Identification of Azospirillum strains by restriction fragment length polymorphism of the 16S rDNA and of the histidine operon

Abstract DNA fingerprints of several Azospirillum strains, belonging to the five known species A. amazonense, A. brasilense, A. halopraeferens, A. irakense and A. lipoferum , were obtained by restriction analysis of the amplified 16S rDNA and by restriction fragment length polymorphism of the histidine biosynthetic genes. Data obtained showed that amplified rDNA restriction analysis is an easy, fast, reproducible and reliable tool for identification of Azospirillum strains, mainly at the species level, whereas restriction fragment length polymorphism could, in some cases, differentiate strains belonging to the same species. Moreover, both analyses gave congruent results in grouping strains and in the assignment of new strains to a given species.     

HindII, HindIII, and HpaI restriction fragment maps of bacteriophage lambda DNA

The site-specific restriction endonucleases isolated from Hemophilus influenzae strains Rc (HincII) and Rd (HindII + III), and Hemophilus parainfluenzae (HpaI) were used to digest bacteriophage lambda DNA into 34, 40, and 15 specific fragments, respectively. The sites cleaved by each of these enzymes were localized on the lambda physical map and the fragments resulting from these cleavages were electrophoretically identified on gels by (1) analysis of the digestion profiles of deletion and transducing derivatives of lambda; and (2) digesting individual fragments produced by one restriction endonuclease with another restriction endonuclease. This paper presents the HindII, HindIII, and HpaI restriction fragment maps for the entire lambda genome, and the data used to derive these maps for the region of the lambda genome between the attachment site (at 57.3% lambda) and the right vegetative end (100% lambda). The data for mapping the left arm of lambda may be found in the accompanying paper (Robinson and Landy, 1977).     

Restriction endonuclease cleavage of satellite DNA in intact bovine nuclei

We have analyzed the efficiency with which specific nucleotide sequences within nucleosomes are recognized and cleaved by DNA restriction endonucleases. A system amenable to this sort of analysis is the cleavage of the bovine genome with the restriction endonuclease EcoRI. Bovine satellite I comprises 7% of the genome and is tandemly repetitious with an EcoRI site at 1400 base pair (bp) intervals within this sequence. The ease with which this restriction fragment can be measured permits an analysis of the accessibility of this sequence when organized in a nucleosomal array.Initial studies indicated that satellite I sequences are organized in a nucleosomal structure in a manner analogous to that observed for total genomic DNA. We then examined the accessibility of the EcoRI cleavage sites in satellite to endonucleolytic cleavage in intact nuclei. We find that whereas virtually all the satellite I sequences from naked DNA are cleaved into discrete 1400 bp fragments, only 33% of the satellite I DNA is liberated as this fragment from intact nuclei. These data indicate that 57% of the EcoRI sites in nuclei are accessible to cleavage and that cleavage can occur within the core of at least half the nucleosomal subunits. Analysis of the products of digestion suggests a random distribution of nucleosomes about the EcoRI sites of satellite I DNA.Finally, the observation that satellite sequences can be cleaved from nuclei to 1400 bp length fragments with their associated proteins provides a method for the isolation of specific sequences as chromatin. Using sucrose gradient velocity centrifugation, we have isolated a 70% pure fraction of satellite I chromatin. Nuclease digestion of this chromatin fraction reveals the presence of nucleosomal subunits and indicates that specific sequences can be isolated in this manner without gross disorganization of their subunit structure.     

Construction and analysis of recombinant lambda phages containing mitochondrial DNA fragments.

Rat mtDNA has a molecular length of about 16 kilobase (kb) pairs and is cleaved into seven fragments by restriction endonuclease EcoRI. These fragments were cloned in Escherichia coli K-12 host using lambda gtWES.lambda B' (lambda gtWES.lambda B, for short, in this paper) as a vector. Recombinant DNAs containing one or a few fragments of the mtDNA were transfected to CaCl2-treated E. coli, and the plaques containing specific recombinant phages were selected. DNA amplified in the recombinanat phage lambda gt.mt was shown to contain the same restriction endonuclease cleavage sites as those found in the mtDNA. Present results permitted the DNA sequencing of any portion of the mitochondrial genome.     

Phylogeny of the genus Azospirillum based on 16S rDNA sequence

Abstract The 16S rDNA of 17 strains of Azospirillum , 14 assigned to one of the known species A. amazonense A. brasilense A. halopraeferens A. irakense and A. lipoferum , and the other three of uncertain taxonomic position, was sequenced after polymerase chain reaction amplification and analysed in order to investigate the phylogenetic relationships at the intra-generic and super-generic level. The phylogenetic analysis confirms that the genus Azospirillum constitutes a phylogenetically separate entity within the a subclass of Proteobacteria and that the five species are well defined. A. brasilense and A. lipoferum are closely related species and form one cluster together with A. halopraeferens ; the pair of species A. amazonense and A. irakense forms a second cluster in which Rhodospirillum centenum is also placed.     

Regulation of nitrogenase activity by ammonium chloride in Azospirillum spp.

Ammonium chloride (greater than or equal to 0.05 mM) effectively and reversibly inhibited the nitrogenase activity of Azospirillum brasilense, Azospirillum lipoferum and Azospirillum amazonense. The glutamine synthetase inhibitor L-methionine-DL- sulfoximine abolished this "switch-off" in A. lipoferum and A. brasilense, but not in A. amazonense. Azaserine, an inhibitor of glutamate synthase, inhibited nitrogenase activity itself. This provides further evidence for glutamine as a metabolite of regulatory importance in the NH4+ switch-off phenomenon. In A. brasilense and A. lipoferum, a transition period before the complete inhibition of nitrogenase activity after the addition of 1 mM ammonium chloride was observed. The in vitro nitrogenase activity also was decreased after treatment with ammonium. During sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a second dinitrogenase reductase (Fe protein) subunit appeared, which migrated in coincidence with the modified subunit of the inactive Fe protein of the nitrogenase of Rhodospirillum rubrum. After the addition of ammonium 32P was incorporated into this subunit of the Fe protein of A. brasilense. In A. amazonense, the inhibition of nitrogenase activity by ammonium was only partial, and no transition period could be observed. The in vitro nitrogenase activity of ammonium-treated cells was not decreased, and no evidence for a modified Fe protein subunit was found. Nitrogenase extracts of A. amazonense were active and had an Fe protein that migrated as a close double band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Intracellular Location and O(2) Sensitivity of Uptake Hydrogenase in Azospirillum spp

Uptake hydrogenase activity of Azospirillum brasilense in vitro (cell-free extract) was very much more sensitive to O(2) than was that of A. amazonense, and the O(2) pressure optima for uptake hydrogenase activities were 0.01 and 0.4 to 3 kPa for A. brasilense and A. amazonense, respectively. The addition of superoxide dismutase did not increase uptake hydrogenase activity of A. brasilense either in vivo or in vitro. The O(2) uptake rates of A. brasilense and A. amazonense were nearly the same. Inhibition of A. brasilense O(2)-dependent uptake hydrogenase activity by O(2) was highly reversible under the conditions tested. O(2) also markedly inhibited in vitro methylene blue-dependent uptake hydrogenase activity of A. brasilense, and this inhibition was highly reversible. It is concluded that the difference in O(2) tolerance of the uptake hydrogenases is not due to a difference in respiratory protection in the two species and may be due to inherent differences in the two enzymes. For the three species, A. brasilense, A. amazonense, and A. lipoferum, almost all the recovered methylene blue-dependent uptake hydrogenase activity was associated with the membrane fraction.     

Identification of sequences necessary for packaging DNA into lambda phage heads

Several species of DNA molecules are packaged into lambda phage heads if they carry the region around the cohesive end site of lambda phage (cos lambda). The minimal functional sequence around cos lambda needed for packaging was examined by cloning in pBR322. The results showed that the minimal region contained 85 bp around cos lambda; 45 bp of the left arm of lambda phage and 40 bp of the right arm. A 75-bp region located to the right of the minimal region seems to enhance packaging. A 223-bp fragment containing these regions can be used as a portable element for plasmid DNA packaging into lambda phage heads. Plasmid ppBest 322, a derivative of pBR322 carrying this portable packager and both amp and tet genes, was constructed. This plasmid is useful for cloning of large DNA fragments.     

pH dependence of uptake hydrogenases in Azospirillum brasilense and A. amazonense

Abstract Methylene blue (MB)-dependent uptake hydrogenase activity of Azospirillum brasilense was at least 30% higher than O₂-dependent activity in this species, whereas for A. amazonense the reverse was true. Both MB-dependent and O₂-dependent uptake hydrogenase activities of A. brasilense showed maxima at pH 7.0 both in vivo and in vitro. For A. amazonense , both in vivo and in vitro, the MB-dependent uptake hydrogenase activity increased roughly linearly as pH values were increased from pH 6.25 to 7.75, whereas the O₂-dependent activity was almost unaffected by pH. The optimal pH for A. amazonense growth was broad and in the range of 5.4 to 6.5.     

Restriction fragment length polymorphism of Azospirillum strains

Abstract: Total DNA of various Azospirillum strains representing different species was digested with restriction enzymes, Southern blotted and hybridized with four A. brasilense probes. Pairwise comparison of the conserved hybridization fragments was applied to calculate sequence divergence and to group the strains using the unweighted pair group method. The resulting dendrogram grouped the strains according to the known species indicating that the analysis of the restriction fragment length polymorphism is an useful tool for characterizing Azospirillum isolates.     

Loss of reiterated DNA sequences during serial passage of human diploid fibroblasts

A specific family of tandemly repeated DNA sequences was found to diminish in the human genome after serial passage of three strains of diploid fibroblasts. Eco RI restriction fragments of 340 and 680 bp were significantly reduced in quantity at late passage as determined by autoradiography of 14C-DNA and also by ethidium bromide fluorescence. The reduction in these closely related DNA sequences was confirmed by saturation hybridization to excess 14H-RNA transcribed from a homogeneous restriction fragment recleaved from the 340 bp DNA. The maximal fraction of DNA hybridizing to the 3H-RNA probe declined by 33-50% over 21-41 population doublings. Divergence and/or methylation of such sequences could not account for these results since the thermal stability of cRNA:DNA duplexes actually increased by 0.3 degrees C at late passage. Total highly repetitive sequences assayed by reassociation kinetics were also substantially reduced at late passage, implying that depletion may be common to many repeat families in DNA. The denaturation temperature for such rapidly reassociated duplexes again increased slightly at late passage, possibly reflecting the minor decreases in DNA methylation which were detected in two of the cell strains. Karyotype analyses demonstrated that over 95% euploidy was maintained, with no specific chromosome loss and no visible deletions at late passage. The depletion of reiterated sequences during repeated cell division is thus attributed to numerous small DNA deletions, which may arise from unequal recombination coupled with selection or from a nonreciprocal mechanism such as excision.     

Primary structure of an EcoRI fragment of lambda imm434 DNA containing regions cI-cro of phage 434 and cII-o of phage lambda.

Digestion of phage lambda imm434 DNA with restriction endonuclease EcoRI yields 7 fragments. The shortest among them (1287 bp) contains the right part of the phage 434 immunity region and the phage DNA portion proximal to it. The complete primary structure of this fragment has been determined using the chemical method of DNA sequencing. Hypothetical amino-acid sequences of proteins coded by the cro gene of phage 434 and the cII gene of phage lambda, as well as NH2-terminal amino-acid sequences of the cI protein of phage 434 and the O protein of phage lambda, have been deduced solely on the basis of the DNA sequence. The fragment studied contains also the pR and probably prm promoters and the oR operator of phage 434. The sequence coding for them differs from the respective DNA sequence of phage lambda.     

Physical map and properties of a 90-MDa plasmid of Azospirillum brasilense Sp7

Homology was previously detected between the DNA restriction fragments containing Rhizobium meliloti nodulation genes and the 90-MDa plasmid, p90, of Azospirillum brasilense Sp7. Two DNA loci from Sp7 genome that complement mutations in the exopolysaccharide synthesis genes, exoB and exoC, of R. meliloti were also shown to be present on the plasmid. A more detailed characterization of the plasmid was undertaken to establish its physical map and to localize the nod homologies and other specific regions. Six loci were mapped, the region homologous to the nodulation genes, nodPQ, of R. meliloti, the exoB and exoC mutation-correcting loci, a locus for Ap resistance, a bla homology region different from the Ap resistance locus, and a region necessary for the maintenance of p90 as an independent replicon. Mobilization into Agrobacterium tumefaciens of p90-Tn5-Mob was obtained at a frequency of 10(-4), with the plasmid helper pJB3JI. Self-transfer of p90 was not demonstrated. Fragments of p90 hybridized with a plasmid of 90 MDa present in most A. brasilense and some A. lipoferum strains, suggesting a plasmid family in Azospirillum.     

A method for the generation of small pre-determined deletions in plasmid DNA: deletion analysis of the tetR region of vector pBR322

A general method is described that allows precise deletion of a chosen restriction fragment(s) from a plasmid having many cleavage sites for that restriction enzyme. The DNA to be deleted is first separated from the rest of the plasmid on a larger DNA fragment contained between two different unique restriction sites. This fragment is then subdigested by the restriction endonuclease of interest, which recognises two or more tetranucleotide (cohesive end or blunt end) sequences on the fragment, and is recloned between the two original unique restriction sites. The method is rapid, efficient, and the results are predictable. Examples are given in which predetermined HpaII (9 bp, 147 bp), TaqI (141 bp) and AluI (15 bp, 403 bp) fragments have been selectively removed from the tetR region of plasmid pBR322.     

Restriction generated oligonucleotides utilizing the two base recognition endonuclease CviJI*.

The conversion of an anonymous DNA sample into numerous oligonucleotides is enzymatically feasible using an unusual restriction endonuclease, CviJI. Depending on reaction conditions, CviJI is capable of digesting DNA at a two or three base recognition sequence. CviJI normally cleaves RGCY sites between the G and C to leave blunt ends. Under 'relaxed' conditions CviJI* cleaves RGCY, and RGCR/YGCY, but not YGCR sites. In theory, CviJI* restriction of pUC19 (2686 bp) should produce 157 fragments, 75% of which are smaller than 20 bp. Instead, 96% of the CviJI* fragments were 18-56 bp long and none of the fragments were smaller than 18 bp. Thermal denaturation of these fragments generates sequence specific oligonucleotides homologous for the cognate template. The enzymatic conversion of anonymous DNA into sequence specific oligomers has implications for several conventional and novel molecular biology procedures.     

Sequence analysis of the major outer membrane protein gene from Chlamydia trachomatis serovar L2.

The structural gene for the major outer membrane protein (MOMP) from Chlamydia trachomatis was cloned and sequenced. A lambda gt11 recombinant (lambda gt11/L2/33) that contains a portion of the MOMP coding sequence was used to probe a lambda 1059 library constructed from DNA obtained from C. trachomatis serovar L2. Selected lambda 1059 recombinants were mapped with endonuclease restriction enzymes. The MOMP gene was mapped to the 5' end of a BamHI fragment of approximately 9 kilobases. Contiguous endonuclease restriction fragments identified within this region permitted the selection of specific fragments for subcloning and DNA sequencing. The MOMP gene consisted of a 1,182-base-pair open reading frame that encoded 394 amino acids and ended with three stop codons. The known amino-terminal amino acid was preceded by 22 amino acids whose sequence was compatible with a leader or signal sequence. The primary structure of MOMP determined from the translated DNA sequence demonstrated nine cysteine residues and a remarkably homogeneous distribution of charged and hydrophobic residues.     

A site-specific endodeoxyribonuclease from Streptomyces albus CMI 52766 sharing site-specificity with Providencia stuartii endonuclease PstI.

A class II site-specific endodeoxyribonuclease (SalPI) was identified in cell-free extracts of Streptomyces albus CMI 52766 after high speed centrifugation and fractionation through Bio Gel AO.5M. SalPI cleaves lambda DNA into at least 18 fragments. Five cleavage sites were located in the linear lambda map by the use of double and triple restriction enzyme digests involving EcoRI, HindIII, SalGI and another new Streptomyces enzyme, SacI. The results were indistinguishable from those previously obtained for a Providencia stuartii enzyme, PstI, by Smith, Blattner & Davies (Nucleic Acids Res. 1976 3, 343). SalPI and PstI were shown by a double digest test to have the same site specificity. None of 34 phages tested was obviously restricted by S. albus CMI 52766, and correspondingly DNA from two of them was not cleaved in vitro by SalPI. DNA from Streptomyces phage that does not form plaques on S. albus CMI 52766, and plasmid SCP2 DNA from S. coelicolor A3 (2), were both cleaved.     

Molecular cloning of unintegrated and a portion of integrated moloney murine leukemia viral DNA in bacteriophage lambda.

A covalently closed circular form of unintegrated viral DNA obtained from NIH 3T3 cells freshly infected with Moloney murine leukemia virus (M-MLV) and a port of the endogenous M-MLV from the BALB/Mo mouse strain have been cloned in bacteriophage lambda. The unintegrated viral DNA was cleaved with restriction endonuclease HindIII and inserted into the single HindIII site of lambda phage Charon 21A. Similarly high-molecular-weight DNA from BALB/Mo mice ws cleaved sequentially with restriction endonucleases EcoRI and HindIII and separated on the basis of size, and one of the two fractions which reacted with an M-MLV-specific complementary DNA was inserted into the HindIII site of Charon 21A. Recombinant clones containing M-MLV-reacting DNA were analyzed by restriction endonuclease mapping, heteroduplexing, and infectivity assays. The restriction endonuclease map of the insert derived from unintegrated viral DNA, lambda x MLV-1, was comparable to published maps. Electron microscope analysis of the hybrid formed between lambda x MLV-1 DNA and 35S genomic M-MLV RNA showed a duplex structure. The molecularly cloned lambda x MLV-1 DNA contained only one copy of the long terminal repeat and was not infectious even after end-to-end ligation of the insert DNA. The insert DNA derived from endogenous M-MLV, lambda x MLVint-1, contained a DNA stretch measuring 5.4 kilobase pairs in length, corresponding to the 5' part of the genomic viral RNA, and cellular mouse DNA sequences measuring 3.5 kilobase pairs in length. The viral part of the insert showed the typical restriction pattern of M-MLV DNA except that a single restriction site, PvuII, in the 5' long terminal repeat was missing. Reconstructed genomes containing the 5' half derived from the integrated viral DNA and the 3' half derived from the unintegrated viral DNA were able to induce XC plaques after transfection in uninfected mouse fibroblasts.     

Analysis of DNA, lipopolysaccharide structure, and some cultural and morphological properties in closely related strains of Azospirillum brasilense

We studied closely related Azospirillum brasilense strains Sp7 and Cd. For probing of their genomes, the fragments of 85-MDa (p85) and 120-MDa (p120) from A. brasilense Sp245 plasmids were hybridized with 115-MDa (p115) and 90-Mda (p90) plasmids of strain Sp7, respectively. Strain Cd was found to lose the 115-Mda plasmid and one of the two EcoRI restriction fragments of the total DNA (localized within p115 and the chromosome) that was homologous to an EcoRI-generated p85 fragment of 2.4 kb. On the contrary, in the total DNA of strain Sp7-S, in spite of the previously established disappearance of the 115-Mda replicon, two fragments homologous to p85 were revealed, as with strain Sp7. It is suggested that the Sp7-S genome contains the total p115 DNA or at least a certain part of it. Strains Sp7 and Cd were found to differ in size and morphology of colonies on solid and semisolid media, in the levels of resistance to a cation surfactant cetavlon, and in the antigen structure of lipopolysaccharides.     

Preferential uptake of restriction fragments from a gonococcal cryptic plasmid by competent Neisseria gonorrhoeae

Factors involved in the specificity of DNA uptake by competent Neisseria gonorrhoeae were examined. Host-controlled modification did not affect uptake. Certain restriction fragments of the 4.2 kb gonococcal cryptic plasmid pFA1 and of the replicative form of the bacteriophage M13 were taken up in preference to others, independent of differences in fragment size. A 600 bp fragment from the 4.2 kb plasmid was cloned into pLES2, a gonococcal-Escherichia coli shuttle vector; the 600 bp fragment was taken up into a DNAase-I-resistant state in preference to the vector fragment. A second 370 bp fragment in pFA1 was also taken up preferentially. The 600 bp and 370 bp fragments share a 10 bp sequence, which is found in pFA1 only on fragments that were taken up readily. However, a fragment from M13 which was efficiently taken up did not contain this 10 bp sequence. In addition, this sequence was not sufficient to direct preferential DNA uptake by gonococci, since a recombinant plasmid containing this 10 bp sequence was not taken up appreciably better than the vector plasmid or another recombinant plasmid containing an unrelated 10 bp sequence. Sequence comparisons of the three restriction fragments which were preferentially taken up did not yield any consensus sequences greater than 7 bp. Although it is likely that efficient uptake of DNA by gonococci is determined by DNA structure, a single short sequence could not be found that accounted for specific uptake.