Several loci on the tumor-inducing plasmid from Agrobacterium tumefaciens were transcriptionally activated in the presence of wounded plant tissue or extracts. The inducible virulence loci were required for efficient tumor formation. In contrast, the plant-inducible locus pinF was not observed to be absolutely essential for virulence. Mutants in pinF showed an attenuated virulence on a variety of dicotyledonous hosts, and this attenuation became more pronounced with decreasing numbers of bacterial cells in the inoculum. The DNA sequence of a 5.5-kilobase region which included the pinF locus from the octopine-type tumor-inducing plasmid A6 was determined. Four open reading frames consistent with the observed transcription of pinF were observed. Two of the open reading frames, pinFI and pinF2, coded for polypeptides with relative molecular weights of 47,519 (pinFI) and 46,740 (pinF2). A comparison of the amino acid sequences of pinFI and pinF2 indicated that they were similar to each other and to known polypeptide sequences for cytochrome P-450 enzymes.Agrobacteriumn tumefaciens stably transforms a variety of dicotyledonous plants by transferring a specific portion of its tumor-inducing plasmid (Ti plasmid) into the plant nucleus (19,25). The association of A. tumefaciens with the host plant and the mobilization of the transferred DNA (T-DNA) requires several trans-acting loci located on both the bacterial chromosome (chv) (4,6,7) and the Ti plasmid (vir) (9, 16, 17, 33). The Oir region of the Ti plasmid contains at least six loci in which mutations yield either an avirulent (virA, virB, virG, virD) or attenuated virulent (OirC, *irE) phenotype (12,17,33,42). All of the Oir loci can be induced by cocultivation of A. tumefaciens with plant cells (31, 34), by plant phenolic compounds such as acetosyringone or ox-hydroxyacetosyringone (32), or by a mixture of phenolic compounds (2). In addition, OirG can be partially induced by acidic conditions and low levels of phosphate alone (41). The product of lirA is postulated to bind acetosyringone and act as either a signal transducer or transport protein (35,39). This hypothesis predicts that the association of acetosyringone with VirA results in an activation of the virG product. Activated VirG is then proposed to transcriptionally activate the other rir loci, whose products mediate the site-specific cleavage of the T-DNA (1, 43) and its efficient transfer to the plant cell by a process which bears certain similarities to bacterial conjugation (36).In addition to the vir loci, a plant-inducible locus, pinF, was identified by Tn3::HoHol mutagenesis of the octopinetype Ti plasmid A6 from A. turmefaciens (33) Fig. 1) double digest of pVK219 was isolated, the 5' overhangs were filled in by using the Klenow fragment of DNA polymerase I and deoxynucleotides, and then it was subcloned into SmaIdigested pUC19. Plasmid DNA from this clone (pDD19.1) containing pinF was digested with BamHI and KpnI (restriction within vector polylinkers) and a 5.4-kb fragment purified from a 0.6% ...

A procedure for the immunoprecipitation of Fe protein from cell extracts was developed and used to monitor the modification of Fe protein in vivo. The subunit pattern of the isolated Fe protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was assayed by Coomassie brilliant blue protein staining and autoradiographic 32P detection of the modifying group. Whole-cell nitrogenase activity was also monitored during Fe protein modification. The addition of ammonia, darkness, oxygen, carbonyl cyanide m-chlorophenylhydrazone, and phenazine methosulfate each resulted in a loss of whole-cell nitrogenase activity and the in vivo modification of Fe protein. For ammonia and darkness, the rate of loss of nitrogenase activity was similar to that for Fe protein modification. The reillumination of a culture incubated in the dark brought about a rapid recovery of nitrogenase activity and the demodification of Fe protein. Cyclic dark-light treatments resulted in matching cycles of nitrogenase activity and Fe protein modification. Carbonyl cyanide m-chlorophenylhydrazone and phenazine methosulfate treatments caused an immediate loss of nitrogenase activity, whereas Fe protein modification occurred at a slower rate. Oxygen treatment resulted in a rapid loss of activity but only an incomplete modification of the Fe protein.

The amino acid concentrations in the phototrophic bacterium Rhodospirillum rubrum were measured during growth under nif-repressing and nif-derepressing conditions. The effects of ammonium, glutamine, darkness, phenazine methosulfate, and the inhibitors methionine sulfoximine and azaserine on amino acid levels of cells were tested. The changes were compared to changes in whole-cell nitrogenase activity and ADP-ribosylation of dinitrogenase reductase. Glutamate was the dominant amino acid under every growth condition. Glutamine levels were equivalent when cells were grown on high-ammonia (nif-repressing) medium or glutamate (nif-derepressing) medium. Thus, glutamine is not the solitary agent that controls nif expression. No other amino acid correlated with nif expression. Glutamine concentrations rose sharply when either glutamate-grown or N-starved cells were treated with ammonia, glutamine, or azaserine. Glutamine levels showed little change upon treatment of the cells with darkness or ammonium plus methionine sulfoximine. Treatment with phenazine methosulfate resulted in a decrease in glutamine concentration. The glutamine concentration varied independently of dinitrogenase reductase ADP-ribosylation, and it is concluded that an increase in glutamine concentration is neither necessary nor sufficient to initiate the modification of dinitrogenase reductase. No other amino acid exhibited changes in concentration that correlated consistently with modification. Glutamine synthetase activity and nitrogenase activity were not coregulated under all conditions, and thus the two regulatory cascades perceive different signal(s) under at least some conditions. Nitrogenase activity in the phototrophic bacterium Rhodospirillum rubrum is regulated at the level of expression and by posttranslational modification (8,10,12,28,33, and references therein). Oxygen and fixed sources of N such as glutamine and ammonia lead to the repression of nitrogenase; when glutamate is provided as the N source, nitrogenase is expressed.The inhibition of nitrogenase activity in vivo by ammonia was first noted by Gest et al. (8). The molecular basis for the inhibition is the ADP-ribosylation of dinitrogenase reductase (the iron protein) at a specific arginyl residue (26). Enzymes that attach (14) and remove (15,23,29) the ADP-ribosyl residue have been isolated from R. rubrum and have been found under a variety of culturing conditions (14,33).Factors that lead to the loss of activity in vivo include ammonia (10, 22, 31), glutamine (22), darkness (12, 40), and phenazine methosulfate (PMS) (12,25). Cells grown on N2 or glutamate as the N source are competent to switch-off nitrogenase activity (switch-off is the term given by Zumft and Castillo [44] to the reversible, in vivo loss of nitrogenase activity), whereas cells grown on limiting-ammonia (i.e., N-starved) medium are incapable of switch-off (38). Nitrogen assimilation in R. rubrum under nif-derepressed conditions is via glutamine synthetase (GS) and glutamate synthase (GOGAT) (3,21,32,36), and ...

Glutamine synthetase from Rhodospirillum rubrum was purified and characterized with respect to its pH optimum and the effect of Mg2+ on its active and inactive forms. Both adenine and phosphorus were incorporated into the inactive form of the enzyme, indicating covalent modification by AMP. The modification could not be removed by phosphodiesterase. Evidence for regulation of the enzyme by oxidation was obtained. Extracts from oxygen-treated cells had lower specific activities than did extracts from cells treated anaerobically. Glutamine synthetase activity was found to decrease in the dark in phototrophically grown cells; activity was recovered on re-illumination.

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