Genomic Manipulations of the Diazotroph Azotobacter vinelandii.

The biological reduction of nitrogen gas to ammonia is limited to a select group of nitrogen-fixing prokaryotes. While nitrogenase is the catalyst of nitrogen fixation in these biological systems, a consortium of additional gene products is required for the synthesis, activation, and catalytic competency of this oxygen-sensitive metalloenzyme. Thus, the biochemical complexity of this process often requires functional studies and isolation of gene products from the native nitrogen-fixing organisms. The strict aerobe Azotobacter vinelandii is the best-studied model bacterium among diazotrophs. This chapter provides a description of procedures for targeted genomic manipulation and isolation of A. vinelandii strains. These methods have enabled identification and characterization of gene products with roles in nitrogen fixation and other related aspects of metabolism. The ability to modify and control expression levels of targeted sequences provides a biotechnological tool to uncover molecular details associated with nitrogen fixation, as well as to exploit this model system as a host for expression of oxygen-sensitive proteins.

A novel bleb-dependent polysaccharide export system in nitrogen-fixing Azotobacter vinelandii subjected to low nitrogen gas levels

The alginate biofilm-producing bacterium Azotobacter vinelandii aerobically fixes nitrogen by oxygensensitive nitrogenases. Here we investigated the bacterial response to nitrogen/oxygen gas mixtures. A. vinelandii cells were cultured in nitrogen-free minimal media containing gas mixtures differing in their ratios of nitrogen and oxygen. The bacteria did not grow at oxygen concentrations >75 % but grew well in the presence of 5 % nitrogen/25 % oxygen. Growth of wild-type and alginate-deficient strains when cultured with 50 % oxygen did not differ substantially, indicating that alginate is not required for the protection of nitrogenases from oxygen damage. In response to decreasing nitrogen levels, A. vinelandii produced greater amounts of alginate, accompanied by the formation of blebs on the cell surface. The encystment of vegetative cells occurred in tandem with the release of blebs and the development of a multilayered exine. Immuno electron microscopy using anti alginate-antibody revealed that the blebs contained alginate molecules. By contrast, alginate-deficient mutants could not form blebs. Taken together, our data provide evidence for a novel bleb dependent polysaccharide export system in A. vinelandii that is activated in response to low nitrogen gas levels (AU)

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Fluorescence in situ hybridization for intracellular localization of nifH mRNA.

Few reports on in situ mRNA detection in bacteria have been published, even though a major aim in environmental microbiology is to link function/activity to the identity of the organisms. This study reports a reliable approach for the in situ detection of nifH mRNA using fluorescence hybridization based on a previously described protocol for pmoA. nifH codes for a dinitrogenase reductase, a key enzyme in dinitrogen fixation. nifH mRNA was hybridized with a digoxigenin-labelled polynucleotide probe. The hybrid was detected with an anti-DIG-antibody labelled with horseradish peroxidase. Subsequently, the signal was amplified by catalyzed reporter deposition (CARD) with fluorochrome-labelled tyramides. Furthermore, the imaged organisms were identified using standard fluorescence in situ hybridization of rRNA. Thus, the approach enabled us specifically to link in situ the information from the dinitrogen fixation activity of an organism to its identity. Unexpectedly, the signals derived from nifH mRNA hybridization showed a distinct uneven pattern within the cells. This indicated that the method used could even give insights about the localization of the detected mRNA within the cell, which is a potential use of mRNA fluorescence in situ hybridization (FISH) that has not been reported up to now for bacterial cells.

Characterization of Azotobacter vinelandii aggregation in submerged culture by digital image analysis.

A simple and accurate method for determining the distribution of sizes of single cells and aggregates of Azotobacter vinelandii by image analysis has been developed. A staining procedure using methylene blue helps to enhance the contrast between aggregates and background without altering aggregate size distribution. Sample dilution affected the distribution of the population and therefore should be avoided. Mixing and aeration conditions during culture play an important role in the aggregation of A. vinelandii. Cells grown under mild mixing conditions (unbaffled flasks) presented a thick slime layer and formed aggregates of up to 35 microm of average equivalent diameter. In contrast, under strong agitation conditions (baffled flasks) practically no aggregates were formed throughout cultivation. The method described can be used for the characterization of aggregation of other microbial cultures.

Role of Azotobacter vinelandii mucA and mucC gene products in alginate production.

Azotobacter vinelandii produces the exopolysaccharide alginate, which is essential for its differentiation to desiccation-resistant cysts. In different bacterial species, the alternative sigma factor sigma(E) regulates the expression of functions related to the extracytoplasmic compartments. In A. vinelandii and Pseudomonas aeruginosa, the sigma(E) factor (AlgU) is essential for alginate production. In both bacteria, the activity of this sigma factor is regulated by the product of the mucA, mucB, mucC, and mucD genes. In this work, we studied the transcriptional regulation of the A. vinelandii algU-mucABCD gene cluster, as well as the role of the mucA and mucC gene products in alginate production. Our results show the existence of AlgU autoregulation and show that both MucA and MucC play a negative role in alginate production.

Inactivation of the ampDE operon increases transcription of algD and affects morphology and encystment of Azotobacter vinelandii.

Transcription of algD, encoding GDP-mannose dehydrogenase, the key enzyme in the alginate biosynthetic pathway, is highly regulated in Azotobacter vinelandii. We describe here the characterization of a Tn5 insertion mutant (AC28) which shows a higher level of expression of an algD::lacZ fusion. AC28 cells were morphologically abnormal and unable to encyst. The cloning and nucleotide sequencing of the Tn5-disrupted locus in AC28 revealed an operon homologous to the Escherichia coli ampDE operon. Tn5 was located within the ampD gene, encoding a cytosolic N-acetyl-anhydromuramyl-L-alanine amidase that participates in the intracellular recycling of peptidoglycan fragments. The ampE gene encodes a transmembrane protein, but the function of the protein is not known. We constructed strains carrying ampD or ampE mutations and one with an ampDE deletion. The strain with a deletion of the ampDE operon showed a phenotype similar to that of mutant AC28. The present work demonstrates that both alginate production and bacterial encystment are greatly influenced by the bacterial ability to recycle its cell wall.

Isolation and characterization of nifDK::kanamycin and nitrogen fixation proficient Azotobacter vinelandii strain, and its implication on the status of multiple chromosomes in Azotobacter.

Several lines of experimental analyses on the ploidy status of Azotobacter vinelandii genome lead to the conclusion that it contains more than 40 copies of its chromosome and therefore it is a polyploid organism. The genetic evidence argues against the existence of polyploidy in these cells since the segregation pattern of genetic markers under lack of selection pressure mimic that of haploids. However, when A. vinelandii was made Nif- by inserting a kanamycin resistance marker gene in the nifDK sequence and the cells were selected for kanamycin resistance and Nif+ phenotype, we were able to score colonies that are both kanamycin resistant and Nif+. Therefore, when the cells were subjected to forced double selection of the same locus, they behaved as if they carried at least two chromosomes, one carrying the kanamycin resistance marker in the nifDK genes and the other carrying the intact nifDK genes. These analyses suggested that at least a diploidy status can be induced in these cells under selection pressure.

COVASIAM: an image analysis method that allows detection of confluent microbial colonies and colonies of various sizes for automated counting.

In this work we introduce the confluent and various sizes image analysis method (COVASIAM), an automated colony count technique that uses digital imaging technology for detection and separation of confluent microbial colonies and colonies of various sizes growing on petri dishes. The proposed method takes advantage of the optical properties of the surfaces of most microbial colonies. Colonies in the petri dish are epi-illuminated in order to direct the reflection of concentrated light coming from a halogen lamp towards an image-sensing device. In conjunction, a multilevel threshold algorithm is proposed for colony separation and counting. These procedures improved the quantification of colonies showing confluence or differences in size. We tested COVASIAM with a sample set of microorganisms that form colonies with contrasting physical properties: Saccharomyces cerevisiae, Aspergillus nidulans, Escherichia coli, Azotobacter vinelandii, Pseudomonas aeruginosa, and Rhizobium etli. These physical properties range from smooth to hairy, from bright to opaque, and from high to low convexities. COVASIAM estimated an average of 95.47% (sigma = 8.55%) of the manually counted colonies, while an automated method based on a single-threshold segmentation procedure estimated an average of 76% (sigma = 16.27) of the manually counted colonies. This method can be easily transposed to almost every image-processing analyzer since the procedures to compile it are generically standard.