Azotobacter vinelandii mutS: nucleotide sequence and mutant analysis.

An Azotobacter vinelandii homolog to the Salmonella typhimurium mutS gene was discovered upstream of the fdxA gene. The product of this gene is much more similar to S. typhimurium MutS than either is to the HexA protein of Streptococcus pneumoniae. An A. vinelandii delta mutS mutant strain was shown to have a spontaneous mutation frequency 65-fold greater than that of the wild type.     

Accumulation of 55Fe-labeled precursors of the iron-molybdenum cofactor of nitrogenase on NifH and NifX of Azotobacter vinelandii

Iron-molybdenum cofactor (FeMo-co) biosynthesis involves the participation of several proteins. We have used (55)Fe-labeled NifB-co, the specific iron and sulfur donor to FeMo-co, to investigate the accumulation of protein-bound precursors of FeMo-co. The (55)Fe label from radiolabeled NifB-co became associated with two major protein bands when the in vitro FeMo-co synthesis reaction was carried out with the extract of an Azotobacter vinelandii mutant lacking apodinitrogenase. One of the bands, termed (55)Fe-labeled upper band, was purified and shown to be NifH by immunoblot analysis. The (55)Fe-labeled lower band was identified as NifX by N-terminal sequencing. NifX purified from an A. vinelandii nifB strain showed a different electrophoretic mobility on anoxic native gels than did NifX with the FeMo-co precursor bound.     

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 associates with insulin receptor substrate-1 and enhances insulin actions and adipogenesis

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is a unique enzyme that associates with the pSer/Thr-Pro motif and catalyzes cis-trans isomerization. We identified Pin1 in the immunoprecipitates of overexpressed IRS-1 with myc and FLAG tags in mouse livers and confirmed the association between IRS-1 and Pin1 by not only overexpression experiments but also endogenously in the mouse liver. The analysis using deletion- and point-mutated Pin1 and IRS-1 constructs revealed the WW domain located in the N terminus of Pin1 and Ser-434 in the SAIN (Shc and IRS-1 NPXY binding) domain of IRS-1 to be involved in their association. Subsequently, we investigated the role of Pin1 in IRS-1 mediation of insulin signaling. The overexpression of Pin1 in HepG2 cells markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events: phosphatidylinositol 3-kinase binding with IRS-1 and Akt phosphorylation. In contrast, the treatment of HepG2 cells with Pin1 siRNA or the Pin1 inhibitor Juglone suppressed these events. In good agreement with these in vitro data, Pin1 knock-out mice exhibited impaired insulin signaling with glucose intolerance, whereas adenoviral gene transfer of Pin1 into the ob/ob mouse liver mostly normalized insulin signaling and restored glucose tolerance. In addition, it was also demonstrated that Pin1 plays a critical role in adipose differentiation, making Pin1 knock-out mice resistant to diet-induced obesity. Importantly, Pin1 expression was shown to be up-regulated in accordance with nutrient conditions such as food intake or a high-fat diet. Taken together, these observations indicate that Pin1 binds to IRS-1 and thereby markedly enhances insulin action, essential for adipogenesis.     

Accumulation of 99Mo-containing iron-molybdenum cofactor precursors of nitrogenase on NifNE,NifH, and NifX of Azotobacter vinelandii

The biosynthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase was investigated using the purified in vitro FeMo-co synthesis system and 99Mo. The purified system involves the addition of all components that are known to be required for FeMo-co synthesis in their purified forms. Here, we report the accumulation of a 99Mo-containing FeMo-co precursor on NifNE. Apart from NifNE, NifH and NifX also accumulate 99Mo label. We present evidence that suggests NifH may serve as the entry point for molybdenum incorporation into the FeMo-co biosynthetic pathway. We also present evidence suggesting a role for NifX in specifying the organic acid moiety of FeMo-co.     

Phospholipids of Azotobacter vinelandii

Analyses of resting cells of Azotobacter vinelandii revealed that numerous phospholipids were present that did not concentrate in the membranous R(3) fraction which carried out electron transport function.     

Ultrastructure of Azotobacter vinelandii

Vegetative cells and cysts of Azotobacter vinelandii 12837 were prepared for electron microscopy by several methods assumed to preserve structural details destroyed by techniques previously reported in the literature. Examination of large numbers of cells and cysts by these methods revealed four structural details not reported previously: intine fibrils, intine vesicles, intine membrane, and microtubules. The intine fibrils form a network in the gel-like homogeneous matrix of the CC2 layer. Intine vesicles which seem to originate in the cell wall complex of the central body are seen in the intine and exine of cysts. Analogous structures are found on vegetative cells. The intine is divided into two chemically distinct areas by the two-layered intine membrane. Microtubules, previously reported only in vegetative cells, were found in cysts.     

Construction of a recF deletion mutant of Azotobacter vinelandii and its characterization

A deletion was engineered in the cloned recF gene by digestion with suitable restriction endonucleases and a tetracycline resistance gene cartridge was inserted. The mutation was subsequently transferred to the Azotobacter vinelandii chromosome by double cross-over under pressure of tetracycline selection. A recF recA mutant was also constructed in a similar manner. The mutations were found to be stable and mutation of the wild-type recF gene was confirmed by Southern blot hybridization. Both the mutants were UV sensitive and recombination deficient. Mutations in genes involved in nitrogen fixation in A. vinelandii are rather frequent and obtained comparatively easily despite of the presence of multiple identical chromosomes in A. vinelandii. It has been speculated that some kind of `homogenotization' process operates which is responsible for the `transmission' of mutation from one chromosome to all the chromosomes. This process is not affected by a mutation in recF or recA or in both recF and recA.     

Genetic analysis of Azotobacter vinelandii mutant strains unable to fix nitrogen.

Transformation was used to perform ratio test crosses with mutant strains of Azotobacter vinelandii unable to fix N2. Mutations that simultaneously eliminated both components of nitrogenase (nif-1 and nif-2) were tightly linked. The nif-45 mutation that resulted in the absence of an active molybdenum cofactor was closer to nif-1 and nif-2 than to any of the other nif mutations. Strains that lacked component I carried mutations that were closely linked to each other. Mutations that probably were located in the structural genes for components I and II appeared to be relatively close to each other on the A. vinelandii genome.     

Azotobacter vinelandii ferredoxin I: cloning, sequencing, and mutant analysis

The structure of Azotobacter vinelandii ferredoxin I (AvFdI) has been extensively characterized by a variety of techniques. Although its physiological function is unknown, it has long been implicated as being involved in electron donation to nitrogenase. Here we report that the AvFdI gene (fdxA) has been cloned from an EcoRI digest lambda library using a synthetic oligonucleotide probe and that its sequence has been determined. The amino acid sequence deduced from the DNA sequence is identical to the previously published protein sequence. Analysis of the promoter region indicates that AvFdI is not a nif specific gene product. A mutant of A. vinelandii has been constructed which is identical to the wild-type, at the DNA level, except that the fdxA gene has been interrupted by insertion of a kanamycin cartridge. This mutant, called LM100, does not synthesize AvFdI but does synthesize the Fe and MoFe proteins of nitrogenase and grows at wild-type rates under N2-fixing conditions. This demonstrates that AvFdI is not required for N2 fixation by A. vinelandii. There is a small acidic protein, which is present in wild-type A. vinelandii, whose level is dramatically increased in LM100. The nature of this protein is under further investigation.     

Excretion of ammonium by a nifL mutant of Azotobacter vinelandii fixing nitrogen.

A mutation in the gene upstream of nifA in Azotobacter vinelandii was introduced into the chromosome to replace the corresponding wild-type region. The resulting mutant, MV376, produced nitrogenase constitutively in the presence of 15 mM ammonium. When introduced into a nifH-lacZ fusion strain, the mutation permitted beta-galactosidase production in the presence of ammonium. The gene upstream of nifA is therefore designated nifL because of its similarity to the Klebsiella pneumoniae nifL gene in proximity to nifA, in mutant phenotype, and in amino acid sequence of the gene product. The A. vinelandii nifL mutant MV376 excreted significant quantities of ammonium (approximately 10 mM) during diazotrophic growth. In contrast, ammonium excretion during diazotrophy was much lower in a K. pneumoniae nifL deletion mutant (maximum, 0.15 mM) but significantly higher than in NifL+ K. pneumoniae. The expression of the A. vinelandii nifA gene, unlike that of K. pneumoniae, was not repressed by ammonium.     

Chemotactic Behavior of Azotobacter vinelandii

Chemotaxis was exhibited by Azotobacter vinelandii motile cells. Exposure of cells to sudden increases in attractant concentration suppressed the frequency of tumbling and resulted in smooth swimming. Cells responded chemotactically to a chemical gradient produced during metabolism. Motility occurred over a temperature range of 25 to 37°C with an optimum pH range of between pH 7.0 and 8.0. The average speed of motile cells was determined to be 74 μm/s or 37 body lengths per s. The speed of cells appeared to increase as a function of attractant concentration. Chemotactic systems for fructose, glucose, xylitol, and mannitol were inducible. A. vinelandii exhibited chemotaxis for a number of compounds, including hexoses, hexitols, pentitols, pentoses, disaccharides, and amino sugars. We conclude from these studies that A. vinelandii exhibits a temporal chemotactic sensing system.     

Excretion of ammonium by a nifL mutant of Azotobacter vinelandii fixing nitrogen.

A mutation in the gene upstream of nifA in Azotobacter vinelandii was introduced into the chromosome to replace the corresponding wild-type region. The resulting mutant, MV376, produced nitrogenase constitutively in the presence of 15 mM ammonium. When introduced into a nifH-lacZ fusion strain, the mutation permitted beta-galactosidase production in the presence of ammonium. The gene upstream of nifA is therefore designated nifL because of its similarity to the Klebsiella pneumoniae nifL gene in proximity to nifA, in mutant phenotype, and in amino acid sequence of the gene product. The A. vinelandii nifL mutant MV376 excreted significant quantities of ammonium (approximately 10 mM) during diazotrophic growth. In contrast, ammonium excretion during diazotrophy was much lower in a K. pneumoniae nifL deletion mutant (maximum, 0.15 mM) but significantly higher than in NifL+ K. pneumoniae. The expression of the A. vinelandii nifA gene, unlike that of K. pneumoniae, was not repressed by ammonium.     

Plasmids of Azotobacter vinelandii.

Four laboratory strains and two isolates of Azotobacter vinelandii were found to contain plasmids. Twenty-five laboratory strains which could fix nitrogen did not have free, covalently closed circular plasmid DNA. The plasmids varied in size from 9 to 52 megadaltons, and each strain yielded only one plasmid. No discernible differences in ability to fix nitrogen were found between plasmid-bearing and cured cultures.     

Endogenous Encystment of Azotobacter vinelandii

When young cells of Azotobacter vinelandii are impinged on membrane filters, washed free of carbon substrate, and placed on a mineral salts basal medium, the culture will proceed to encyst although at a slower rate than if n-butanol were supplied as a substrate. The endogenous cysts are depleted in polyβ-hydroxybutyrate and have a narrower intine but show an increased resistance to desiccation and are susceptible to lysis by chelating agents. Membrane-supported cells reveal details of the encystment process such as the formation of a zone within the capsule prior to exine formation and the early deposition of exine structures.     

Characterization of an Escherichia coli rotA mutant, affected in periplasmic peptidyl-prolyl cis/trans isomerase

The rotA gene of Escherichia coli encodes a peptidyl-prolyl cis/trans isomerase (PPlase), which is supposed to catalyse protein folding in the periplasm. To investigate the importance of the enzyme, the rotA gene was cloned and a chromosomal deletion mutant was created. The rotA mutant was normally viable. No residual PPlase activity could be detected in the periplasmic fraction of the mutant. Comparison of the patterns of periplasmic and outer membrane proteins by SDS-PAGE revealed no differences in protein composition between the rotA mutant and its parental strain. Similarly, the kinetics of periplasmic protein folding and outer membrane protein assembly appeared unaffected by the rotA mutation. Our results show that the periplasmic PPlase of E. coli is not essential and that the protein does not play an important role in protein folding.