Genome size and complexity in Azotobacter chroococcum

All of eight strains of Azotobacter chroococcum examined contained between two and six plasmids ranging from 7 to more than 200 MDal in size. Strain MCC-1, a derivative of NCIMB 8003, was cured of various of the four largest of its five plasmids and the phenotypes of the strains compared. all fixed nitrogen and exhibited uptake hydrogenase activity. No differences were observed in carbon source utilization or antibiotic, heavy metal or UV resistance. The genome sizes of two strains of A. chroococcum were determined by two-dimensional electrophoresis. Strain CW8, an isolate from local soil containing two small plasmids of 6 and 6.5 MDAl contained unique DNA sequences equivalent to 1.78 x 10(6) (+/- 20%) bp (1.2 x 10(9) Dal). In strain MDC-1, a derivative of MCC-1, containing a 190 MDal and 7 MDal plasmid, the genome size was 1.94 x 10(6) (+/- 20%) bp. In exponential batch cultures, both contained 20 to 25 genome equivalents per cell. MCD-1 exhibited complex UV kill kinetics with a marked plateau of resistance; CW8 showed a simple response inconsistent with the possibility of organization of its DNA into identical chromosome copies capable of independent segregation.     

The genome of Rhizobium leguminosarum has recognizable core and accessory components

Background

Rhizobium leguminosarum is an α-proteobacterial N₂-fixing symbiont of legumes that has been the subject of more than a thousand publications. Genes for the symbiotic interaction with plants are well studied, but the adaptations that allow survival and growth in the soil environment are poorly understood. We have sequenced the genome of R. leguminosarum biovar viciae strain 3841.

Results

The 7.75 Mb genome comprises a circular chromosome and six circular plasmids, with 61% G+C overall. All three rRNA operons and 52 tRNA genes are on the chromosome; essential protein-encoding genes are largely chromosomal, but most functional classes occur on plasmids as well. Of the 7,263 protein-encoding genes, 2,056 had orthologs in each of three related genomes (Agrobacterium tumefaciens, Sinorhizobium meliloti, and Mesorhizobium loti), and these genes were over-represented in the chromosome and had above average G+C. Most supported the rRNA-based phylogeny, confirming A. tumefaciens to be the closest among these relatives, but 347 genes were incompatible with this phylogeny; these were scattered throughout the genome but were over-represented on the plasmids. An unexpectedly large number of genes were shared by all three rhizobia but were missing from A. tumefaciens.

Conclusion

Overall, the genome can be considered to have two main components: a 'core', which is higher in G+C, is mostly chromosomal, is shared with related organisms, and has a consistent phylogeny; and an 'accessory' component, which is sporadic in distribution, lower in G+C, and located on the plasmids and chromosomal islands. The accessory genome has a different nucleotide composition from the core despite a long history of coexistence.     

Complete Genomic Structure of the Cultivated Rice Endophyte Azospirillum sp. B510

We determined the nucleotide sequence of the entire genome of a diazotrophic endophyte, Azospirillum sp. B510. Strain B510 is an endophytic bacterium isolated from stems of rice plants (Oryza sativa cv. Nipponbare). The genome of B510 consisted of a single chromosome (3 311 395 bp) and six plasmids, designated as pAB510a (1 455 109 bp), pAB510b (723 779 bp), pAB510c (681 723 bp), pAB510d (628 837 bp), pAB510e (537 299 bp), and pAB510f (261 596 bp). The chromosome bears 2893 potential protein-encoding genes, two sets of rRNA gene clusters (rrns), and 45 tRNA genes representing 37 tRNA species. The genomes of the six plasmids contained a total of 3416 protein-encoding genes, seven sets of rrns, and 34 tRNAs representing 19 tRNA species. Eight genes for plasmid-specific tRNA species are located on either pAB510a or pAB510d. Two out of eight genomic islands are inserted in the plasmids, pAB510b and pAB510e, and one of the islands is inserted into trnfM-CAU in the rrn located on pAB510e. Genes other than the nif gene cluster that are involved in N₂ fixation and are homologues of Bradyrhizobium japonicum USDA110 include fixABCX, fixNOQP, fixHIS, fixG, and fixLJK. Three putative plant hormone-related genes encoding tryptophan 2-monooxytenase (iaaM) and indole-3-acetaldehyde hydrolase (iaaH), which are involved in IAA biosynthesis, and ACC deaminase (acdS), which reduces ethylene levels, were identified. Multiple gene-clusters for tripartite ATP-independent periplasmic-transport systems and a diverse set of malic enzymes were identified, suggesting that B510 utilizes C₄-dicarboxylate during its symbiotic relationship with the host plant.     

Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120.

The nucleotide sequence of the entire genome of a filamentous cyanobacterium, Anabaena sp. strain PCC 7120, was determined. The genome of Anabaena consisted of a single chromosome (6,413,771 bp) and six plasmids, designated pCC7120alpha (408,101 bp), pCC7120beta (186,614 bp), pCC7120gamma (101,965 bp), pCC7120delta (55,414 bp), pCC7120epsilon (40,340 bp), and pCC7120zeta (5,584 bp). The chromosome bears 5368 potential protein-encoding genes, four sets of rRNA genes, 48 tRNA genes representing 42 tRNA species, and 4 genes for small structural RNAs. The predicted products of 45% of the potential protein-encoding genes showed sequence similarity to known and predicted proteins of known function, and 27% to translated products of hypothetical genes. The remaining 28% lacked significant similarity to genes for known and predicted proteins in the public DNA databases. More than 60 genes involved in various processes of heterocyst formation and nitrogen fixation were assigned to the chromosome based on their similarity to the reported genes. One hundred and ninety-five genes coding for components of two-component signal transduction systems, nearly 2.5 times as many as those in Synechocystis sp. PCC 6803, were identified on the chromosome. Only 37% of the Anabaena genes showed significant sequence similarity to those of Synechocystis, indicating a high degree of divergence of the gene information between the two cyanobacterial strains.     

Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain (ATCC 12228)

Staphylococcus epidermidis strains are diverse in their pathogenicity; some are invasive and cause serious nosocomial infections, whereas others are non-pathogenic commensal organisms. To analyse the implications of different virulence factors in Staphylococcus epidermidis infections, the complete genome of Staphylococcus epidermidis strain ATCC 12228, a non-biofilm forming, non-infection associated strain used for detection of residual antibiotics in food products, was sequenced. This strain showed low virulence by mouse and rat experimental infections. The genome consists of a single 2499 279 bp chromosome and six plasmids. The chromosomal G + C content is 32.1% and 2419 protein coding sequences (CDS) are predicted, among which 230 are putative novel genes. Compared to the virulence factors in Staphylococcus aureus, aside from delta-haemolysin and beta-haemolysin, other toxin genes were not found. In contrast, the majority of adhesin genes are intact in ATCC 12228. Most strikingly, the ica operon coding for the enzymes synthesizing interbacterial cellular polysaccharide is missing in ATCC 12228 and rearrangements of adjacent genes are shown. No mec genes, IS256, IS257, were found in ATCC 12228. It is suggested that the absence of the ica operon is a genetic marker in commensal Staphylococcus epidermidis strains which are less likely to become invasive.     

Complete genome sequence of the ammonia-oxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea

Nitrosomonas europaea (ATCC 19718) is a gram-negative obligate chemolithoautotroph that can derive all its energy and reductant for growth from the oxidation of ammonia to nitrite. Nitrosomonas europaea participates in the biogeochemical N cycle in the process of nitrification. Its genome consists of a single circular chromosome of 2,812,094 bp. The GC skew analysis indicates that the genome is divided into two unequal replichores. Genes are distributed evenly around the genome, with approximately 47% transcribed from one strand and approximately 53% transcribed from the complementary strand. A total of 2,460 protein-encoding genes emerged from the modeling effort, averaging 1,011 bp in length, with intergenic regions averaging 117 bp. Genes necessary for the catabolism of ammonia, energy and reductant generation, biosynthesis, and CO(2) and NH(3) assimilation were identified. In contrast, genes for catabolism of organic compounds are limited. Genes encoding transporters for inorganic ions were plentiful, whereas genes encoding transporters for organic molecules were scant. Complex repetitive elements constitute ca. 5% of the genome. Among these are 85 predicted insertion sequence elements in eight different families. The strategy of N. europaea to accumulate Fe from the environment involves several classes of Fe receptors with more than 20 genes devoted to these receptors. However, genes for the synthesis of only one siderophore, citrate, were identified in the genome. This genome has provided new insights into the growth and metabolism of ammonia-oxidizing bacteria.     

Complete Genome Sequence of the Marine, Chemolithoautotrophic, Ammonia-Oxidizing Bacterium Nitrosococcus oceani ATCC 19707

The gammaproteobacterium Nitrosococcus oceani (ATCC 19707) is a gram-negative obligate chemolithoautotroph capable of extracting energy and reducing power from the oxidation of ammonia to nitrite. Sequencing and annotation of the genome revealed a single circular chromosome (3,481,691 bp; G+C content of 50.4%) and a plasmid (40,420 bp) that contain 3,052 and 41 candidate protein-encoding genes, respectively. The genes encoding proteins necessary for the function of known modes of lithotrophy and autotrophy were identified. Contrary to betaproteobacterial nitrifier genomes, the N. oceani genome contained two complete rrn operons. In contrast, only one copy of the genes needed to synthesize functional ammonia monooxygenase and hydroxylamine oxidoreductase, as well as the proteins that relay the extracted electrons to a terminal electron acceptor, were identified. The N. oceani genome contained genes for 13 complete two-component systems. The genome also contained all the genes needed to reconstruct complete central pathways, the tricarboxylic acid cycle, and the Embden-Meyerhof-Parnass and pentose phosphate pathways. The N. oceani genome contains the genes required to store and utilize energy from glycogen inclusion bodies and sucrose. Polyphosphate and pyrophosphate appear to be integrated in this bacterium's energy metabolism, stress tolerance, and ability to assimilate carbon via gluconeogenesis. One set of genes for type I ribulose-1,5-bisphosphate carboxylase/oxygenase was identified, while genes necessary for methanotrophy and for carboxysome formation were not identified. The N. oceani genome contains two copies each of the genes or operons necessary to assemble functional complexes I and IV as well as ATP synthase (one H⁺-dependent F₀F₁ type, one Na⁺-dependent V type).     

Presence of a Vanadium Nitrogenase in Azotobacter paspali

There have been no previous studies on the genetics of Azotobacter paspali, an aerobic bacterium which forms a highly specific diazotrophic association with Bahia grass (Paspalum notatum). We constructed A. paspali strains defective in the molybdenum nitrogenase so that alternative N₂ases could be studied. The cosmid vector pTBE and genomic DNA fragments (~50 kb) of A. paspali ATCC 23367 were used to construct a gene library in Escherichia coli. Recombinant cosmids containing sequences homologous to molybdenum nitrogenase nifDK structural genes were identified by hybridization. A 2.9-kb fragment bearing the putative nifDK genes of A. paspali was subcloned and mutagenized in vitro by the insertion of a kanamycin resistance gene cassette. The mutation was recombined into the chromosome of A. paspali with the suicide vector pCU101. One resultant mutant strain, AP2, was incapable of diazotrophic growth in a molybdenum-containing medium (Nif^-) without vanadium but grew well in a molybdenum-deficient medium with vanadium. The nitrogenase system in AP2 reduced acetylene to ethylene and produced ethane as 2.4% of the total products. Molybdenum levels as low as 10 nM prevented the diazotrophic growth of AP2, even in the presence of vanadium at levels up to 10 μM. These results are consistent with the existence of a vanadium nitrogenase system in A. paspali.     

Structural analysis of four large plasmids harboring in a unicellular cyanobacterium, Synechocystis sp. PCC 6803.

The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 consists of a single chromosome and several plasmids of different sizes, and the nucleotide sequences of the chromosome and three small plasmids (5.2 kb, 2.4 kb, and 2.3 kb) have already been sequenced. We newly determined the nucleotide sequences of four large plasmids, which have been identified in our laboratory (pSYSM:120 kb, pSYSX:106 kb, pSYSA:103 kb, and pSYSG:44 kb). Computer-aided analysis was performed to explore the genetic information carried by these plasmids. A total of 397 potential protein-encoding genes were predicted, but little information was obtained about the functional relationship of plasmids to host cell, as a large portion of the predicted genes (77%) were of unknown function. The occurrence of the potential genes on plasmids was divergent, and parA was the only gene common to all four large plasmids. The distribution data of a Cyanobacterium-specific sequence (HIP1: 5'-GCGATCGC-3') suggested that respective plasmids could have originated from different cyanobacterial strains.     

Complete genome sequence of the marine, chemolithoautotrophic, ammonia-oxidizing bacterium Nitrosococcus oceani ATCC 19707

The gammaproteobacterium Nitrosococcus oceani (ATCC 19707) is a gram-negative obligate chemolithoautotroph capable of extracting energy and reducing power from the oxidation of ammonia to nitrite. Sequencing and annotation of the genome revealed a single circular chromosome (3,481,691 bp; G+C content of 50.4%) and a plasmid (40,420 bp) that contain 3,052 and 41 candidate protein-encoding genes, respectively. The genes encoding proteins necessary for the function of known modes of lithotrophy and autotrophy were identified. Contrary to betaproteobacterial nitrifier genomes, the N. oceani genome contained two complete rrn operons. In contrast, only one copy of the genes needed to synthesize functional ammonia monooxygenase and hydroxylamine oxidoreductase, as well as the proteins that relay the extracted electrons to a terminal electron acceptor, were identified. The N. oceani genome contained genes for 13 complete two-component systems. The genome also contained all the genes needed to reconstruct complete central pathways, the tricarboxylic acid cycle, and the Embden-Meyerhof-Parnass and pentose phosphate pathways. The N. oceani genome contains the genes required to store and utilize energy from glycogen inclusion bodies and sucrose. Polyphosphate and pyrophosphate appear to be integrated in this bacterium's energy metabolism, stress tolerance, and ability to assimilate carbon via gluconeogenesis. One set of genes for type I ribulose-1,5-bisphosphate carboxylase/oxygenase was identified, while genes necessary for methanotrophy and for carboxysome formation were not identified. The N. oceani genome contains two copies each of the genes or operons necessary to assemble functional complexes I and IV as well as ATP synthase (one H(+)-dependent F(0)F(1) type, one Na(+)-dependent V type).     

A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi

We have determined that Borrelia burgdorferi strain B31 MI carries 21 extrachromosomal DNA elements, the largest number known for any bacterium. Among these are 12 linear and nine circular plasmids, whose sequences total 610 694 bp. We report here the nucleotide sequence of three linear and seven circular plasmids (comprising 290 546 bp) in this infectious isolate. This completes the genome sequencing project for this organism; its genome size is 1 521 419 bp (plus about 2000 bp of undetermined telomeric sequences). Analysis of the sequence implies that there has been extensive and sometimes rather recent DNA rearrangement among a number of the linear plasmids. Many of these events appear to have been mediated by recombinational processes that formed duplications. These many regions of similarity are reflected in the fact that most plasmid genes are members of one of the genome's 161 paralogous gene families; 107 of these gene families, which vary in size from two to 41 members, contain at least one plasmid gene. These rearrangements appear to have contributed to a surprisingly large number of apparently non-functional pseudogenes, a very unusual feature for a prokaryotic genome. The presence of these damaged genes suggests that some of the plasmids may be in a period of rapid evolution. The sequence predicts 535 plasmid genes >/=300 bp in length that may be intact and 167 apparently mutationally damaged and/or unexpressed genes (pseudogenes). The large majority, over 90%, of genes on these plasmids have no convincing similarity to genes outside Borrelia, suggesting that they perform specialized functions.     

Comparative Genome Analysis of Wheat Blue Dwarf Phytoplasma,an Obligate Pathogen That Causes Wheat Blue Dwarf Disease in China

Wheat blue dwarf (WBD) disease is an important disease that has caused heavy losses in wheat production in northwestern China. This disease is caused by WBD phytoplasma, which is transmitted by Psammotettix striatus. Until now, no genome information about WBD phytoplasma has been published, seriously restricting research on this obligate pathogen. In this paper, we report a new sequencing and assembling strategy for phytoplasma genome projects. This strategy involves differential centrifugation, pulsed-field gel electrophoresis, whole genome amplification, shotgun sequencing, de novo assembly, screening of contigs from phytoplasma and the connection of phytoplasma contigs. Using this scheme, the WBD phytoplasma draft genome was obtained. It was comprised of six contigs with a total size of 611,462 bp, covering ∼94% of the chromosome. Five-hundred-twenty-five protein-coding genes, two operons for rRNA genes and 32 tRNA genes were identified. Comparative genome analyses between WBD phytoplasma and other phytoplasmas were subsequently carried out. The results showed that extensive arrangements and inversions existed among the WBD, OY-M and AY-WB phytoplasma genomes. Most protein-coding genes in WBD phytoplasma were found to be homologous to genes from other phytoplasmas; only 22 WBD-specific genes were identified. KEGG pathway analysis indicated that WBD phytoplasma had strongly reduced metabolic capabilities. However, 46 transporters were identified, which were involved with dipeptides/oligopeptides, spermidine/putrescine, cobalt and Mn/Zn transport, and so on. A total of 37 secreted proteins were encoded in the WBD phytoplasma chromosome and plasmids. Of these, three secreted proteins were similar to the reported phytoplasma virulence factors TENGU, SAP11 and SAP54. In addition, WBD phytoplasma possessed several proteins that were predicted to play a role in its adaptation to diverse environments. These results will provide clues for research on the pathogenic mechanisms of WBD phytoplasma and will also provide a perspective about the genome sequencing of other phytoplasmas and obligate organisms.     

Efficient transposition of IS204-derived plasmids in Streptomyces coelicolor

In order to study functional gene expression in Streptomyces coelicolor, a mini-transposon encoding the apramycin resistance gene aac(3)IV within its inverted repeat (IR) boundaries was constructed based on IS204, which was previously identified in the genome of Nocardia asteroides YP21. The mini-transposon and IS204 transposase gene were then put on a kanamycin-resistant conjugative plasmid pDZY101 that can only replicate in Escherichia coli. After mating with S. coelicolor A3(2) M145, resistant colonies arose efficiently on both apramycin and kanamycin plates. Plasmid rescue indicated that entire plasmids were inserted into the M145 genome with cleavage at an inverted repeat junction formed by the right inverted repeat (IRR) and the last 18 bp of the transposase gene, while the left inverted repeat (IRL) was untouched. Southern blot analysis of the mutants using an aac(3)IV gene probe showed that transposition of plasmid pDZY101 was genetically stable, with a single-copy insertion within the S. coelicolor M145 genome. Several mutagenesis libraries of S. coelicolor M145 were constructed using plasmid pDZY101 derivatives and the transposon insertion site was determined. The correlation between novel mutant phenotypes and previously uncharacterized genes was established and these transposon locations were widely scattered around the genome.     

Stimulation of Agrobacterium tumefaciens Growth by Azotobacter vinelandii Ferrisiderophores

Azotobacter vinelandii stimulated the growth of Agrobacterium tumefaciens H2, H23, H24, H27, and ATCC 15955 on media containing insoluble iron sources. The Azotobacter vinelandii siderophores appeared to promote Agrobacterium tumefaciens growth by solubilizing mineral iron, and the ferrisiderophores so formed then acted as iron sources for Agrobacterium tumefaciens. Agrobactin, the Agrobacterium siderophore, appeared to be inefficient in solubilizing mineral iron directly.     

Rapid cloning and bioinformatic analysis of spinach Y chromosome-specific EST sequences

The genome of spinach single chromosome complement is about 1000 Mbp, which is the model material to study the molecular mechanisms of plant sex differentiation. The cytological study showed that the biggest spinach chromosome (chromosome 1) was taken as spinach sex chromosome. It had three alleles of sex-related X, X ^m and Y. Many researchers have been trying to clone the sex-determining genes and investigated the molecular mechanism of spinach sex differentiation. However, there are no successful cloned reports about these genes. A new technology combining chromosome microdissection with hybridization-specific amplification (HSA) was adopted. The spinach Y chromosome degenerate oligonucleotide primed-PCR (DOP-PCR) products were hybridized with cDNA of the male spinach flowers in florescence. The female spinach genome was taken as blocker and cDNA library specifically expressed in Y chromosome was constructed. Moreover, expressed sequence tag (EST) sequences in cDNA library were cloned, sequenced and bioinformatics was analysed. There were 63 valid EST sequences obtained in this study. The fragment size was between 53 and 486 bp. BLASTn homologous alignment indicated that 12 EST sequences had homologous sequences of nucleic acids, the rest were new sequences. BLASTx homologous alignment indicated that 16 EST sequences had homologous protein-encoding nucleic acid sequence. The spinach Y chromosome-specific EST sequences laid the foundation for cloning the functional genes, specifically expressed in spinach Y chromosome. Meanwhile, the establishment of the technology system in the research provided a reference for rapid cloning of other biological sex chromosome-specific EST sequences.     

Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti.

The complete nucleotide sequence of the genome of a symbiotic bacterium Mesorhizobium loti strain MAFF303099 was determined. The genome of M. loti consisted of a single chromosome (7,036,071 bp) and two plasmids, designated as pMLa (351,911 bp) and pMLb (208, 315 bp). The chromosome comprises 6752 potential protein-coding genes, two sets of rRNA genes and 50 tRNA genes representing 47 tRNA species. Fifty-four percent of the potential protein genes showed sequence similarity to genes of known function, 21% to hypothetical genes, and the remaining 25% had no apparent similarity to reported genes. A 611-kb DNA segment, a highly probable candidate of a symbiotic island, was identified, and 30 genes for nitrogen fixation and 24 genes for nodulation were assigned in this region. Codon usage analysis suggested that the symbiotic island as well as the plasmids originated and were transmitted from other genetic systems. The genomes of two plasmids, pMLa and pMLb, contained 320 and 209 potential protein-coding genes, respectively, for a variety of biological functions. These include genes for the ABC-transporter system, phosphate assimilation, two-component system, DNA replication and conjugation, but only one gene for nodulation was identified.     

Complete genome sequence of Riemerella anatipestifer reference strain

Riemerella anatipestifer is an infectious pathogen causing serositis in ducks. We had the genome of the R. anatipestifer reference strain ATCC 11845 sequenced. The completed draft genome consists of one circular chromosome with 2,164,087 bp. There are 2,101 genes in the draft, and its GC content is 35.01%.     

Plasmid Characterization and Chromosome Analysis of Two netF+ Clostridium perfringens Isolates Associated with Foal and Canine Necrotizing Enteritis

The recent discovery of a novel beta-pore-forming toxin, NetF, which is strongly associated with canine and foal necrotizing enteritis should improve our understanding of the role of type A Clostridium perfringens associated disease in these animals. The current study presents the complete genome sequence of two netF-positive strains, JFP55 and JFP838, which were recovered from cases of foal necrotizing enteritis and canine hemorrhagic gastroenteritis, respectively. Genome sequencing was done using Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000. The JFP55 and JFP838 genomes include a single 3.34 Mb and 3.53 Mb chromosome, respectively, and both genomes include five circular plasmids. Plasmid annotation revealed that three plasmids were shared by the two newly sequenced genomes, including a NetF/NetE toxins-encoding tcp-conjugative plasmid, a CPE/CPB2 toxins-encoding tcp-conjugative plasmid and a putative bacteriocin-encoding plasmid. The putative beta-pore-forming toxin genes, netF, netE and netG, were located in unique pathogenicity loci on tcp-conjugative plasmids. The C. perfringens JFP55 chromosome carries 2,825 protein-coding genes whereas the chromosome of JFP838 contains 3,014 protein-encoding genes. Comparison of these two chromosomes with three available reference C. perfringens chromosome sequences identified 48 (~247 kb) and 81 (~430 kb) regions unique to JFP55 and JFP838, respectively. Some of these divergent genomic regions in both chromosomes are phage- and plasmid-related segments. Sixteen of these unique chromosomal regions (~69 kb) were shared between the two isolates. Five of these shared regions formed a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. These results provide significant insight into the basis of canine and foal necrotizing enteritis and are the first to demonstrate that netF resides on a large and unique plasmid-encoded locus.     

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.