Rhodobacter capsulatus contains two PII-like proteins, GlnB and GlnK, which play central roles in controlling the synthesis and activity of nitrogenase in response to ammonium availability. Here we used the yeast two-hybrid system to probe interactions between these PII-like proteins and proteins known to be involved in regulating nitrogen fixation. Analysis of defined protein pairs demonstrated the following interactions: GlnBNtrB, GlnB-NifA1, GlnB-NifA2, GlnB-DraT, GlnK-NifA1, GlnK-NifA2, and GlnK-DraT. These results corroborate earlier genetic data and in addition show that PII-dependent ammonium regulation of nitrogen fixation in R. capsulatus does not require additional proteins, like NifL in Klebsiella pneumoniae. In addition, we found interactions for the protein pairs GlnB-GlnB, GlnB-GlnK, NifA1-NifA1, NifA2-NifA2, and NifA1-NifA2, suggesting that fine tuning of the nitrogen fixation process in R. capsulatus may involve the formation of GlnBGlnK heterotrimers as well as NifA1-NifA2 heterodimers. In order to identify new proteins that interact with GlnB and GlnK, we constructed an R. capsulatus genomic library for use in yeast two-hybrid studies. Screening of this library identified the ATP-dependent helicase PcrA as a new putative protein that interacts with GlnB and the Ras-like protein Era as a new protein that interacts with GlnK.

Abstract. Posttranscriptional steps in the production of mRNA include well characterized polyadenylation and splicing reactions, but it is also necessary to understand how RNA is transported within the nucleus from the site of its transcription to the nuclear pore, where it is translocated to the cytoplasmic compartment. Determining the localization of RNA within the nucleus is an important aspect of understanding RNA production and may provide clues for investigating the trafficking of RNA within the nucleus and the mechanism for its export to the cytoplasm. We have previously shown that late phase adenovirus-infected cells contain large clusters of snRNP and non-snRNP splicing factors; the presence of these structures is correlated with high levels of viral late gene transcription. The snRNP clusters correspond to enlarged interchromatin granules present in late phase infected cells. Here we show that polyadenylated RNA and spliced tripartite leader exons from the viral major late transcription unit are present in these same late phase snRNP-containing structures. We find that the majority of the steady state viral RNA present in the nucleus is spliced at the tripartite leader exons. Tripartite leader exons are efficiently exported from the nucleus at a time when we detect their accumulation in interchromatin granule clusters. Since the enlarged interchromatin granules contain spliced and polyadenylated RNA, we suggest that viral RNA may accumulate in this late phase structure during an intranuclear step in RNA transport.T HE production of eukaryotic mRNAs can be considered in several steps; these include transcription, polyadenylation, splicing, and export of mRNA from the nucleus to the cytoplasm. Export involves movement of the RNA within the nucleus from the site of transcription to the nuclear pore, followed by transport of the message through the nuclear pore and into the cytoplasm. Although it is convenient to think of these posttranscriptional processes as separate steps, they are likely to be inter-related during the production of RNA in vivo. All of these reactions occur within the structural framework of the nucleus; thus the nuclear organization of gene expression activities has recently received considerable attention (for reviews see Carter, 1994;Wansink et al., 1994;.The nucleus has a high degree of organization with the nucleolus as the most obvious and well characterized nuclear compartment (Scheer et al., 1993 Fakan, 1994). These include perichromatin fibrils, interchromatin granules, and coiled bodies. Perichromatin fibrils are strongly labeled after short pulses of [3H]uridine indicating that transcription occurs at these structures. Perichromatin fibrils are thought to be the in situ forms of newly synthesized hnRNA. Interchromatin granules are not strongly labeled by a pulse of [3H]uridine and are therefore not likely to be locations of transcription, but several studies have shown that polyadenylated RNA is present in these structures (Carter et al., 1991(Carter et al., , 1993Visa et al....

Growth of Rhodobacter capsulatus with molecular dinitrogen as the sole N source via the alternative Fe-only nitrogenase requires all seven gene products of the anfHDGK-1-2-3 operon. In contrast to mutant strains carrying lesions in the structural genes of nitrogenase (anfH, anfD, anfG, and anfK), strains defective for either anf1, anf2, or anf3 are still able to reduce the artificial substrate acetylene, although with diminished activity. To obtain further information on the role of Anf1, we screened an R. capsulatus genomic library designed for use in yeast two-hybrid studies with Anf1 as bait. Two genes, which we propose to call ranR and ranT (for genes related to alternative nitrogenase), coding for products that interact with Anf1 were identified. A ranR mutant exhibited a phenotype similar to that of an anf1 mutant strain (no growth with N 2 in the absence of molybdenum, but significant reduction of acetylene via the Fe-only nitrogenase), whereas a ranT mutant retained the ability to grow diazotrophically, but growth was clearly delayed compared to the parental strain. In contrast to the situation for anf1, expression of neither ranR nor ranT was regulated by ammonium or molybdenum. A putative role for Anf1, RanR, and RanT in the acquisition and/or processing of iron in connection with the Fe-only nitrogenase system is discussed.

The Rhizobium meliloti fdxN gene, which is part of the nifA-nifB-fdxN operon, is absolutely required for symbiotic nitrogen fixation. The deduced sequence of the FdxN protein is characterized by two cysteine motifs typical of bacterial-type ferredoxins. The Fix-phenotype of an R. meliloti fdxN::[Tc] mutant could be rescued by the R. leguminosarum fdxN gene, whereas no complementation was observed with nif-associated genes encoding ferredoxins from Bradyrhizobium japonicum, Azotobacter vinelandii, A. chroococcum and Rhodobacter capsulatus. In addition to these heterologous genes, several R. meliloti fdxN mutant genes constructed by site-directed mutagenesis were analyzed. Not only a cysteine residue within the second cysteine motif (position 42), which is known to coordinate the Fe-S cluster in homologous proteins, but also a cysteine located down-stream of this motif (position 61), was found to be essential for the activity of the R. meliloti FdxN protein. Changing the amino acid residue proline in position 56 into methionine resulted in a FdxN mutant protein with decreased activity, whereas changes in positions 35 (Asp35Glu) and 45 (Gly45Glu) had no significant effect on the function of the FdxN mutant proteins. In contrast to bacterial-type ferredoxins, which contain two identical cysteine motifs of the form C-X2-C-X2-C-X3-C, nif-associated ferredoxins, including R. meliloti FdxN, are characterized by two different cysteine motifs. Six "additional" amino acids separate the second (Cys42) and the third cysteine (Cys51) in the C-terminal motif (C-X2-C-X8-C-X3-C).(ABSTRACT TRUNCATED AT 250 WORDS)

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