Characterization of the iron superoxide dismutase gene of Azotobacter vinelandii: sodB may be essential for viability.

Azotobacter vinelandii contains two superoxide dismutases (SODs), a cytoplasmic iron-containing enzyme (FeSOD), and a periplasmic copper/zinc-containing enzyme (CuZnSOD). In this study, the FeSOD was found to be constitutive, while the activity of CuZnSOD increased as the culture entered the stationary phase. Total SOD (units/mg protein) in stationary phase cells grown under nitrogen-fixing conditions was not significantly different from those grown under non-nitrogen-fixing conditions. The gene encoding FeSOD (sodB) was isolated from an A. vinelandii cosmid library. A 1-kb fragment containing the coding region and 400 base pairs of upstream sequence was cloned and sequenced. The nucleotide sequence and the deduced amino acid sequence had a high degree of homology with other bacterial FeSODs, particularly with P. aeruginosa. Attempts to construct a sodB mutant by recombination of a sodB::kan insertion mutation into the multicopy chromosome of A. vinelandii were unsuccessful even in the presence of SOD mimics or nutritional supplements. These results suggest that FeSOD may be essential for the growth and survival of A. vinelandii, and that the periplasmic CuZnSOD cannot replace the function of FeSOD.

Isolation of nitrogen-fixing bacteria containing molybdenum-independent nitrogenases from natural environments.

Seven diazotrophs that grow well under Mo-deficient, N(2)-fixing conditions were isolated from a variety of environments. These isolates fall in the gamma subdivision of the class Proteobacteria and have genes that encode the Mo nitrogenase (nitrogenase 1) and the V nitrogenase (nitrogenase 2). Four of the isolates also harbor genes that encode the iron-only nitrogenase (nitrogenase 3).

Identification of genes unique to Mo-independent nitrogenase systems in diverse diazotrophs.

A number of nitrogen-fixing bacteria were screened using PCR for genes (vnfG and anfG) unique to the V-containing nitrogenase (vnf) and the Fe-only nitrogenase (anf) systems. Products with sequences similar to that of vnfG were obtained from Azotobacter paspali and Azotobacter salinestris genomic DNAs, and products with sequences similar to that of anfG were obtained from Azomonas macrocytogenes, Rhodospirillum rubrum, and Azotobacter paspali DNAs. Phylogenetic analysis of the deduced amino acid sequences of anfG and vnfG genes shows that each gene product forms a distinct cluster. Furthermore, amplification of an internal 839-bp region in anfD and vnfD yielded a product similar to anfD from Heliobacterium gestii and a product similar to vnfD from Azotobacter paspali and Azotobacter salinestris. Phylogenetic analysis of NifD, VnfD, and AnfD amino acid sequences indicates that AnfD and VnfD sequences are more closely related to each other than either is to NifD. The results of this study suggest that Azotobacter salinestris possesses the potential to express the vanadium (V)-containing nitrogenase (nitrogenase 2) and that R. rubrum, Azomonas macrocytogenes, and H. gestii possess the potential to express the Fe-only nitrogenase (nitrogenase 3). Like Azotobacter vinelandii, Azotobacter paspali appears to have the potential to express both the V-containing nitrogenase and the Fe-only nitrogenase.

Regulation of the transcriptional activators AnfA and VnfA by metals and ammonium in Azotobacter vinelandii.

Transcription of the genes encoding molybdenum (Mo)-independent nitrogenases 2 and 3 of Azotobacter vinelandii requires the activators VnfA and AnfA, respectively. The effect of NH4+, Mo, or V (vanadium) was tested on the expression of vnfA-lacZ and anfA-lacZ transcriptional fusions. Mo repressed expression of both fusions whereas NH4+ and V repressed the anfA-lacZ fusion, but not the vnfA-lacZ fusion. Thus the repressive effect on transcription of the anfHDGKOR operon by NH4+, Mo, or V is mediated through their effect on transcription of anfA and the repressive effect of Mo on the vnfHFd and vnfDGK operons is mediated through Mo repression of vnfA transcription. Mo-dependent repression of anfA transcription is influenced but not entirely mediated by the Mo-responsive regulator ModE.

Phenotypic characterization of a tungsten-tolerant mutant of Azotobacter vinelandii.

A tungsten-tolerant mutant strain (CA6) of Azotobacter vinelandii first described in 1980 (P. E. Bishop, D. M. L. Jarlenski, and D. R. Hetherington, Proc. Natl. Acad. Sci. USA 77:7342-7346, 1980) has been further characterized. Results from growth experiments suggest that both nitrogenases 1 and 3 are utilized when CA6 grows in N-free medium containing Na2MoO4. Strain CA6.1.71, which lacks both nitrogenases 2 and 3, grew as well as strain CA in N-free medium containing Na2MoO4 after an initial lag. This indicates that nitrogenase 1 is fully functional in strain CA6. nifH-lacZ and anfH-lacZ transcriptional fusions were expressed in CA6 in the presence of Na2MoO4. Thus, in contrast to wild-type strain CA, transcription of the anfHDGK gene cluster in strain CA6 is not repressed by Mo. Expression of the vnfD-lacZ fusion was the same in both strains CA and CA6. In agreement with the results obtained with lac fusions, subunits of both nitrogenases 1 and 3 were found in protein extracts of CA6 cells grown in N-free medium containing Na2MoO4. However, CA6 cells, cultured in the presence of Na2WO4, accumulated nitrogenase 3 proteins without detectable amounts of nitrogenase 1 proteins. This indicates that expression of Mo-independent nitrogenase 3 is the basis for the tungsten tolerance phenotype of strain CA6. A measure of Mo accumulation as a function of time showed that accumulation by strain CA6 was slower than that for strain CA. When Mo accumulation was studied as a function of Na2MoO4 concentration, the two strains accumulated similar amounts of Mo in the concentration range of 0 to 1 microM Na2MoO4 during a 2-h period. Within the range of 1 to 5 microM Na2MoO4, Mo accumulation by strain CA increased linearly with increasing concentration whereas no further increases were observed for strain CA6. These results are consistent with the possibility that the tungsten tolerance mutation carried by CA6 is in a Mo transport system.

Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii.

In Azotobacter vinelandii, the anfHDGK operon encodes the subunits for the third nitrogenase complex. Two open reading frames (orf1 and orf2) located immediately downstream of anfK were shown to be required for diazotrophic growth under Mo- and V-deficient conditions. We have designated orf1 and orf2 anfO and anfR, respectively. Strains (CA115 and CA116) carrying in-frame deletions in anfO and anfR accumulate the subunits for nitrogenase 3 under Mo-deficient diazotrophic conditions. AnfO and AnfR are required for nitrogenase 3-dependent diazotrophic growth and 15N2 incorporation but not for acetylene reduction. AnfO contains a putative heme-binding domain that exhibits similarity to presumed heme-binding domains of P-450 cytochromes. Amino acid substitutions of Cys-158 show that this residue is required for fully functional AnfO as measured by diazotrophic growth under Mo- and V-deficient conditions. The nucleotide sequence of the region located immediately downstream of anfR has been determined. A putative rho-independent transcription termination site has been identified 250 bp from the 3' end of anfR. A third open reading frame (orf3), located downstream of anfR, does not appear to be required for diazotrophic growth under Mo- and V-deficient conditions.

The genes encoding the delta subunits of dinitrogenases 2 and 3 are required for mo-independent diazotrophic growth by Azotobacter vinelandii.

vnfG and anfG encode the delta subunits of alternative nitrogenases 2 and 3 in Azotobacter vinelandii, respectively. As a first step towards elucidating the role of these subunits, diazotrophic growth and acetylene reduction studies were conducted on mutants containing alterations in the genes encoding these subunits. Mutants containing a stop codon (C36stop) or an in-frame deletion in anfG were unable to grow in N-free, Mo-deficient medium (Anf-). Mutants in which cysteine 36 of AnfG (a residue conserved between VnfG and AnfG) was changed to Ala or Ser were Anf+. Thus, this conserved cysteine is not essential for the function of AnfG in dinitrogenase 3. A mutant with a stop codon in vnfG (C17stop) grew after a lag of 25 h in N-free, Mo-deficient medium containing V2O5. However, a Nif- Anf- strain with this mutation was unable to grow under these conditions. This shows that the vnfG gene product is required for nitrogenase 2-dependent growth. Strains with mutations in vnfG and anfG reduced acetylene to different degrees. This indicates that the delta subunits are not required for acetylene reduction by nitrogenases 2 and 3.

Effect of amino acid substitutions in a potential metal-binding site of AnfA on expression from the anfH promoter in Azotobacter vinelandii.

AnfA, an activator required for transcription of the structural genes encoding nitrogenase 3 (anfHDGK) in Azotobacter vinelandii, has a potential metal-binding site [(S19)H(C21)FTGE(C26)R] in its N terminus. Growth studies and expression of an anfH-lacZ fusion in mutants containing amino acid substitutions in this site indicate that Ser-19 is not required for AnfA activity whereas Cys-21 and Cys-26 are required. Residual expression of the anfH-lacZ fusion in AnfA- mutants was found to be due to activation by VnfA, the activator required for expression of genes encoding nitrogenase 2.

Mo-independent nitrogenase 3 is advantageous for diazotrophic growth of Azotobacter vinelandii on solid medium containing molybdenum.

Competition experiments between wild-type Azotobacter vinelandii and a mutant lacking Mo-independent nitrogenase 3 indicate that nitrogenase 3 provides an advantage during diazotrophic growth on agar media containing 100 to 500 nM Na2MoO4 but not in liquid media under the same conditions. Expression of nitrogenase 3 in wild-type cells growing on agar surfaces was verified with an anfH-lacZ fusion and by detection of nitrogenase 3 subunits. These results show that nitrogenase 3 is important for diazotrophic growth on agar medium at molybdenum concentrations that are not limiting for Mo-dependent diazotrophic growth in liquid medium.

Characterization of transcripts expressed from nitrogenase-3 structural genes of Azotobacter vinelandii.

Five major anfH-hybridizing mRNA species accumulated in Azobacter vinelandii cells derepressed for nitrogenase-3 (an alternative nitrogenase, which appears to lack Mo and V). Using anfH-, anfD-, anfG-, anfK-, and orflorf2-specific probes and mutant strains of A. vinelandii these mRNA species have been identified as encoding anfHDGKorflorf2 (6.0 kb), anfHDGK (4.3 kb), anfHD (2.6 kb), vnfHorfFd (1.3 kb), and vnfH and (or) anfH (1.0 kb). A 0.6-kb mRNA species, which hybridized only to the orflorf2-specific probe, and a 3.5-kb mRNA species, which hybridized to anfD or anfK, also accumulated under these conditions. Northern blot analysis and S1 nuclease mapping indicate that transcription of the anf structural gene cluster initiates at a unique nif consensus promoter situated 127 base pairs upstream from the anfH coding region. Observation of anfH-hybridizing mRNA species that accumulate in strains derepressed for nitrogen fixation demonstrates that transcription of the anfHDGKorflorf2 cluster is normally repressed by Mo, V, and NH4+, whereas transcription of the vnfHorfFd cluster does not require the presence of V and is repressed only by Mo, but not NH4+. Analysis of the accumulation of mRNAs in a tungsten-tolerant strain revealed that Mo and V repression of anf transcription must occur by different mechanisms.

Regulation of nitrogenase-2 in Azotobacter vinelandii by ammonium, molybdenum, and vanadium.

Under diazotrophic conditions in the absence of molybdenum and in the presence of vanadium, Azotobacter vinelandii reduces N2 to NH4+ by using nitrogenase-2, a V-containing enzyme complex encoded by vnfH (the gene for dinitrogenase reductase-2), and vnfDGK (the genes for dinitrogenase-2 subunits). Accumulation of the vnfHorfFd and vnfDGK transcripts occurred under Mo-deficient conditions in the presence and absence of V; however, in the case of vnfDGK, the protein products only accumulated in the presence of V. This suggests that V is required for translation of the vnfDGK transcripts. In addition, expression of vnfH-lacZ and vnfD-lacZ transcriptional fusions was only partially repressed in the presence of NH4+. Transcripts hybridizing with vnfH (1.4 and 1.0 kb), vnfDG (3.4 and 1.8 kb), and vnfK (3.4 kb) were detected in RNA extracted from wild-type cells cultured with NH4+ in the presence or absence of V. However, nitrogenase-2 subunits were not detected in extracts of cells derepressed for nitrogenase-2 in the presence of NH4+. These results indicate that this nitrogen source acts at the posttranscriptional level as well as at the transcriptional level. vnf transcripts were not detected in the presence of Mo (with or without NH4+).

Nucleotide sequence and mutational analysis of the vnfENX region of Azotobacter vinelandii.

The nucleotide sequence (3,600 bp) of a second copy of nifENX-like genes in Azotobacter vinelandii has been determined. These genes are located immediately downstream from vnfA and have been designated vnfENX. The vnfENX genes appear to be organized as a single transcriptional unit that is preceded by a potential RpoN-dependent promoter. While the nifEN genes are thought to be evolutionarily related to nifDK, the vnfEN genes appear to be more closely related to nifEN than to either nifDK, vnfDK, or anfDK. Mutant strains (CA47 and CA48) carrying insertions in vnfE and vnfN, respectively, are able to grow diazotrophically in molybdenum (Mo)-deficient medium containing vanadium (V) (Vnf+) and in medium lacking both Mo and V (Anf+). However, a double mutant (strain DJ42.48) which contains a nifEN deletion and an insertion in vnfE is unable to grow diazotrophically in Mo-sufficient medium or in Mo-deficient medium with or without V. This suggests that NifE and NifN substitute for VnfE and VnfN when the vnfEN genes are mutationally inactivated. AnfA is not required for the expression of a vnfN-lacZ transcriptional fusion, even though this fusion is expressed under Mo- and V-deficient diazotrophic growth conditions.

The gene encoding dinitrogenase reductase 2 is required for expression of the second alternative nitrogenase from Azotobacter vinelandii.

Under diazotrophic conditions in the absence of molybdenum (Mo) and vanadium (V), Azotobacter vinelandii reduces N2 to NH4+ by using nitrogenase 3 (encoded by anfHDGK). However, dinitrogenase reductase 2 (encoded by vnfH) is also expressed under these conditions even though this protein is a component of the V-containing alternative nitrogenase. Mutant strains that lack dinitrogenase reductase 2 (VnfH-) grow slower than the wild-type strain in N-free, Mo-, and V-deficient medium. In this medium, these strains synthesize dinitrogenase reductase 1 (a component of the Mo-containing nitrogenase encoded by nifH), even though this component is not normally synthesized in the absence of Mo. Strains that lack both dinitrogenase reductases 1 and 2 (NifH-VnfH-) are unable to grow diazotrophically in Mo- and V-deficient medium. In this medium, NifH- VnfH- strains containing an anfH-lacZ transcriptional fusion exhibited less than 3% of the beta-galactosidase activity observed in the wild type with the same fusion. Beta-Galactosidase activity expressed by VnfH- mutants containing the anfH-lacZ fusion ranged between 57 and 78% of that expressed by the wild type containing the same fusion. Thus, expression of dinitrogenase reductase 2 seems to be required for transcription of the anfHDGK operon, although, in VnfH-mutants, dinitrogenase reductase 1 appears to serve this function. Active dinitrogenase reductase 1 or 2 is probably required for this function since a nifM deletion mutant containing the anfH-lacZ fusion was unable to synthesize beta-galactosidase above background levels. An anfA deletion strain containing the anfH-lacZ fusion exhibited beta-galactosidase activity at 16% of that of the wild type containing the same fusion. However, in the presence of NH4+, the beta-galactosidase activity expressed by this strain more than doubled. This indicates that AnfA is required not only for normal levels of anfHDGK transcription but also for NH4+ -and, to a lesser extent, Mo-mediated repression of this transcription.

Sensitivity of digoxigenin and biotin labelled probes for detection of human papillomavirus by in situ hybridisation.

The sensitivity of digoxigenin and biotin labelled DNA probes for the detection of human papillomavirus (HPV) by dot blotting and in situ hybridisation was compared in tissues from cervical, laryngeal, and anogenital neoplasia. Probes were either labelled with digoxigenin by the random primer technique and detected with anti-digoxigenin antibody, or labelled with biotin by nick translation and detected with streptavidin, both methods having a common final visualisation procedure using alkaline phosphatase. Digoxigenin labelled probes proved two to 10-fold more sensitive by quantitative dot blotting and four-fold more sensitive in detecting HPV 16 DNA in a series of 31 anal carcinomas, compared with biotinylated probes. The digoxigenin method also produced less non-specific background staining of tissue sections than biotin labelled probes. It is concluded that digoxigenin DNA labelling and detection provides a simple, reliable, and efficient alternative to the use of biotin or radioactive isotopes for the detection of HPV DNA by in situ hybridisation. Digoxigenin labelled probes also offer the possibility of double labelling in situ hybridisation procedures when used with biotin labelled probes to provide simultaneous identification of different DNA sequences.

An immunohistological study of condylomata acuminata.

Using an indirect immunoperoxidase method and a panel of monoclonal antibodies, anogenital warts removed from 86 individuals (28 heterosexual men, 47 homosexual men and 11 women) were studied. A constant feature was the presence of a stromal infiltrate of lymphocytes, in which CD4+ and CD8+ cells appeared in a ratio of about 5:1. Cells expressing interleukin-2 receptors were found in the stroma, amounting to 10% of the CD3+ cells. Leu 6+ epidermal dendritic cells (Langerhans' cells) were present in each wart; their density and morphology were similar to that in adjacent, apparently normal, skin. The mean number of Langerhans' cells that expressed HLA-DQ was lower than that of HLA-DR + cells. In 40 warts, HLA-DR was expressed on the surface of keratinocytes. Leu 7+ and Leu 11b+ cells were found in the epidermis of 17 and five warts, respectively. B-cells were noted in only five warts. These results are discussed in relation to persistence of wart virus infection.

Nucleotide sequences and mutational analysis of the structural genes for nitrogenase 2 of Azotobacter vinelandii.

The nucleotide sequence (6,559 base pairs) of the genomic region containing the structural genes for nitrogenase 2 (V nitrogenase) from Azotobacter vinelandii was determined. The open reading frames present in this region are organized into two transcriptional units. One contains vnfH (encoding dinitrogenase reductase 2) and a ferredoxinlike open reading frame (Fd). The second one includes vnfD (encoding the alpha subunit of dinitrogenase 2), vnfG (encoding a product similar to the delta subunit of dinitrogenase 2 from A. chroococcum), and vnfK (encoding the beta subunit of dinitrogenase 2). The 5'-flanking regions of vnfH and vnfD contain sequences similar to ntrA-dependent promoters. This gene arrangement allows independent expression of vnfH-Fd and vnfDGK. Mutant strains (CA80 and CA11.80) carrying an insertion in vnfH are still able to synthesize the alpha and beta subunits of dinitrogenase 2 when grown in N-free, Mo-deficient, V-containing medium. A strain (RP1.11) carrying a deletion-plus-insertion mutation in the vnfDGK region produced only dinitrogenase reductase 2.

An immunohistological study of spontaneous regression of condylomata acuminata.

An immunohistological study of four men whose perianal warts were undergoing spontaneous regression was undertaken, and the results compared with those obtained from non-regressing condylomata from six men. CD4+ and CD8+ cells were noted in the stroma of each wart, but there was no clear difference in the density of the infiltrate between regressing and non-regressing warts. Natural killer cells (CD16+ and CD57+) were only noted in the stroma and epidermis of regressing warts. Possible immunological mechanisms of regression of condylomata acuminata are discussed.

Immunological study of condylomata acuminata in men infected with the human immunodeficiency virus.

As condylomata acuminata often persist in individuals infected with the human immunodeficiency virus (HIV), an immunohistological study of warts from infected men was undertaken to further knowledge about human papillomavirus persistence in this group. Using an indirect immunoperoxidase method and a panel of monoclonal antibodies, the phenotypes of cells were studied in cryostat sections of perianal or anal warts removed from 14 HIV-infected men (10 homosexual and 4 heterosexual) and from 16 non-infected men (10 homosexual and 6 heterosexual). Although the median numbers of CD1+, CD3+ and CD4+ cells per unit area were similar in each group of individuals, the number of CD8+ cells was significantly higher in HIV-infected homosexual men when compared with non-infected individuals and HIV-infected heterosexual men. The median CD4+ cell count in the peripheral blood was significantly higher in HIV-infected heterosexual men than in HIV-infected homosexual men (P less than 0.05). These findings may reflect differences in duration of HIV infection between the two groups. There was no significant difference in the proportion of cells expressing interleukin-2 receptors between HIV-infected and non-infected individuals. Natural killer (CD16+) cells were not identified in any of the condylomata.

Clinical course of anogenital warts in men infected with human immunodeficiency virus.

Fifty four men with anogenital warts were studied; 22 had concurrent infection with the human immunodeficiency virus (HIV). The median duration of the warts before and after the start of treatment of seven HIV infected and 10 non-infected heterosexual men was similar. In homosexual men, however, the duration of the lesions in 15 HIV infected patients was greater before and after treatment than in 22 non-HIV infected men. As the median number of CD4+ cells in the peripheral blood was significantly lower in homosexual than heterosexual men infected with HIV, the difference in the course of anogenital warts in homosexual compared with heterosexual men may reflect different degrees of immunosuppression.

Two nifA-like genes required for expression of alternative nitrogenases by Azotobacter vinelandii.

Two nifA-like genes, designated anfA and vnfA, have been identified in Azotobacter vinelandii. The anfA gene is located upstream from the nitrogenase-3 structural gene cluster (anfHDGK) and is preceded by a sequence that is potentially part of a ntrA-dependent promoter. The product of anfA appears to be required for expression of nitrogenase-3, since cells of the anfA deletion strain CA66 were unable to synthesize this nitrogenase when derepressed in N-free, Mo- and V-deficient medium. The vnfA gene was identified after determination of the nucleotide sequence of DNA flanking the Tn5 insertion in mutant strain CA46. Two open reading frames (ORF1 and ORF2) were found located upstream from the vnfA gene, and a nifE-like ORF, preceded by a possible ntrA-dependent promoter, was found downstream from this gene. It is not known whether vnfA is expressed only under N2-fixing conditions. However, potential ntrA-dependent promoters were found immediately upstream from vnfA (within the 3' end of ORF2) and immediately downstream from ORF1. The region spanning ORF1 and ORF2 contained an A + T-rich sequence that was also found immediately upstream from the potential ntrA-dependent promoter of anfA. The product of vnfA appears to be required for the synthesis of nitrogenase-2, since cells of strain CA46 synthesized only nitrogenase-1 and -3 but not nitrogenase-2 when grown in the presence of vanadium. The product of nifA, which is required for synthesis of nitrogenase-1, is not required for synthesis of either nitrogenase-2 or nitrogenase-3. However, growth data indicate that nifA is required for a factor (or factors) necessary for maximal diazotrophic growth under Mo- and V-deficient conditions.