Isolation, sequencing, and mutagenesis of the nifF gene encoding flavodoxin from Azotobacter vinelandii

The nifF gene encoding flavodoxin from Azotobacter vinelandii OP was cloned and its DNA sequence determined. It is located adjacent to, or possibly within, the major nif cluster and it is preceded by nif-specific regulatory elements. Southern hybridization analysis revealed that there is only a single copy of the nifF gene on the A. vinelandii OP genome. Mutant strains were constructed which have an insertion mutation or an insertion and a deletion mutation within the nifF gene coding sequence. These mutant strains are capable of diazotrophic growth, indicating that flavodoxin is not the unique physiological electron donor to nitrogenase. The results of nifF-lacZYA gene fusion experiments and Northern hybridization analyses indicated that the nifF gene is both transcribed and translated under nitrogen fixing and non-nitrogen fixing conditions. However, under nitrogen fixing conditions a substantial increase in both nifF synthesis and in accumulation of an approximately 800-base pair nifF-encoding mRNA species was observed. Furthermore, strains mutated within the nifF gene have only 70% of the wild type in vivo nitrogenase activity as determined by whole cell acetylene reduction assays. These data demonstrate that the nifF-encoded flavodoxin of A. vinelandii OP, although not essential for nitrogen fixation, is required for maximum in vivo nitrogenase activity.     

Posttranslational modification of Klebsiella pneumoniae flavodoxin by covalent attachment of coenzyme A, shown by 31P NMR and electrospray mass spectrometry, prevents electron transfer from the nifJ protein to nitrogenase. A possible new regulatory mechanism for biological nitrogen fixation.

A strain of Escherichia coli (71-18) that produces ca. 15% of its soluble cytoplasmic protein as a flavodoxin, the Klebsiella pneumoniae nifF gene product, has been constructed. The flavodoxin was purified using FPLC and resolved into two forms, designated KpFldI and KpFldII, which were shown to have identical N-terminal amino acid sequences (30 residues) in agreement with that predicted by the K. pneumoniae nifF DNA sequence. 31P NMR, electrospray mass spectrometry, UV-visible spectra, and thiol group estimations showed that the single cysteine residue (position 68) of KpFldI is posttranslationally modified in KpFldII by the covalent, mixed disulfide, attachment of coenzyme A. KpFldII was inactive as an electron carrier between the K. pneumoniae nifJ product (a pyruvate-flavodoxin oxidoreductase) and K. pneumoniae nifH product (the Fe-protein of nitrogenase). This novel posttranslational modification of a flavodoxin is discussed in terms of the regulation of nitrogenase activity in vivo in response to the level of dissolved O2 and the carbon status of diazotrophic cultures.     

The base sequence of the nifF gene of Klebsiella pneumoniae and homology of the predicted amino acid sequence of its protein product to other flavodoxins.

The nucleotide sequence of a 629 base-pair segment of DNA spanning the nifF gene of Klebsiella pneumoniae is presented. The structural gene comprises 531 base-pairs (175 codons, excluding the translational initiator and terminator) encoding an acidic polypeptide of 18950 Da. The nifF product thus belongs to the long-chain class of flavodoxins. It shows some sequence homology to the short-chain flavodoxins from Desulfovibrio vulgaris, Clostridium MP and Megasphaera elsdenii, and much stronger homology to long-chain flavodoxins from Azotobacter vinelandii and Anacystis nidulans. The long chain flavodoxins thus seem to constitute a well-conserved sub-group. The homology with the A. vinelandii flavodoxin is particularly strong, which may reflect their common function in nitrogen fixation.     

Roles of nifF and nifJ gene products in electron transport to nitrogenase in Klebsiella pneumoniae.

Crude extracts of the wild-type Klebsiella pneumoniae reduced C2H2 with either pyruvate or formate as reductant (specific activity, 3 nmol min-1 mg of protein-1), whereas crude extracts of nifF mutant were almost inactive (specific activity, 0.05). However, activity in the latter extracts was stimulated by adding Azotobacter chroococcum flavodoxin (specific activity, 10). Thus, nifF mutants may lack an electron transport factor. Crude extracts of nifJ mutants had about 20% of the wild-type level of active MoFe protein, and thus nifJ has a presumptive role in maintaining active MoFe protein. Studies on pyruvate or formate as reductants for nitrogenase in extracts of the nifJ mutants suggest in addition a role in electron input to nitrogenase for the following reasons. (i) Nitrogenase activity with these reductants was very low (specific activity, 0.06) and was not stimulated by extra MoFe protein or the flavodoxin. (ii) Activity was increased by adding a crude extract of a mutant lacking the structural nif genes (specific activity, 1) or a crude extract of the nifF mutant (specific activity, 4).     

Cloning and characterization of the Escherichia coli lit gene, which blocks bacteriophage T4 late gene expression.

Escherichia coli lit mutations inhibit gene expression late in infection by bacteriophage T4. We cloned the lit gene from wild-type E. coli and three independent lit mutants. We present evidence that lit mutations [renamed lit(Con) mutations] cause overproduction of the lit gene product and that overproduction of this product causes the inhibition of gene expression. We also present evidence that the lit gene product is nonessential for E. coli growth, although the gene is common to most E. coli K-12 strains.     

Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets

Identification of genes that affect the product accumulation phenotype of recombinant strains is an important problem in industrial strain construction and a central tenet of metabolic engineering. We have used systematic (model-based) and combinatorial (transposon-based) methods to identify gene knockout targets that increase lycopene biosynthesis in strains of Escherichia coli. We show that these two search strategies yield two distinct gene sets, which affect product synthesis either through an increase in precursor availability or through (largely unknown) kinetic or regulatory mechanisms, respectively. Exhaustive exploration of all possible combinations of the above gene sets yielded a unique set of 64 knockout strains spanning the metabolic landscape of systematic and combinatorial gene knockout targets. This included a global maximum strain exhibiting an 8.5-fold product increase over recombinant K12 wild type and a twofold increase over the engineered parental strain. These results were further validated in controlled culture conditions.     

The Salmonella typhimurium LT2 uvrD gene is regulated by the lexA gene product.

The uvrD gene product apparently plays a role in the repair of UV damage, in mismatch repair, and in genetic recombination. A lower level of expression of the Salmonella typhimurium LT2 uvrD gene was observed in maxicells prepared from an Escherichia coli strain that contained a lexA+ plasmid than in maxicells prepared from an E. coli strain that lacked functional LexA protein. These results suggest that the uvrD+ gene is repressed by the LexA protein and is thus a member of the set of genes whose expression is increased by "SOS"-inducing treatments.     

Identification of a new inhibitor of essential division gene ftsZ as the kil gene of defective prophage Rac.

A gene function carried by a plasmid, causing arrest of cell division in Escherichia coli, has been identified as the product of a short open reading frame of the prophage Rac, previously designated orfE, expressed only under conditions of prophage induction. Because Rac carries a killing function expressed under conditions of zygotic induction, an orfE-defective Rac+ strain was constructed. This strain had lost the killing function, indicating that orfE is kil. Division inhibition by kil was specifically relieved by overexpression of essential division gene ftsZ. The kil gene product acts independently of the min operon, and its effects are increased in conditions of high cyclic AMP (cAMP) receptor protein-cAMP complex levels in the cell. Furthermore, at high levels of expression, kil product distorts the rod shape of the cells. These features distinguish kil-encoded protein from the inhibitory product of gene dicB, which occupies a similar genetic location in Kim (Qin), another defective prophage of Escherichia coli.     

Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii.

Determination of a 28,793-base-pair DNA sequence of a region from the Azotobacter vinelandii genome that includes and flanks the nitrogenase structural gene region was completed. This information was used to revise the previously proposed organization of the major nif cluster. The major nif cluster from A. vinelandii encodes 15 nif-specific genes whose products bear significant structural identity to the corresponding nif-specific gene products from Klebsiella pneumoniae. These genes include nifH, nifD, nifK, nifT, nifY, nifE, nifN, nifX, nifU, nifS, nifV, nifW, nifZ, nifM, and nifF. Although there are significant spatial differences, the identified A. vinelandii nif-specific genes have the same sequential arrangement as the corresponding nif-specific genes from K. pneumoniae. Twelve other potential genes whose expression could be subject to nif-specific regulation were also found interspersed among the identified nif-specific genes. These potential genes do not encode products that are structurally related to the identified nif-specific gene products. Eleven potential nif-specific promoters were identified within the major nif cluster, and nine of these are preceded by an appropriate upstream activator sequence. A + T-rich regions were identified between 8 of the 11 proposed nif promoter sequences and their upstream activator sequences. Site-directed deletion-and-insertion mutagenesis was used to establish a genetic map of the major nif cluster.     

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.     

Activation of an elastase precursor by the lasA gene product of Pseudomonas aeruginosa.

To study the role of the lasA gene product in the secretion of enzymatically active elastase by Pseudomonas aeruginosa, we constructed mutants by gene replacement with in vitro-derived insertion and deletion mutations in the cloned lasA gene. lasA mutants were deficient in the production of elastolytic activity. A membrane-associated, higher-molecular-weight (approximately 47,000) precursor of elastase was observed in both the wild-type and the lasA mutants. Unlike the wild-type strain, the lasA mutant accumulated the 47,000-molecular weight elastase species in the soluble fraction of the cell, suggesting that the lasA gene product has a role in elastase secretion. Although lasA mutants were deficient in elastolytic activity, they produced a proelastase with a mature molecular weight (approximately 37,000) that still retained general proteolytic activity. Final yields of elastase-related material were approximately the same in both the wild-type strain and lasA mutant supernatants. The lasA gene was expressed in Escherichia coli, and the approximate molecular weight of the lasA gene product was 31,000. Extracts of E. coli containing the lasA gene product were shown in vitro to activate the proelastase produced by P. aeruginosa lasA mutants to an enzyme with elastolytic activity. Thus the lasA gene product has a direct effect on broadening the substrate specificity of secreted proelastase, as well as a second role (direct or indirect) in the secretion of elastase.     

The unconventional myosin encoded by the myoA gene plays a role in Dictyostelium motility.

The myoA gene of Dictyostelium is a member of a gene family of unconventional myosins. The myosin Is share homologous head and basic domains, but the myoA gene product lacks the glycine-, proline-, alanine-rich and src homology 3 domains typical of several of the other myosin Is. A mutant strain of Dictyostelium lacking a functional myoA gene was produced by gene targeting, and the motility of this strain in buffer and a spatial gradient of the chemoattractant cyclic AMP was analyzed by computer-assisted methods. The myoA- cells have a normal elongate morphology in buffer but exhibit a decrease in the instantaneous velocity of cellular translocation, an increase in the frequency of lateral pseudopod formation, and an increase in turning. In a spatial gradient, in which the frequency of pseudopod formation is depressed, myoA- cells exhibit positive chemotaxis but still turn several times more frequently than control cells. These results demonstrate that the other members of the unconventional myosin family do not fully compensate for the loss of functional myoA gene product. Surprisingly, the phenotype of the myoA- strain closely resembles that of the myoB- strain, suggesting that both play a role in the frequency of pseudopod formation and turning during cellular translocation.     

Bacillus subtilis ypgA gene is fni, a nonessential gene encoding type 2 isopentenyl diphosphate isomerase

We previously identified the fni gene of Streptomyces sp. strain CL190 as type 2 isopentenyl diphosphate (IPP) isomerase, which needs both FMN and NADPH for enzyme activity. An fni gene homolog, ypgA, was detected in the database of the Bacillus subtilis genome. However, the ypgA product was about 140 amino acids shorter in the N-terminal than the Streptomyces fni gene product. A database search found three new putative start codons in 129, 225, and 411 bases upstream of the original start codon of the ypgA gene. The longest gene product, which was named ypgA3, showed the most significant homology to the Streptomyces fni gene product. The ypgA3 gene was expressed with an N-terminal His-tag in Escherichia coli and the purified soluble protein was characterized in detail. The ypgA3 protein converted IPP to its isomer dimethylallyl diphosphate in the presence of both FMN and NADPH. The enzyme also catalyzed the reverse reaction in the presence of both the cofactors. Disruption of the ypgA3 gene was not lethal to B. subtilis. These results indicate that Bacillus ypgA3 gene is fni, a nonessential gene encoding type 2 IPP isomerase.