Characterization of two genes (hupD and hupE) required for hydrogenase activity in Azotobacter chroococcum

In Azotobacter chroococcum the hydrogenase structural genes (hupSL) cover about 2.8 kb of a 15-kb region associated with hydrogen-uptake (Hup) activity. Two other genes in this region, hupD and hupE, were located 8.9 kb downstream of hupL and were shown to be essential for hydrogenase activity by insertion mutagenesis. A fragment of DNA beginning 3.4 kb downstream of hupL was able to complement the hupE mutant, supporting earlier evidence for a promoter downstream of hupSL. Hybridization experiments showed that hupD and hupE share some similarity with a region of Alcaligenes eutrophus DNA which is apparently involved in the formation of catalytically active hydrogenase. The hupD gene encodes a 379-amino acid, 41.4-kDa polypeptide while hupE codes for a 341-amino acid, 36.1-kDa product. The predicted amino acid sequences of the hupD and hupE genes are homologous to the Escherichia coli hypD and hypE gene products, respectively. A polar mutation in hupD had no effect on beta-galactosidase activity in a strain also carrying a hupL-lacZ fusion, indicating that hupD and hupE are probably not involved in regulating hydrogenase structural gene expression.     

Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme

The alginate lyase-encoding gene (algL) of Azotobacter chroococcum was localized to a 3.1-kb EcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.     

Molecular characterization of structural genes coding for a membrane bound hydrogenase in Methylococcus capsulatus (Bath)

The first gene cluster encoding for a membrane bound [NiFe] hydrogenase from a methanotroph, Methylococcus capsulatus (Bath), was cloned and sequenced. The cluster consisted of the structural genes hupS and hupL and accessory genes hupE, hupC and hupD. A DeltahupSL deletion mutant of Mc. capsulatus was constructed by marker exchange mutagenesis. Membrane associated hydrogenase activity disappeared. The membrane associated hydrogenase appeared to have a hydrogen uptake function in vivo.     

Secondary structure and calcium-induced folding of the Clostridium thermocellum dockerin domain determined by NMR spectroscopy

Assembly of the cellulosome, a large, extracellular cellulase complex, depends upon docking of a myriad of enzymatic subunits to homologous receptors, or cohesin domains, arranged in tandem along a noncatalytic scaffolding protein. Docking to the cohesin domains is mediated by a highly conserved domain, dockerin (DS), borne by each enzymatic subunit. DS consists of two 22-amino-acid duplicated sequences, each bearing homology to the EF-hand calcium-binding loop. To compare the DS structure with that of the EF-hand helix-loop-helix motif, we analyzed the solution secondary structure of the DS from the cellobiohydrolase CelS subunit of the Clostridium thermocellum cellulosome using multidimensional heteronuclear NMR spectroscopy. The effect of Ca(2+)-binding on the DS structure was first investigated by using 2D (15)N-(1)H HSQC NMR spectroscopy. Changes in the spectra during Ca(2+) titration revealed that Ca(2+) induces folding of DS into its tertiary structure. This Ca(2+)-induced protein folding distinguishes DS from typical EF-hand-containing proteins. Sequential backbone assignments were determined for 63 of 69 residues. Analysis of the NOE connectivities and H(alpha) chemical shifts revealed that each half of the dockerin contains just one alpha-helix, comparable to the F-helix of the EF-hand motif. Thus, the structure of the DS Ca(2+)-binding subdomain deviates from that of the canonical EF-hand motif.     

Diversity and expression of cyanobacterial hupS genes in pure cultures and in a nitrogen-limited phototrophic biofilm

A PCR was developed for conserved regions within the cyanobacterial small subunit uptake hydrogenase (hupS) gene family. These primers were used to PCR amplify partial hupS sequences from 15 cyanobacterial strains. HupS clone libraries were constructed from PCR-amplified genomic DNA and reverse-transcribed mRNA extracted from phototrophic biofilms cultivated under nitrate-limiting conditions. Partial hupS gene sequences derived from cyanobacteria, some of which were not previously known to contain hup genes were used for phylogenetic analysis. Phylogenetic trees constructed with partial hupS genes were congruent with those based on 16S rRNA genes, indicating that hupS sequences can be used to identify cyanobacteria expressing hup. Sequences from heterocystous and nonheterocystous cyanobacteria formed two separate clusters. Analysis of clone library data showed a discrepancy between the presence and the activity of cyanobacterial hupS genes in phototrophic biofilms. The results showed that the hupS gene can be used to characterize the diversity of natural populations of diazotrophic cyanobacteria, and to characterize gene expression patterns of individual species and strains.     

Cyanamide: a new substrate for nitrogenase

(1) Cyanamide (N identical to C-NH2) has been shown to be a substrate for purified Mo-nitrogenases of Klebsiella pneumoniae and Azotobacter chroococcum, with apparent Km values near 0.8 mM. (2) Reduction products were CH4, CH3NH2 and NH3 formed by pathways requiring 6 or 8 electrons: N identical to CNH2 + 6e + 6H+----CH3NH2 + NH3; N identical to CNH2 + 8e + 8H+----CH4 + 2NH3 (3) Acetylene reduction and hydrogen evolution were inhibited more than 75% by cyanamide (10 mM). Cyanamide also inhibited total electron flux at nitrogenase protein component ratios (Fe/MoFe) near 10. (4) Cyanamide was also a substrate for the recently isolated Va-nitrogenase of A. chroococcum, but with an apparent Km of 2.6 mM showed weaker binding and an 8-fold lower Vmax than did either Mo-nitrogenase. (5) The component ratios of nitrogenase proteins favouring CH4 formation was 3.5 Fe/MoFe protein and 1 Fe/VaFe protein.     

Identification of a gene family regulated by transforming growth factor-beta

We have identified two related genes whose mRNAs are increased after treatment with transforming growth factor-beta (TGF-beta 1). Mouse AKR-2B cells were treated with TGF-beta 1 in the presence of cyclohexamide and a cDNA library was subjected to differential screening. Several TGF-beta-induced genes (beta IG) were isolated and two of these, beta IG-M1 and beta IG-M2, were characterized. beta IG-M1 and beta IG-M2 RNAs were significantly increased after TGF-beta 1 treatment and both were superinduced in the presence of cyclohexamide. cDNA sequence analysis of beta IG-M1 showed that it encoded a 379-amino-acid protein which was 81% homologous to CEF-10, a v-src and TPA-inducible gene, and identical to cyr61, a gene induced by serum in growth-arrested BALB-3T3 cells. cDNA sequence analysis of beta IG-M2 showed that it encoded a 348-amino-acid protein that was 50% homologous to beta IG-M1. Thirty-eight cysteine residues are conserved between beta IG-M1 and beta IG-M2, which are clustered at the amino and carboxy ends: The middle regions of the two proteins are cysteine free and display the highest degree of nonhomology. Both proteins contain an amino-terminal cysteine-rich motif common to insulin-like growth factor binding proteins and a carboxy-terminal domain with strong homology to a motif found near the carboxy-terminal of the malarial circumsporozoite protein which may be involved in cell adhesion. The regulation of mRNA encoding these proteins by TGF-beta 1 suggests that they may be involved in mediating some of the pleiotropic effects of this multipotent modulator of cell growth and differentiation.     

The hupTUV operon is involved in negative control of hydrogenase synthesis in Rhodobacter capsulatus.

The hupT, hupU, and hupV genes, which are located upstream from the hupSLC and hypF genes in the chromosome of Rhodobacter capsulatus, form the hupTUV operon expressed from the hupT promoter. The hupU and hupV genes, previously thought to belong to a single open reading frame, encode HupU, of 34.5 kDa (332 amino acids), and HupV, of 50.4 kDa (476 amino acids), which are >/= 50% identical to the homologous Bradyrhizobium japonicum HupU and HupV proteins and Rhodobacter sphaeroides HupU1 and HupU2 proteins, respectively; they also have 20 and 29% similarity with the small subunit (HupS) and the large subunit (HupL), respectively, of R. capsulatus [NiFe]hydrogenase. HupU lacks the signal peptide of HupS and HupV lacks the C-terminal sequence of HupL, which are cleaved during hydrogenase processing. Inactivation of hupV by insertional mutagenesis or of hupUV by in-frame deletion led to HupV- and Hup(UV)- mutants derepressed for hydrogenase synthesis, particularly in the presence of oxygen. These mutants were complemented in trans by plasmid-borne hupTUV but not by hupT or by hupUV, except when expressed from the inducible fru promoter. Complementation of the HupV- and Hup(UV)- mutants brought about a decrease in hydrogenase activity up to 10-fold, to the level of the wild-type strain B10, indicating that HupU and HupV participate in negative regulation of hydrogenase expression in concert with HupT, a sensor histidine kinase involved in the repression process. Plasmid-borne gene fusions used to monitor hupTUV expression indicated that the operon is expressed at a low level (50- to 100-fold lower than hupS).     

Electron-paramagnetic-resonance studies on the redox properties of the molybdenum-iron protein of nitrogenase between +50 and -450 mV.

The midpoint potentials, Em, for the oxidation of the characteristic e.p.r. signal with g values near 4.3, 3.7 and 2.01, of the nitrogenase Mo-Fe proteins from a number of bacteria were measured. They were 0mV for Clostridium pasteurianum, -42mV for Azotobacter chroococcum and Azotobacter vinelandii, -95mV for Bacillus polymyxa and -180mV for Klebsiella pneumoniae Mo-Fe proteins at pH 7.9. The oxidations were thermodynamically reversible for the proteins from A. chroococcum, A. vinelandii and K. pneumoniae and the Em was independent of protein activity for this last protein. The protein from C. pasteurianum required a lower potential for reduction than for oxidation, and the oxidation of the protein from B. polymyxa was only 70% reversible. The apparent Em of the latter protein was decreased by 40mV in the presence of 60mM-MgCl2. The pH-dependence of the Em of the protein from K. pneumoniae was interpreted in terms of a single ionization, not directly associated with the e.p.r.-active centre, with a pKa of 7.0 in the oxidized form of the protein and a pH-independent region at low pH (Em = 118 +/- 6.3 mV). Approx. 20% increase in activity after oxidation was observed for the proteins from B. polymyxa, A. chroococcum and K. pneumoniae. The significance of the above results and their relationship to other published data are discussed.