Identification of gene products from the Azotobacter vinelandii nifBfdxNnifOQ operon

The Azotobacter vinelandii nifBfdxNnifOQ operon is required for synthesis of the nitrogenase iron-molybdenum cofactor. To further characterize the roles of its gene products, specific antibodies against NifB and NifO were generated, and the NifB, NifO and NifQ gene products were visualized and identified in nitrogen-fixing A. vinelandii cell extracts by a combination of two-dimensional gel electrophoresis of radiolabelled extracts and immunological detection methods. The three proteins showed apparent pI and M_r values similar to those expected from sequence data, except for NifO, which showed an apparent M_r of ca. 23 kDa (vs. 16 kDa expected).     

Identification of a cis-acting replication element within the poliovirus coding region

The replication of poliovirus, a positive-stranded RNA virus, requires translation of the infecting genome followed by virus-encoded VPg and 3D polymerase-primed synthesis of a negative-stranded template. RNA sequences involved in the latter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, other than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-loop in the region encoding the 2C protein. Covariance analysis suggested the structure was well conserved in the Enterovirus genus of the Picornaviridae. Site-directed mutagenesis, disrupting the structure without affecting the 2C product, destroyed genome viability and suggested that the structure was required in the positive sense for function. Recovery of revertant viruses suggested that integrity of the structure was critical for function, and analysis of replication demonstrated that nonviable mutants did not synthesize negative strands. Our conclusion, that this RNA secondary structure constitutes a novel poliovirus cis-acting replication element (CRE), is supported by the demonstration that subgenomic replicons bearing lethal mutations in the native structure can be restored to replication competence by the addition of a second copy of the 61-nt wild-type sequence at another location within the genome. This poliovirus CRE functionally resembles an element identified in rhinovirus type 14 (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) and the cardioviruses (P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560-11565, 1999) but differs in sequence, structure, and location. The functional role and evolutionary significance of CREs in the replication of positive-sense RNA viruses is discussed.     

Identification and Characterization of two nitrogen fixation regulatory regions, nifA and nfrX, in Azotobacter vinelandii and Azotobacter chroococcum

Five Tn5-induced Nif- mutants of Azotobacter vinelandii were characterized as regulatory mutants because they were restored to Nif+ by the introduction of constitutively expressed nifA from Klebsiella pneumoniae. The mutants fell into two different classes on the basis of hybridization to a Rhizobium leguminosarum nifA gene probe and by complementation with cosmids isolated from pLAFRI gene banks of A. vinelandii and Azotobacter chroococcum. One mutant, MV3, was located in or near a nifA gene. The others, MV12, MV16, MV18 and MV26, defined a new regulatory gene, which has been called nfrX. The lack of expression of different nif-lacZ fusions confirmed the regulatory phenotype of all five mutant strains. The ability of both nifA and nfrX mutants to grow on nitrogen-free medium with vanadium, but not on medium with molybdenum, suggests that neither gene is required for expression of the alternative V-containing nitrogenase of A. vinelandii. A fragment carrying Tn5 and flanking DNA from MV3 was used as a probe to isolate the nifA region of A. chroococcum. Ligation of two adjacent EcoRI fragments of A. chroococcum yielded an intact nifA gene that activated expression of nifH-lac fusions and also restored MV3 to Nif+. The four nfrX mutants were complemented by pLAFR1 cosmids pLV163 and pLC121. The nfrX gene was subcloned from pLV163 and located within a 3.2 kb fragment. To determine whether nfrX might be found in other nitrogen-fixing organisms, DNA from 13 different species was hybridized to an nfrX probe. The failure to observe hybridization suggests that nfrX may be specific to nif regulation in Azotobacter.     

Characterization of a cis-acting regulatory element in the protein coding region of thymidylate synthase mRNA

Thymidylate synthase (TS) functions as an RNA-binding protein by interacting with two different sequences on its own mRNA. One site is located in the 5′-upstream region of human TS mRNA while the second site is located within the protein coding region corresponding to nt 434–634. In this paper, a 70 nt RNA sequence, corresponding to nt 480–550, was identified that binds TS protein with an affinity similar to that of full-length TS mRNA and TS434–634 RNA. In vitro translation studies confirmed that this sequence is critical for the translational autoregulatory effects of TS. To document in vivo biological significance, TS sequences contained within this region were cloned onto the 5′-end of a luciferase reporter plasmid and transient transfection experiments were performed using H630 human colon cancer cells. In cells transfected with p644/TS434–634 or p644/TS480–550, luciferase activity was decreased 2.5-fold when compared to cells transfected with p644 plasmid alone. Luciferase mRNA levels were identical for each of these conditions as determined by RNase protection and RT–PCR analysis. Immunoprecipitation of TS ribonucleoprotein complexes revealed a direct interaction between TS protein and TS480–550 RNA in transfected H630 cells. Treatment with 5-fluorouridine resulted in a nearly 2-fold increase in luciferase activity only in cells transfected with p644/TS434–634 and p644/TS480–550. This study identifies a 70 nt TS response element in the protein coding region of TS mRNA with in vitro and in vivo translational regulatory activity.     

Identification and restriction endonuclease mapping of the threonine operon regulatory region.

A DNA fragment carrying the thrP region of Escherichia coli has been identified and characterized. Starting from a secondary site lysogen of bacteriophage λ, where the position of the prophage is either within thrP or between thrP and thrP structural genes (Gardner et al., 1974; Gardner &; Smith, 1975) it has been possible to isolate transducing phages which carry bacterial DNA from either side of the prophage. By Int-Xis mediated site-specific recombination we have generated recombinant transducing phages which carry an intact thrP region. A phage carrying a regulatory mutation, which maps in the thrP region, has also been constructed.Mapping with several restriction endonucleases has allowed us to construct a map of the cleavage sites of the thrP region. A 1700 base-pair HaeIII fragment carrying the secondary attachment site (attΔOΔ′) was isolated and its position was localized within a 180 base-pair TaqI-HhaI restriction fragment. The location of attΔOΔ′ in the HaeIII fragment suggests that the entire thrP region is carried by this fragment.     

Complementation of a pleiotropic Nif-Gln regulatory mutant of Rhodospirillum rubrum by a previously unrecognized Azotobacter vinelandii regulatory locus

A spontaneous pleiotropic Nif^- mutation in Rhodospirillum rubrum has been partially characterized biochemically and by complementation analysis with recombinant plasmids carrying Azotobacter vinelandii DNA in the vicinity of ORF12 [Jacobson et al. (1989) J. Bacteriol 171:1017–1027]. In addition to being unable to grow on N₂ as a nitrogen source the phenotypic characterization of this and other metronidazole enriched spontaneous mutants showed (a) no nitrogenase activity, (b) the absence of NifHDK polypeptides, (c) a slower growth rate on NH _inf4 ^sup+ , (d) approximately 50% higher glutamine synthetase (GS) activity than the wild-type, which was repressible, (e) an inability to switch-off GS activity in response to an NH _inf4 ^sup+ up-shift, and (f) an inability to modify (³²P-label) the GS polypeptide. The apparent relationship between the absence of nifHDK expression and the absence of GS adenylylation cannot be explained in terms of the current model for nif gene regulation. However, R. rubrum transconjugants receiving A. vinelandii DNA which originated immediately upstream from nifH, restored all aspects of the wild-type phenotype. These data suggest a here-to-fore unrecognized relationship between nif expression and GS switch-off (adenylylation) activity, and the existence of a previously unidentified regulatory locus in Azotobacter that complements this mutation.     

Identification of a tissue-specific regulatory element within the murine CD14 gene.

We previously isolated and sequenced the 5'-flanking region of the mouse CD14 (mCD14) gene (Matsuura, K., Setoguchi, M., Nasu, N., Higuchi, Y., Yoshida, S., Akizuki, S., and Yamamoto, S. (1989) Nucleic Acids Res. 17, 2132). To define the regulatory elements that control expression of the mCD14 gene, we analyzed the structure of the 5' end of the gene, including a region further upstream of that determined previously. Sequentially 5'-deleted, chimeric, and point mutated clones were tested for the ability to stimulate chloramphenicol acetyltransferase. An 8-base pair sequence, TGATTCAC, at position -255, which resembled the consensus sequence of the 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE), enhanced the expression of the chloramphenicol acetyltransferase gene in macrophage (aHINS-B3) and non-macrophage (glioblastoma G203 and myeloma NS1) cells. The enhancing ability of the TRE-like sequence (TLS), however, was markedly reduced in G203 cells but not in aHINS-B3 cells when the TLS was followed by the sequence immediately downstream. The TLS and sequence immediately downstream were capable of binding nuclear proteins which were unique to aHINS-B3 cells and macrophages, suggesting that these unique protein regulate the specific expression of the mCD14 gene. Binding of AP-1 to the TLS was also found in aHINS-B3 and G203 cells. Although it is uncertain whether AP-1 is involved in expression of the mCD14 gene, the effect of AP-1 in non-macrophage cells was inhibited by a nuclear protein which binds to the sequence immediately downstream of the TLS.     

The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase.

The roles of the product of the hoxZ gene immediately downstream of the hydrogenase gene (hoxKG) in Azotobacter vinelandii were investigated by constructing and characterizing a mutant with the center of the hoxZ gene deleted. The strain lacking the functional hoxZ gene product exhibited a low rate of H2 oxidation with O2 as the electron acceptor relative to that of the wild-type strain. Nevertheless, when the enzyme was exogenously activated and methylene blue was used as the electron acceptor from hydrogenase, rates of H2 oxidation comparable to those in the wild-type strain were observed. These results suggest that the gene product of hoxZ plays a role in activating and maintaining hydrogenase in a reduced active state. The product of hoxZ could also be the linkage necessary for transfer of electrons from H2 to the electron transport chain.     

Translational regulatory signals within the coding region of the bacteriophage lambda cIII gene.

Six independent mutations which enhance the lysogenic response were analyzed. The mutations cause single-base substitutions at three sites within the cIII coding sequence, one of which does not change the amino acid code. The mutations allow for elevated translation of the cIII gene, possibly via changes in the mRNA secondary structure.     

Evidence for two dinitrogenase reductases under regulatory control by molybdenum in Azotobacter vinelandii

Evidence for an alternative nitrogen fixation system which is expressed under conditions of molybdenum deficiency has been reported in Azotobacter vinelandii (Bishop, P.E., Jarlenski, D.M.L. and Hetherington, D.R., Proc. Natl. Acad. Sci. U.S.A. (1980) 77, 7342–7346). In the present report we describe the existence of activity for a dinitrogenase reductase-like enzyme (alternative reductase) in Mo-deficient cell-free extracts of Nif^? mutant strains of A. vinelandii which lack either conventional dinitrogenase reductase (strains UW1 and UW3) or contain a defective enzyme (strain UW91) under conditions of Mo-sufficiency. Nitrogenase activities were determined by the acetylene reduction method in a complementation assay where extracts of strain UW91 served as a source of dinitrogenase and extracts of strains UW1, UW3 or UW91 served as a source of alternative reductase. Strains that lack dinitrogenase reductase activity in the presence of Mo, were shown to have alternative reductase activity under Mo-deficient conditions. Two-dimensional gel electrophoretic analysis showed these extracts to contain a protein of similar mobility as the conventional dinitrogenase reductase. Molybdenum and tungsten repressed the formation of the alternative reductase whereas vanadium mimicked Mo deprivation. In conclusion, the results with the Nif^? strains provide evidence for the presence of two reductase activities, one of which is expressed in the presence of Mo (dinitrogenase reductase) and the other in the absence of Mo (alternative reductase).