Alan G. Atherly

Plasmids of Rhizobium and their role in symbiotic nitrogen fixation

Publisher Summary This chapter discusses the plasmids of Rhizobium and their role in symbiotic nitrogen fixation. Rhizobia are gram-negative soil bacteria which fix nitrogen in a symbiotic association with plants of the family Leguminosae; however, this classical definition must be extended now to include nodulation and nitrogen fixation on a nonlegume, Parasponia . Rhizobium parasponia can infect this tropical tree and fix nitrogen in a manner very similar to that observed in legumes. In both the cases, establishment of the symbiosis starts with invasion of plant root or stem by free-living rhizobia followed by a series of steps that result in the formation of a nodule. It is in these nodules that nitrogen fixation takes place. Both the plant and the bacteria undergo differentiation that is regulated by gene expression. Members of the genus Rhizobium are of great economic importance because of their ability to fix nitrogen. The genus has somewhat informally been separated into those species that are fast growers and those that are slow growers. An understanding of the genes of Rhizobium involved in plant symbiosis and nitrogen fixation has moved very rapidly with the fast-growing strains ( Rhizobium leguminosarum , Rhizobium trifolii , Rhizobium meliloti , and R. parasponia and R.fredii ).

In situ detection of transposition of the maize controlling element (Ac) in transgenic soybean tissues

The development of a transposon mutagenesis system in soybean would aid in the isolation of unknown genes. The maize controlling element (Ac) has, therefore, been introduced into the soybean (Glycine max (L.) Merr.) genome byAgrobacterium-mediated transformation.Ac was inserted into the untranslated leader region of the bacterial ß-glucuronidase gene (GUS) such that the excision ofAc resulted in restoration of the GUS gene activity. Excision events of theAc element were monitored by detecting blue cells or sectors in transgenic soybean tissues. Using the GUS gene assay and with hybridization data, we have demonstrated that theAc element transposes in transgenic soybean calli, leaves, stems, and roots.

Studies on the metabolic role of peptidyl-tRNA hydrolase

SummaryA mutant strain of Eschrichia coli that is temperature-sensitive for growth stopped protein biosynthesis at 43° C after a brief lag (Fig. 1). Cell-free extracts from the strain showed no specific defect in aminoacyl-tRNA synthetases, binding initiator tRNA to ribosomes (Table 1), protein chain elongation (Tables 2, 5) or protein chain termination (Tables 3, 4) at high temperature.The partially purified enzyme peptidyl-tRNA hydrolase, however, was temperature-sensitive (Table 6); the mutant hydrolase was inactivated rapidly at 43° C (Table 7). Mixing experiments ruled out the presence, in the mutant enzyme preparation, of an inhibitor and also demonstrated, on the mutant enzyme, a protective effect by wild type enzyme that was not shown by general coli proteins (Tables 8, 9).Interrupted mating allowed the temperature-sensitive growth phenotype to be mapped near to and before trp (Figs. 4, 5). Co-transsduction, mediated by bacteriophage P1, with trp+ (frequency 7.5%) located the marker at 24 min on the coli map. All transductants for temperature-sensitive growth also had temperature-sensitive peptidyl-tRNA hydrolase activity in crude sonicates (Table 10). We provisionally conclude that the temperature-sensitive protein synthesis and growth are caused by a single genetic change in the structural gene (pth) for peptidyl-tRNA hydrolase.After shift to 43° C the polysomes of the mutant cells broke down into 70S particles (Figs. 2, 3). A defect in protein biosynthesis thus appeared to be located after termination and before reformation of new polysomes.The metabolic role of peptidyl-tRNA hydrolase is discussed in the light of these experiments.

Analysis of rice (Oryza sativa L.) genome using pulsed-field gel electrophoresis and rare-cutting restriction endonucleases.

TheOryza sativa (rice) genome is small (600 to 900 megabase pairs) when compared to that of other monocotyledonous plants. Rice was the first of the major cereals to be successfully transformed and regenerated. An RFLP map with approximately 300 markers is readily available, and the DNA content per map unit is only two to three times that ofArabidopsis thaliana. Rice is also the main staple food for the majority of peoples in the world. We developed techniques for the preparation of intact genomic DNA from Indica and Japonica subspecies of rice, used statistical methods to determine which restriction endonucleases are rare-cutting, and used pulsed-field gel electrophoresis (PFE) to separate large fragments of rice DNA. Southern hybridization to blotted rice PFE gels was used to show that the digests were complete. The long-term goal of our work is to generate an integrated genetic/physical map for the rice genome, as well as helping to establish rice as a model for map-based gene cloning and genome analysis.

The presence of repeated DNA sequences and a partial restriction map of the pSym of Rhizobium fredii USDA193.

The large, 350-kb Sym (symbiotic) plasmid pRjaUSDA193 of Rhizobium fredii was examined to determine the frequency of repeated sequences present and to produce a physical and genetic map of a large region of the plasmid. A novel hybridization method, the Southern Cross, revealed that the plasmid pRjaUSDA193 contained many repeated sequences and assisted in restriction enzyme mapping of a 100-kb region containing nod genes. A cosmid clone bank was prepared with the broad-host-range cosmid pVK102. The restriction enzymes HindIII, HpaI, and KpnI were used to construct a physical map of overlapping clones. Labeled nod gene sequences were used to determine their location in the mapped region.

Inhibition of deoxyribonuclease activity associated with soybean chloroplasts

The effects of spermidine, pH, ethylene diamine tetracetic acid (EDTA), and adenosine triphosphate (ATP) on deoxyribonuclease (DNase) activity associated with the chloroplasts of soybean (Glycine max (L.) Merr.) were investigated. Chloroplast DNase activity was found to be partially inhibited by either 10 mM spermidine, 20 mM EDTA, or 20 mM ATP. DNase activity was also partially inhibited at non-neutral pHs. Nearly complete inhibition was achieved with use of 30 mM EDTA, pH 10, or a combination of 10 mM spermidine and 10 mM EDTA.

Specific inhibition of ribosomal RNA synthesis in escherichia coli by tetracycline

Five Hundred μg/ml tetracycline was found to inhibit the accumulation of RNA in Escherichia coli cells. RNA synthesis was not totally inhibited, however. On the contrary, by using DNA-RNA hybridization, it was found that 500 μg/ml tetracycline specifically inhibits ribosomal RNA (rRNA) synthesis but allows messenger RNA synthesis to continue. Also, tetracycline specifically inhibits initiation of ribosomal RNA synthesis as determined from rate studies. The inhibition of rRNA synthesis at 500 μg/ml tetracycline was not due to several trivial explanations, that is, stimulation of ppGpp synthesis, inhibition of nucleoside triphosphate synthesis, nor was it due to sequestering of magnesium ion necessary for RNA polymerase action. Also, 500 μg/ml tetracycline was not inhibitory to RNA synthesis in permeabilized cells capable of normal rates of rRNA synthesis, similar to data previously obtained using ppGpp. Thus, it is proposed that tetracycline stops rRNA synthesis by inhibiting a ribosomal specific transcription factor; this factor is lost or destroyed during cell permeabilization. It is further proposed that tetracycline acts in a manner analogous to the normal rRNA regulator molecule (very likely ppGpp) at the transcriptional level.

The formation of R-prime deletion mutants and the identification of the symbiotic genes in Rhizobium fredii strain USDA191

SummaryR-prime plasmids were formed between the plasmid of Rhizobium fredii strain USDA191 containing nodulation and nitrogen-fixation genes, pRjaUSDA191c, and pRL180, and RP1 derivative. R. fredii USDA191 contains four HindIII fragments that hybridize with an 8.7 kb EcoRI fragment that contains nodulation genes from R. meliloti. These four fragments are on pRjaUSDA191c and are 15.5 kb, 12.5 kb, 6.8 kb, and 5.2 kb in size. A series of R-primes generated in E. coli of pRjaUSDA191c were transferred into a Nod- Nif- derivative of strain USDA191 to determine which nodulation region is necessary for nodule formation. Transconjugants containing the 12.5 kb and the 6.8 kb HindIII fragments on segments of pRjaUSDA191c produced nodules on soybean plants. However, transconjugants containing the 12.5 kb HindIII fragment alone were unable to form nodules, suggesting that the 6.8 kb HindIII fragment or the 6.8 kb and the 12.5 kb HindIII fragments together were needed for nodule formation. The 6.8 kb HindIII fragment was subcloned into the vector pVK102 and transferred into transconjugants containing no sequences homologous to R. meliloti nodulation DNA or to transconjugants containing only the 12.5 kb HindIII fragment. Nodules were formed on soybeans only when both the 12.5 kb and the 6.8 kb HindIII fragments were present in R. frediistrain USDA191.

Preparation of chloroplast DNA-enriched DNA samples from soybean

W e have been studying organelle genomes of cytoplasmically inherited mutants of soybean (Glycine max [L.] Merr.) by comparing restriction fragment patterns between mutant plants and their normal sibs. We also have begun to construct a cytoplasmic catalog of existing soybean cultivars and Plant Introductions based upon restriction fragment pattern groupings of their cpDNA. Because of the logistics of such an endeavor, we needed to develop a consistent procedure for the rapid isolation of restrictable, undegraded cpDNA from small amounts of fresh tissue. We found that, although the procedure of Murray and Thompson (1980) yielded good amounts of relatively undegraded DNA, the background nuclear DNA often completely obscured the soybean cpDNA restriction fragment pattern.

Natural premature protein synthesis termination can be reduced in Escherichia coli by decreased translation rates

SummaryPeptidyl tRNA hydrolase is an essential enzyme for normal growth inasmuch as a mutant strain of Escherichia coli with a temperature-sensitive hydrolase cannot continue protein synthesis at the non-permissive temperature. In the absence of hydrolase peptidyl tRNA rapidly accumulates. Why peptidyl tRNA should be formed is the subject of this report. The rapid rate of protein synthesis is likely one mechanism of formation of peptidyl tRNA. A strA mutant of the hydrolase (pth-1) mutant strain that has a 40% reduction in amino acid polymerization rate can grow at 42° C. StrA mutants with normal polymerization rates, however, cannot grow at 42° C when pth-1 is present. Furthermore, addition of low levels of chloramphenicol (2–4 μg/ml) but not several other tested drugs, phenotypically suppressed pth-1 at 42° C. Chloramphenicol, at these concentrations, was found to reduce the amino acid polymerization rate 30–40%. On the other hand, no evidence could be found that amino acyl tRNA selection errors are incorporated into pseudo revertants of the pth-1 strain.