Cloning of a phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Streptomyces lividans and Escherichia coli.

Phosphinothricin-tripeptide (Ptt), also known as bialaphos, contains phosphinothricin (Pt), a potent inhibitor of glutamine synthetase. A 4.0-kb Bam HI fragment coding for Ptt resistance was cloned in Streptomyces lividans TK23.

Metabolism and preservation of fresh and stored erythrocytes in blood treated with gentian violet.

1. The metabolism of the erythrocytes in the gentian violet (GV)-treated blood from ten donors was studied weekly from time zero to the 28th day of storage in order to evaluate the efficacy of the use of GV in the chemoprophylaxis of transfusional Chagas' disease. 2.

Rhizobium meliloti nifN (fixF) gene is part of an operon regulated by a nifA-dependent promoter and codes for a polypeptide homologous to the nifK gene product.

An essential gene for symbiotic nitrogen fixation (fixF) is located near the common nodulation region of Rhizobium meliloti. A DNA fragment carrying fixF was characterized by hybridization with Klebsiella pneumoniae nif DNA and by nucleotide sequence analysis. The fixF gene was found to be related to K.

The Streptomyces ghanaensis low copy plasmid pSG2 and its use for vector construction.

A plasmid, pSG2, was isolated from Streptomyces ghanaensis and characterized by electron microscopy, buoyant density measurement, and restriction enzyme analysis. The length of 13.8 kb, single restriction sites for HindIII, EcoRV and PvuII and the possibility of deleting non-essential regions of the plasmid made pSG2 a suitable basic replicon for vector development.

Tight linkage of glnA and a putative regulatory gene in Rhizobium leguminosarum.

Rhizobium leguminosarum, biovar viceae, strain RCC1001 contains two glutamine synthetase activities, GSI and GSII. We report here the identification of glnA, the structural gene for GSI. A 2 kb fragment of DNA was shown to complement the Gln- phenotype of Klebsiella pneumoniae glnA mutant strains.

Organization and partial sequence of a DNA region of the Rhizobium leguminosarum symbiotic plasmid pRL6JI containing the genes fixABC, nifA, nifB and a novel open reading frame.

By hybridization and heteroduplex studies the fixABC and nifA genes of the Rhizobium leguminosarum symbiotic plasmid pRL6JI have been identified. DNA sequencing of the region containing nifA showed an open reading frame of 1557 bp encoding a protein of 56, 178 D. Based on sequence homology, this ORF was confirmed to correspond to the nifA gene.

Development of a plasmid-cloning system for Streptomyces viridochromogenes Tü494.

A plasmid-cloning system was developed for Streptomyces viridochromogenes Tü494, a producer of the tripeptide antibiotic phosphinothricyl-alanyl-alanine (PTT). Parameters affecting protoplast formation and transformability of S. viridochromogenes were investigated in detail.

Physical characterization of Rhizobium meliloti megaplasmids.

Intact megaplasmids of Rhizobium meliloti 2011 have been isolated and visualized by electron microscopy. The contour lengths of 64 megaplasmid molecules were determined. One definite class of molecules of 400 micron length and a range of larger molecules with lengths of up to 560 micron was observed.

Molecular cloning into Tn5 and integration in the Pseudomonas aeruginosa chromosome: a tool for heterologous gene expression.

The DNA primase gene of the promiscuous IncP-1 conjugative plasmid RP1, encoding two polypeptides of 118 and 80 kDa, was inserted into the transposon Tn5 in Escherichia coli. The derivative transposon, Tn2523, was then transposed to a temperature-sensitive replication mutant of the promiscuous IncP-1 conjugative plasmid R68 at permissive temperature and the plasmid transferred to Pseudomonas aeruginosa strain PAO. The latter strain was then grown at non-permissive temperature to identify transposition of Tn2523 into the P.

Mapping and expression of a regulatory nitrogen fixation gene (fixD) of Rhizobium meliloti.

A 3.5-kb HindIII fragment from the main nif/fix (nitrogen fixation) gene cluster of Rhizobium meliloti was characterized by studying its expression in Escherichia coli minicells. A coding region for two polypeptides of 68 K and 66 K was mapped using Tn5 insertions and hybrid fusion polypeptides.

Characterization of a Rhizobium meliloti fixation gene (fixF) located near the common nodulation region.

Rhizobium meliloti 2011 DNA from pRmSL26, a plasmid which is known to carry genes involved in the early stages of nodulation, was used to construct Tn5 mutations by site-directed Tn5 mutagenesis. Tn5 mutations located within an 8.7 kilobase EcoRI fragment defined two adjacent loci encoding functions for nodulation (nod) and symbiotic N2 fixation (fix).

Extension of the host range of Escherichia coli vectors by incorporation of RSF1010 replication and mobilization functions.

The broad-host-range vectors pSUP104, pSUP106, pSUP204, pSUP304, and pSUP404 are based on conventional Escherichia coli vectors (such as pBR325 and pACYC184) which have been modified to include the mobilization and broad-host-range replication functions of the IncQ plasmid RSF1010. These vector plasmids now can be maintained in a wide range of bacterial genera including Rhizobium, Agrobacterium, and Pseudomonas. They are efficiently mobilized by RP4 and thus are of particular interest for bacteria refractory to transformation.

The development of plasmid-free strains of Agrobacterium tumefaciens by using incompatibility with a Rhizobium meliloti plasmid to eliminate pAtC58.

Agrobacterium tumefaciens strains LBA275 and LBA290 were cured of their cryptic plasmid pAtC58 by the introduction of the Rhizobium meliloti plasmid pRme41a, which is incompatible with pAtC58. pRme41a and pTiC58, the resident Ti plasmid of LBA275, were subsequently eliminated by growth at supraoptimal temperature (40 degrees C). The resulting plasmid-free Agrobacterium strains, UBAPF1 and UBAPF2, have proved extremely useful for the study of Rhizobium plasmids.

Morphological and molecular characterization of several actinophages isolated from soil which lyse Streptomyces cattleya or S. venezuelae.

Several lytic and lysogenic actinophages were isolated from soil samples infected with Streptomyces cattleya and S. venezuelae. The morphologies and some biological properties of the phages, and the physico-chemical characteristics of their DNAs, were compared.

Expression of Rhizobium meliloti genes for nitrogen fixation in Escherichia coli minicells: Mapping of the subunit of the nitrogenase reductase.

An EcoRI fragment of Rhizobium meliloti M2011 which shows homology to Klebsiella pneumoniae DNA carrying nifH and nifD was cloned in both orientations into the Cm gene of plasmid pACYC184 and expressed in Escherichia coli minicells. Fragment specific polypeptides of Mr 12 500, 21 000, 30 000, and 31 000 could be identified. By transposon mutagenesis it was shown that two of them (Mr 12 500 and 21 000) are fusion products with parts of the chloramphenicol acetyltransferase.

Expression of plant tumor-specific proteins in minicells of Escherichia coli: a fusion protein of lysopine dehydrogenase with chloramphenicol acetyltransferase.

Fragment EcoRI 7 from Ti-plasmid pTi Ach5, a part of the T-DNA in octopine tumors, was cloned in both orientations into pACYC184 and expressed in E. coli minicells. The cells synthesized four proteins from four different coding regions on EcoRI 7.

ISR1: an insertion element isolated from the soil bacterium Rhizobium lupini.

The insertion element ISR1 was isolated from the soil bacterium R. lupini. In this strain, ISR1 shows a very strong affinity to plasmid RP4.

The tetracycline resistance transposons Tn1721 and Tn1771 have three 38-base-pair repeats and generate five-base-pair direct repeats.

The 10.7 kilobase (kb) tetracycline resistance transposons Tn1721 and Tn1771, isolated from disparate sources, are completely homologous on the basis of heteroduplex analyses. Both transposable elements are capable of forming multiple duplications of a 5.

Identification of the repressor and repressor bypass (antirepressor) polypeptides of bacteriophage P1 synthesized in infected minicells.

P1 infected minicells synthesize approximately 50 phage-encoded polypeptides. Phage expression is temporally controlled, demonstrating phage polypeptides synthesized both early and late after infection. The P1 repressor, gpcl1 (Mr = 33,000), repressor bypass polypeptide, gpreb A (Mr = 27,500) and cistron 10 product, (gp10) (Mr = 64,000), have been identified by infection of minicells with P1 amber mutants.

Adenine + thymine content of different genes located on the broad host range plasmid RP4.

The genetic map of plasmid RP4 was correlated with its adenine+thymine (AT) map. For this purpose, RP4 DNA was digested with one or both of the restriction enzymes EcoRI and HindIII and the resulting linear RP4 molecules and fragments were partially denatured, examined in the electron microscope and their AT maps were determined using a computer program. From these AT maps the EcoRI and HindIII restriction sites were located on the AT map of RP4.

[Applied gene technology using biological N2-fixation for an example (author's transl)].

About biological nitrogen fixation most information on the genetic and enzymatic level is known from investigations with Klebsiella pneumoniae. The gene region for N2-fixation (nif) was transferred from K.pneumoniae to the enterobacterium Escherichia coli.