peruvianum using N-mitroso-N-methylurea (NMU) mutagenesis. From these calli lincomycin-resistant plants were regenerated. For L. peruvianum it was shown that the resistant plants could be divided in two classes with respect to their resistance to lincomycin and its derivative clindamycin. The first class comprised plants which were resistant to 500 mg/l lincomycin and showed no shoot or root formation in the presence of clindamycin; the second class consisted of plants resistant to 2000 mg/l lincomycin and these plants were able to form shoots and roots on clindamycin containing media. Lincomycin is an inhibitor of peptidyltransferase; chloroplast encoded parts of this enzymatic function are sensitive for this antibiotic. Reciprocal crosses between our lincomycin resistant and wild type L. peruvianum plants indicated a maternal inheritance of the mutation.