Isolation and characterization of Azospirillum brasilense loci that correct Rhizobium meliloti exoB and exoC mutations.

The occurrence in Azospirillum brasilense of genes that code for exopolysaccharide (EPS) synthesis was investigated through complementation studies of Rhizobium meliloti Exo- mutants. These mutants are deficient in the synthesis of the major acidic EPS of Rhizobium species and form empty, non-nitrogen-fixing root nodules on alfalfa (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We demonstrated that the exoC mutation of R. meliloti could be corrected for EPS production by several cosmid clones of a clone bank of A. brasilense ATCC 29145. However, the EPS produced differed in structure from the wild-type R. meliloti EPS, and the symbiotic deficiency of the exoC mutation was not reversed by any of these cosmid clones. The exoB mutation could be corrected not only for EPS production but also for the ability to form nitrogen-fixing nodules on alfalfa by one particular cosmid clone of A. brasilense. Tn5 insertions in the cloned DNA were isolated and used to construct Azospirillum mutants with mutations in the corresponding loci by marker exchange. It was found that these mutants failed to produce the wild-type high-molecular-weight EPS, but instead produced EPSs of lower molecular weight.

Transposon Mutagenesis of Azospirillum brasilense and Azospirillum lipoferum: Physical Analysis of Tn5 and Tn5-Mob Insertion Mutants.

Tn5-induced insertion mutants were generated in Azospirillum brasilense Sp7 and A. lipoferum SpBr17 by mating with Escherichia coli strains carrying suicide plasmid vectors. The sources of Tn5 were the suicide plasmids pGS9 and pSUP2021. Kanamycin-resistant Azospirillum colonies appeared from crosses with E. coli at maximum frequencies of 10 per recipient cell. Transposon Tn5 also conferred streptomycin resistance on Azospirillum colonies as was observed earlier for Rhizobium sp. Eight Tn5-induced Km SmA. brasilense Sp7 mutants with reduced nitrogen-fixing capacity were isolated. The potential use of Tn5-Mob for labeling and mobilization of Azospirillum-indigenous plasmids was demonstrated by isolating Tn5-Mob insertions in the megaplasmids of A. brasilense Sp7.

Nucleotide sequence of an insertion sequence (IS) element identified in the T-DNA region of a spontaneous variant of the Ti-plasmid pTiT37.

We have identified and determined the nucleotide sequence of an IS element (IS136) of Agrobacterium tumefaciens. This is the first IS element isolated and sequenced from a nopaline type Ti-plasmid. Our IS element has 32/30 bp inverted repeats with 6 mismatches, is 1,313 bp long and generates 9 bp direct repeats upon integration. IS136 has 3 main open reading frames (ORF's). Only ORF1 (159 codons) is preceded by sequences that are proposed to serve functional roles in transcriptional and translational initiation. No DNA sequence homology was found between IS136 and IS66, an IS element isolated from an octopine type Ti-plasmid.

The density distribution of gene loci over the genetic map of Escherichia coli: its structural, functional and evolutionary implications.

A quantitative analysis was carried out on the dispersion of gene loci over the E. coli genetic map. Therefore, the map was divided into regions characterized by an homogeneous gene density. This created a distribution pattern of gene loci that contained a symmetry axis located near to the origin of DNA replication. The pattern could be subdivided into a set of 22 functional domains containing gene loci whose products revealed a biochemical or functional relatedness. A correlation was found between the boundary positions of these domains and the distribution of F plasmid- and DNA insertion sites over the E. coli chromosome. The structural, functional and evolutionary implications of these findings are discussed.

Compact Escherichia coli nucleoids in a highly supercoiled conformation.

Escherichia coli nucleoids were visualized in a compact, highly supercoiled conformation at 1 M NaCl. After the salt concentration was lowered to 0.15 M NaCl, the nucleoids uncoiled in beaded fibers in which RNA-mediated structural domains could be distinguished.

Nucleoid release from Escherichia coli cells.

The time course of morphological changes during lysis of Escherichia coli cells was examined with respect to an undisturbed release of nucleoids. The addition of detergents to plasmolyzed, osmotic sensitive cells resulted in the immediate reversal of plasmolysis followed by the appearance of rod-shaped ghost cells without any detectable spheroplast formation. Electron microscopic examination of the rod-shaped ghost cells revealed a zonal gap in the cell envelope, allowing the free release of the nucleoid. Due to the high ionic strength, a suitable cell lysis was shown to require higher incubation temperatures. However, in the absence of an appropriate control this may result in the sphering and vesiculation of ghost cell envelopes and even the unfolding of released nucleoids. To avoid this unfavorable consequence of lysis at high temperatures, a microscopic examination on the course of rod-shaped ghost formation is suggested.

Fracture faces in the cell envelope of Escherichia coli.

Freeze-fracturing of Escherichia coli cells in the presence of 30% (v/v) glycerol resulted in a double cleavage of the cell envelope exposing two convex and two concave fracture faces ([Formula: see text], [Formula: see text] and [Formula: see text], [Formula: see text]) with characteristic patterns. Complementary replicas revealed the relationship of the fracture faces to their corresponding fracture planes. The inner fracture plane splits the plasma membrane at one particular level. Apparently the outer fracture plane was located in the outer part of the wall, as it was separated by a layer ([Formula: see text]) from the fractured profile (CW1) presumably corresponding to the murein layer. The outer fracture plane did alternate toward the cell periphery, exposing complementary smooth areas ([Formula: see text] and [Formula: see text]). When cells were freeze-fractured in the absence of glycerol, the outer cell surface appeared as an etching face rather than a fracture face. A schematic representation of the relative location of the different fracture faces in the E. coli cell envelope is given.