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Streptomyces coelicolor “Muller” is known to excrete the lysozyme N-acetylmuramidase. Culture filtrates of this strain form a characteristic halo on agar plates containing freeze-dried Micrococcus luteus cells (lysoplate technique). The halo consists of a clear inner zone and a turbid outer ring. Simulation experiments showed that the turbid outer ring is most probably produced by lysozyme whereas the clear inner zone can be considered to be due to an additional protease action. Using the lysoplate technique UV- and NTG-mutagenized strains of S. coelicolor “Muller” were screened for mutants defective in lysozyme production. Two mutants, SC11 and SC12, were identified. The mutant SC11 was selected for complementation studies. First, a transformation system was established. The use of a soft-agar overlay method was necessary to yield high regeneration rates of SC11 protoplasts. The plasmid vector pEB15 could be transferred into this mutant strain with an efficiency of 105 transformants/μg DNA. The high efficiency allowed shot-gun cloning experiments. Genomic DNA of S. coelicolor “Muller” digested with Sau3A and inserted into pEB15 was introduced into the mutant SC11. A complemented mutant was identified. A 2.9 kilobase pair (kb) DNA fragment was found which restored the lysozyme production of both mutants, SC11 and SC12. According to the diameter of the produced halos the complemented mutant SC11 was suggested to produce more lysozyme than the wildtype strain.

A DNA region showing homology to Klebsiella pneumoniae nifA and nifB is duplicated in Rhodobacter capsulatus. The two copies of this region are called nifA/nifB copy I and nifA/nifB copy II. Deletion mutagenesis demonstrated that either of the two copies is sufficient for growth in nitrogen-free medium. In contrast, a double deletion mutant turned out to be deficient in nitrogen fixation. The complete nucleotide sequence of a 4838 bp fragment containing nifA/nifB copy I was determined. Two open reading frames coding for a 59,653 (NifA) and a 49,453 (NifB) dalton protein could be detected. Comparison of the amino acid sequences revealed that the R. capsulatus nifA and nifB gene products are more closely related to the NifA and NifB proteins of Rhizobium meliloti and Rhizobium leguminosarum than to those of K. pneumoniae. A rho-independent termination signal and a typical nif promoter region containing a putative NifA binding site and a consensus nif promoter are located within the region between the R. capsulatus nifA and nifB genes. The nifB sequence is followed by an open reading frame (ORF1) coding for a 27721 dalton protein in nifA/nifB copy I. DNA sequence analysis of nifA/nifB copy II showed that both copies differ in the DNA region downstream of nifB and in the noncoding sequence in front of nifA. All other regions compared, i.e. the 5' part of nifA, the intergenic region and the 3' part of nifB, are identical in both copies.

The occurrence of endogenous plasmid DNA in Rhizobium meliloti is reported. R. meliloti strains carrying RP 4 were used to work out a suitable plasmid isolation method. By sarcosyl lysis RP 4 plasmid DNA as well as cryptic plasmid DNA was isolated. Cryptic plasmid DNA possesses a buoyant density of 1.717 g cm-3 and shows a heterogeneity in contour length. Large plasmid molecules of 130 μm length were detected in the electron microscope. We also found that RP4 is able to restore the nitrogen fixation capacity of an ineffective R. meliloti strain.

After isolation, the DNA of simian virus 40 appeared as a negative supertwist (form I) or as an open circle with at least one single-strand scission (form II). Under the denaturation conditions usually applied, such as heating in the presence of formaldehyde or application of alkali, form I molecules could appear as "relaxed" circles without single-strand scissions (form I') containing denatured sites not visible under the electron microscope. Form II molecules, under these denaturation conditions, showed partial or complete strand separations allowing the construction of denaturation maps. By using a modified denaturation procedure, i.e., heating of isolated SV40 DNA in the presence of dimethyl sulfoxide and formaldehyde followed by keeping the DNA in this denaturation solution at room temperature for periods up to 3 weeks, partially denatured relaxed circles without single-strand scissions were produced (form I'D) in addition to completely denatured form II molecules. The absence of single-strand scissions in form I'D molecules was demonstrated by a second heat treatment, which did not change the configuration of this molecular form. Form I'D molecules, in contrast to form I', contained denatured sites clearly discerible under the electron microscope. This combined application of two subsequent denaturation steps (denaturation by heating followed by denaturation at room temperature and neutral pH) showed that the molecular configuration I'D originated in two steps. The heating procedure produced molecules not distinquishable by electron microscopy from form I. In contrast to form I, these molecules were assumed to possess "preformed" denaturation sites (form I). Further treatment of form I molecules with denaturation solution at room temperature finally transformed them into convalently closed, relaxed, partially denatured circles exhibiting strand separations easily measurable on electron micrographs (form I'D). Denaturation maps of form I'D molecules were constructed by computer and compared with denaturation maps derived from partially denatured form II molecules. From these denaturation maps it can be concluded that the melting of base pairs occurring during the transition of simian virus 40 DNA form I into form I'D also preferentially happened at sites rich in the bases adenosine and thymine.

SUMMARY: 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. Electron micrographs indicated that all the phage heads were of an icosahedral form, but head size and length of the tail varied. Two of the phages had a broad host range; the other isolates could lyse only a limited number of species. The molecular sizes of the phage DNAs were between 32-2 and 98-5 kb as estimated by electron microscopy and restriction enzyme analysis. The same study also indicated that one of the DNA species contained cohesive ends. The G + C content of the DNAs ranged between 45-1 and 74-2 mol % as estimated from melting studies. Sedimentation velocity experiments implied that several of the phage DNAs were probably heavily glycosylated or methylated. These modifications might explain the partial or slow digestion of some of the DNAs by several of the 23 restriction enzymes tested. Protoplasts of the appropriate Streptomyces strains could be efficiently transfected with phage DNA in the presence of 25% (w/v) polyethylene glycol (mol. wt 6000).

A set of plasmid vectors was developed which allows fast sequencing by the chemical degradation method. These high-copy-number vectors are derivatives of the plasmid pUC8 containing different multiple-purpose cloning sites flanked by unique recognition sequences for the restriction enzymes BstEII, Tth111I and Eco81I as sites for end-labelling DNA. Due to their partially asymmetric recognition sequences, each of these three restriction sites can be singly end-labelled by a filling-in reaction with selected nucleotides. This allows easy single end-labelling of any cloned DNA fragment for sequencing by the chemical degradation method without any isolation and purification step after the labelling reaction. In addition, the nucleotide sequence of the complementary strand from the same end can be determined by the dideoxy chain termination procedure using the universal M13 primers. In most of the new vectors, the reading frame of the lacZ' gene is retained, allowing identification of cloned fragments.

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. Comparison of three nifA proteins (Klebsiella pneumoniae, Rhizobium meliloti, Rhizobium leguminosarum) revealed only a weak relationship in their N-terminal regions, whereas the C-terminal parts exhibited strong homology. Sequence analysis also showed that the R. leguminosarum nifA gene is followed by nifB and preceded by fixC with an open reading frame inserted in between. This novel ORF of 294 bp was found to be highly conserved also in R. meliloti. No known promoter and termination signals could be defined on the sequenced R. leguminosarum fragment.

Transposon Tn5 was introduced into Rhodopseudomonas capsulata strain BIO by using in vitro constructed plasmids that are mobilizable by broad host range plasmid RP4 but do not replicate outside E. coli. Mutants deficient in nitrogen fixation were identified by retesting Tn5 containing Rh. capsulata strains for their ability to grow on N2 gas as sole source of nitrogen.

ISR1 is a small transposable element, identified in Rhizobium class IV strains by its high frequent mutagenic insertion into plasmid RP4. Hybridization studies showed that ISR1 is present in multiple copies in Rhizobium class IV strains. Nucleotide sequence analysis revealed that ISR1 has a length of 1260 bp and is characterized by perfect inverted repeats of 13 nucleotides followed by a stretch of 28 29 nucleotides with imperfect homology. The insertion under study generated a target site duplication of 4 bp. ISR1 carries a large open reading frame, encoding a putative polypeptide of 278 amino acids (ORFA∗), and three smaller ones in antiparallel direction (ORFs A1, A2, A3). Two of them are completely covered by the large open reading frame. No significant homology to 17 other known insertion sequence elements could be detected, either at nucleotide or at amino acid levels.

A. General Introduction.- The Historical Background of the Bacteria-Plant Interaction.- The Rhizobium-Plant Interaction.- La Piste des Opines.- B. The Rhizobium-Plant Interaction.- I. The Rhizobium meliloti-Medicago sativa System.- Studies on Rhizobium meliloti Plasmids and on Their Role in the Control of Nodule Formation and Nitrogen Fixation: The pSym Megaplasmids and the Other Large Plasmids.- Studies on Rhizobium meliloti Plasmids and on Their Role in the Control of Nodule Formation and Nitrogen Fixation: A Strategy for Region-Specific Transposon Mutagenesis of pSym Megaplasmid.- Analysis of Symbiotic Nitrogen Fixation Genes Carried by the Rhizobium meliloti Mega-Plasmid.- Characterization of Rhizobium meliloti Genetic Loci Essential for Symbiotic Nitrogen Fixation.- Mapping and Regulation of the Structural Genes nifK, nifD, and nifH of Rhizobium meliloti.- Early Nodulation Genes of Rhizobium meliloti.- Symbiotic Nitrogen Fixation Genes of Rhizobium meliloti.- Vector Plasmids for in-Vivo and in-Vitro Manipulations of Gram-Negative Bacteria.- II. The Rhizobium leguminosarum-Pisum sativum System.- Genetic Analysis of pRL1 JI, a Symbiotic Plasmid of Rhizobium leguminosarum.- Natural Variation in Rhizobium Plasmids.- Analysis of Nodule-Specific Plant and Bacteroid Proteins in Pea Plants Inoculated by Transposon Mutagenized Rhizobium leguminosarum.- III. The Rhizobium japonicum-Glycine max System.- Rhizobia Use Free-Living N2 Fixation to Promote Growth by a Mechanism of Crossfeeding.- The Nitrogenase Genes of Rhizobium japonicum.- The Structure and Organization of the Soybean Leghemoglobin Genes.- The Role of Plant Genes in Soybean-Rhizobium Interactions.- IV. The Interaction of Different Rhizobium Species with Plants.- General Organization of Nitrogen Fixation Genes in Rhizobium phaseoli.- Identification of Nitrogen Fixation and Nodulation Genes on a Large Plasmid from a Broad Host Range Rhizobium sp..- Identification, Broad Host Range Mobilization and Mutagenesis of a Rhizobium trifolii Sym::R68.45 Cointegrate Plasmid.- Genetic Analysis of the Symbiotic Regions in Rhizobium trifolii and Rhizobium parasponia.- Molecular Anatomy of the Symbiotic Region in Rhizobium trifolii and Rhizobium parasponia.- Stem Nodulation in Aeschynomene: A Model System for Bacterium-Plant Interactions.- C. The Agrobacterium-Plant Interaction.- I. The Tumor-Inducing Plasmids of Agrobacterium.- Onc- and vir-Genes Located on the Ti-Plasmid of Agrobacterium tumefaciens.- Plasmid Genes Essential for the Interactions of Agrobacteria and Rhizobia with Plant Cells.- Genetic Analysis of Host Range Expression by Agrobacterium.- The Biology of Genetic Transformation of Higher Plants by Agrobacterium rhizogenes.- Expression of the T-Region of Octopine Plasmid pTi Ach5 into Protein in Bacterial Systems.- II. The Role of T-DNA in Transformed Plant Cells.- Liposomes as a Carrier of Ti Plasmid into Protoplasts.- Plant Protoplast Transformation by Agrobacterium tumefaciens and its Ti Plasmid DNA.- The Study of Integration of Tumor-Inducing DNA from the Nopaline Ti Plasmid of Agrobacterium tumefaciens.- Transcription of the Ti-Plasmid in Crown Gall Tumors.- TR Genes Involved in Agropine Production.- Transcription of T-DNA in Octopine and Nopaline Crown Gall Tumours.- Genetic Analysis of T-DNA and Regeneration of Transformed Plants.- D. Plant Pathogenic Bacteria and Related Aspects.- The Genetics of the Cherry Pathogen, Pseudomonas syringae pathovar morsprunorum.- Towards the Genetical Analysis of Pathogenicity of Xanthomonas campestris.- Molecular Analysis of Virulence Genes in Pseudomonas solanacearum.- Cloning of Genes Involved in Bacterial Ice Nucleation and Fluorescent Pigment/Siderophore Production.- The Genetics of Indoleacetic Acid Production and Virulence in Pseudomonas savastanoi.- Azospirillum Genetics.- Genome Rearrangement of the Rhizobiaceae.

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). We investigated the organization and regulation of the fix locus and the characteristics of alfalfa nodules induced by these Fix- mutants. By monitoring expression in Escherichia coli minicells, we determined that the fix locus encoded a 36-kilodalton polypeptide. The gene corresponding to this locus was designated fixF. Morphological and ultrastructural studies of the ineffective nodules formed by R. meliloti fixF mutants showed infected host cells similar to those of the wild type. The ineffective nodules were able to accumulate leghemoglobin, but at lower levels than those found in the wild-type nodules. Expression of the nifHDK operon was unaffected by Tn5 insertions in the fixF gene. Expression of the fixF gene was monitored in E. coli by using translational lacZ fusions. It was shown that transcription of the fixF gene in E. coli could be activated by Klebsiella pneumoniae nifA and the R. meliloti nifA-like regulatory gene products. Expression of the fixF gene was also studied in free-living and symbiotic R. meliloti cells. It was found that the fixF gene was transcribed in the symbiotic state.

SUMMARYPlasmids R68 and R68.45 were transferred fromPseudomonas aeruginosatoEscherichia coliby conjugation. R68.45 was able to mobilize theE. colichromosome from different origins at a frequency of about lO−6/donor cell. With R68, no transfer of chromosomal genes could be detected. Plasmid R68.45 differs from its parent R68 only by an additional DNA segment, 2120 bp long, located close to the kanamycin resistance gene. By restriction enzyme analysis it was shown that the additional DNA segment of R68.45 is a duplication of a pre-existing DNA region of R68. The duplicated region is characterized by the following sequence of restriction sites: A–310 bp–SmaI–70 bp–PstI–795 bp–PstI–15 bp–KpnI–540 bp–HpaI–370 bp–B.The endpoints A and B of the duplicated region were determined by a heteroduplex experiment betweenHindIII linearized molecules of R68 and R68.45. It is proposed that this duplication found in R68.45 is responsible for its chromosome mobilizing ability.

It is evident that the knowledge about biological nitrogen fixation was enormously increased when genetic systems in free-living and symbiotic nitrogen fixing bacteria were developed and used for the analysis of their nif (nitrogen fixation) genes. Due to its close relationship to Eschericia coli, Klebsiella pneumoniae, a facultatively anaerobic procaryotic microorganism, was initially selected to analyze nif genes (Streicher et al., 1971; Dixon, Postgate, 1971). For other Gram-negative nitrogen fixing microorganisms, genetic systems were recently developed and are now used to compile information in the field of nif genetics. To review all the information available would certainly exceed the scope of this paper. Therefore, we concentrate on three different nitrogen fixing species which are currently being analyzed in our laboratory: the facultative anaerobe Klebsiella pneumoniae, the phototrophic Rhodopseudomonas capsulata and the symbiotic nitrogen fixing Rhizobium meliloti. For these three species, the genetic techniques developed will be outlined. It is our special intention to show that break-throughs are often achieved following the development of new methodology.

Mutants of Rhizobium meliloti strain 2011 were induced by general Tn5 transposition using pBR325-Mob-Tn5 as a mobilising vector and inoculated on sterilized seedlings of Medicago sativa (alfalfa). After 3 or 4 weeks plants were examined for nodulation and also tested by the acetylene reduction assay. Of about 2400 mutants screened by this procedure, 23 showed defects or significant morphological and physiological changes in their symbiotic properties. In total, nine mutants failed to induce nodule formation. Among these there were five auxotrophic mutants (two for leucine, two for valine/isoleucine and one mutant with two Tn_5 insertions but unknown auxotrophic markers). Further auxotrophic mutants (arginine, adenine and cysteine) induced small nodules which sometimes were tumorlike in their appearance. These mutants did not fix nitrogen. Among the ineffective mutants, there were three prototrophic ones with an altered colony type.

A denaturation map of mitochondrial DNA from the wild type strain 5256 of Neurospora crassa was constructed by computer analysis of the contour length distribution of single- and double-stranded regions of nineteen circular and three full length linear molecules after partial denaturation. The data suggest that mitochondrial DNA in this strain is a homogeneous population of a circular molecule of molecular weight 41 - 10(6) with an asymmetric distribution of AT-rich regions, and that linear molecules derive from this genome by random breaks during isolation.

The molecular relationships of the IncP1 plasmids RP1, RP4, R68 and RK2 were tested by electron microscopic examination of heteroduplexes. In several hybridization experiments molecules were detected which had a 7.8% portion of incomplete reannealing. This 'heterologous region' could be explained by the typical renaturation behaviour of the transposon Tn1. The identity of the Tn1 transposon present in RP1 and RP4 was proved by heteroduplex experiments with lambda phage DNA containing this transposon. These results indicated that the plasmids RP1 and RP4 are identical. Additional heteroduplex experiments between plasmids R68.45 and RP8 and between R68.45 and RK2 were performed. R68.45, a derivative of R68, has a small DNA insertion and RP8 can be regarded as a large insertion mutant of RP4; both insertions were used as single-stranded hybridization markers. From the hybrid molecules formed, it was deduced that R68 and RK2 are identical with RP1 and RP4 as far as molecular structure is revealed by the technique used.

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 5.3 kb portion encompassing the resistance genes (tet region). A model accounting for both, recA-independent translocation and recA-dependent amplification, postulates two direct and one inverted repeat as essential constituents of the transposons. DNA sequence analyses of Tn1721 and Tn1771 have substantiated this model. They demonstrated three identical 38 base pair repeats identically in both transposons dividing them into a "minor transposon" and a tet region. Identical sequences of at least 87 base pairs providing recombination "hot spots" for gene duplication have been found at the ends of the repetitious tet region. Translocation of Tn1721 and Tn1771 generates five base pair direct repeats at the respective sites of insertion. On the basis of the heteroduplex molecules and sequences analyzed the two transposons are identical.

Plasmid profiles were determined for twenty four strains of R. trifolii. All the strains harboured at least three plasmids and as many as eight were present in some strains. Southern hybridisation with cloned R. meliloti nif genes was used to identify indigenous Nif plasmids. Each strain carried a single Nif plasmid and these plasmids varied in size from 112 Mdal to at least 300 Mdal.

Rhizobium meliloti induces the formation of root nodules on its symbiotic partner alfalfa (Medicago sativa). The morphogenesis of the nodule is a complex interaction between the host plant and the invading bacteria. Genetic analysis of the microsymbiont has revealed interesting details about this symbiotic association. A very large plasmid (pSYM) has bee identified in R. meliloti which was found to carry symbiotic genes, such as common nodulation, host specificity and fixation genes (Rosenberg et al 1981; Banfalvi et al 1981; Kondorosi et al 1982). Recently, it was found that R. meliloti strain 2011 contains two Megaplasmids (Banfalvi et al 1985; Simon 1984) of 1200 and 1500 Kd (Burkardt et al submitted). The smaller plasmid is identical to the symbiotic plasmid pSYM whereas the larger plasmid is involved in exopolysaccharide biosynthesis (Hynes et al 1986). In this paper we report the isolation and characterization of infection mutants which are altered in acidic exopolysaccharide production. The mapping of the infection genes is described and experiments are presented showing that infection and nodulation mutants can complement each other on a cellular level.

The complete nucleotide sequence (24,206 base-pairs) of the Klebsiella pneumoniae gene region for nitrogen fixation (nif) is presented. Coding regions corresponding to the 19 known nif genes (including nifW and nifZ) could be identified. An additional open reading frame of 216 base-pairs, called nifT, was detected between nifK and nifY. Search for transcriptional signal structures revealed some unusual features: (1) several possible NifA-binding motifs are present in the intergenic regions between nifJ and nifH as well as between nifX and nifU; (2) a perfect NifA-binding motif, preceding the nifENX promoter, is located within an inverted repeat structure; (3) structures resembling the consensus nif promoter are found within the coding regions of nifW and nifZ and, together with a NifA-binding motif, in nifN. Typical rho-independent termination structures were detected only downstream from the nifHDKTY and the nifBQ operons. Analysis of the deduced amino acid sequences revealed the presence of two Cys-X2-Cys-X2-Cys-X3-Cys-Pro clusters in the pyruvate-flavodoxin oxidoreductase NifJ. This arrangement of cysteine residues is normally present only in ferredoxins. A high degree of homology between the two gene products (NifE and NifN) involved in iron-molybdenum cofactor biosynthesis and the two nitrogenase component I structural proteins (NifD and NifK) was found. All four proteins are characterized by the conserved motif His-Gly-X2-Gly-Cys, which may play a role in binding the iron-molybdenum cofactor.

The his and nif genes of the P1 type plasmid pRD1 were lost at a high frequency in a recA+ but not in a recA- Escherichia coli host during growth in a non-selective medium. 92% of the His- Nif- segregants after 6 subcultures retained the genetic markers of the precursor plasmid RP4, while the remainder lost all of the pRD1 markers with the concomitant loss of ccc-DNA. Plasmids purified from the His- Nif- segregants resembled RP4 in the physical and genetic properties examined. The contour length of pRD1 DNA molecules from a recA- strain was 49 micrometer corresponding to a molecular weight of 101 Mdals and the buoyant density was 1.715 g/cm3. In contrast, the contour lengths of plasmid molecules isolated from a recA+ host carrying pRD1 fell into 3 size classes of 49, 19 and 2 micrometer corresponding to molecular weights of 101, 39 and 4 Mdals respectively and two DNA species of buoyant density 1.715 and 1.719 g/cm3 were observed.

Two deletion mutants pAP1 (MW 82 Mdals) and pAP2 (MW 64 Mdals) were isolated by P1 transduction of the plasmid pRD1 (MW 101 (Mdals). These plasmid mutants still carry the his-nif region of K. pneumoniae. They are selftransmissible and mediate resistance to ampicillin, kanamycin and tetracycline. Comparing the HindIII maps of pRD1, pAP1 and pAP2 showed that pAP1 was derived from pRD1 by an 8 μm deletion and pAP2 by two deletions — the same 8 μm deletion and a further 9 μm deletion. The plasmids pAP1 and pAP2 helped us to locate the his-nif region of pRD1 on 3 adjacent HindIII fragments (number 5, 4 and 3 according to gelelectrophoresis). The molecular weights of these fragments were 8.2, 10 and 15 Mdals. These 3 fragments were cloned separately on the multicopy plasmid vehicle pWL625 giving rise to the hybrid plasmids pWK1 (pWL625+HindIII fragment 4), pWK2 (pWL625+HindIII fragment 5). None of these hybrid plasmids conferred nitrogen fixation capacity on E. coli C cells. By combining HindIII fragment 4 and 3 in the same alignment as in pRD1 and cloning them together on pWL625 the hybrid plasmid pWK120 (pLW625+HindIII fragments 4 and 3) was found to carry the entire nif region. An E. coli C strain harbouring the plasmid pWK120 grew on nitrogen free medium and reduced acetylene. The plasmid pWK 120 had a contourlength of 17 μm, a buoyant density of 1.715 g/ml and a copy number up to 65.

A cDNA library has been prepared using poly A+mRNA from polysomes of root nodules of broadbean (Vicia faba L.). Several leghemoglobin (Lb) cDNA clones were identified by hybridization to a known Lb-sequence from pea. Sequencing of these clones revealed the existence of at least 3 different Lb genes in broadbean. Hybrid-released translation experiments confirmed the existence of a multigene family of leghemoglobins in this species. A comparison of the sequences of the untranslated 3′ end of these transcrits revealed several polyadenylation signals as well as a highly conserved sequence of 16 base pairs (bp). The central part of this sequence is conserved between the Lb genes of different species and is present as an inverted repeat in the coding region.

The insertion element ISR1 was isolated from the soil bacterium R. lupini. In this strain, ISR1 shows a very strong affinity to plasmid RP4. It causes RP4 mutations at the strikingly high frequency of 10(-2) to 10(-1), either by the integration itself or by generating deletions. In E. coli, ISR1 seems to be inactive. No evidence could be obtained for a promoter site on ISR1 or for an ISR1-encoded protein. Our results indicate, however, an ISR1-specific termination signal for either transcription or translation.

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. Two of the proteins (Mr 25.000 and 26.000) were expressed with promoters from the Ti-plasmid fragment, while transcription for the two other proteins (Mr 18.000 and 74.000) started with a promoter on pACYC184. The Mr 18.000 protein represented a fusion product between chloramphenicol acetyltransferase (CAT) on pACYC184 and a part of lysopine dehydrogenase (LpDH), the enzyme synthesizing octopine and lysopine in plant tumor cells. The results suggest that E. coli minicells are a valuable system to study the proteins coded for by the T-region of Ti-plasmids.

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. pSG2 has a copy number of about four. Plasmid pSG2 was fused to a pACYC184 derivative modified to harbour a thiostrepton resistance gene. The resulting plasmid, designated pSW1, is a 16.6 kb shuttle plasmid which replicates in Escherichia coli and in several Streptomyces strains, including S. ghanaensis, S. lividans and S. viridochromogenes. Replacement of a BglII-fragment of plasmid pSG2 by a fragment encoding thiostrepton resistance resulted in a low copy 12.2 kb Streptomyces plasmid. This plasmid, designated pSW2, is a Streptomyces broad host range plasmid.

Replication of the Streptomyces ghanaensis plasmid pSG5 was shown to be temperature sensitive. The pSG5 replicon is stably inherited at temperatures below 34° C, but is lost at incubation temperatures above this. A family of cloning vectors was constructed using the pSG5 minimal replicon and different marker genes. The vectors obtained are small in size, have an intermediate copy number, possess a broad host range and are compatible with some other streptomycete vector systems. By increasing the incubation temperature, these vectors can be eliminated from their host cells very efficiently. The suitability of the pSG5 vector family for mutating chromosomal genes by gene disruption was demonstrated: pBN10, a pSG5 derivative containing an internal fragment of the phosphinothricyl-alanyl-alanine (PTT) resistance gene pat, was integrated into the chromosomal pat gene of the PTT-producer Streptomyces viridochromogenes thus inactivating PTT resistance. The integrated pBN10 plasmid was rescued from the chromosome, together with an adjacent fragment carrying DNA of the PTT biosynthetic cluster.

We have isolated R.meliloti Tn5-induced mutants which failed to induce nodules on Medicago sativa (alfalfa). Using the mobilizable strain E.coli S-17-1, we transferred the plasmid pRmSL26 into these Nod− mutants. Transconjugants possessing pRmSL26 were tested for nodulation (Table I). For a further characterization of the positively complemented mutants, we subcloned different fragments from pRmSL26 into the vector pSUPlo4. Two hybrids were constructed (pMA10, pMA11), and then conjugated into Nod− mutants. In one case (2526) pMA10 restored the ability to form active nodules (Table I). Furthermore, we analysed this region in homogenotization experiments using the 5.3 kb PstI fragment cloned into the vector pSUP202. The resultant hybrid, pMA22, was mutagenized with Tn5 and then mobilized into wild type R.meliloti 2011. Transconjugants with phenotype Nm + Tc− Cm−, were isolated and tested on plants. One Tn5 insert showed Nod− phenotype (Figure I, A). From these results, we assumed that the region which complemented the Nod− mutant 2526 should be located in the right side of the cloned fragment in pMA10. The fact that mutant 1142 were complemented by the whole pRmSL26 but not by pMA10 or pMA11, indicates that regions other than that cloned in pMAlO contain essential genes for early steps of nodulation. In addition, in heteroduplex experiments between a R.trifolii sequence cloned in pRt8002, and the 8.7 kb EcoRI fragment from pRmSL26 we observed a homologous region extending 3.0 kb into the right side of pMA10.

We have constructed derivatives of the transposon Tn5 carrying the mob site (oriT) of plasmid RP4, and an nptI-sacB-sacR cassette [Ried and Collmer, Gene 57 (1987) 239-246]. The mob site, in conjunction with the antibiotic-resistance markers carried on the transposons, allows identification of transposon inserts in cryptic plasmids by mobilisation to other strains. The sacB-sacR genes allow direct selection for the loss or curing of plasmids, because only strains which no longer contain an active sacB gene are able to grow on media containing sucrose. We have tested these transposons in four strains of Rhizobium leguminosarum and two strains of Rhizobium meliloti, and have been able to demonstrate curing of several large cryptic plasmids, and generation of large deletions in many other plasmids. This method has enabled us to show that the R. leguminosarum plasmids pRL12JI and pR1eVF39f carry auxotrophic markers, and that the plasmid pR1eVF39c carries genes which affect colony morphology.

SUMMARYTwo spontaneously formed R-plasmids (pFS401 and pFS402) originating from the multiple drug-resistantEscherichia colistrain UR12644 were found to carry transposable drug-resistance elements. Incompatibility between these two plasmids was used to select for transposition. An ampicillin transposon (Tn1781) residing on pFS401 and a tetracycline transposon (Tn1771) present on pFS402 were independently translocated to the endogenous RTF-plasmid pFS2. Molecular weight determinations of pFS2::Tn1781(Ap) and pFS2::Tn1771(Tc) revealed a value of 2·9 Mdal for Tn1781 and 7·1 Mdal for Tn1771. The arrangement of 3PstI and 1BamHI restriction endonuclease sites was found to be characteristic for the ampicillin transposon whereas the restriction map of Tn1771 features a nearly symmetrical location of 3EcoRI cleavage sites, two of them close to the termini and one in the middle of the transposon. A model is presented suggesting the existence of repetitive DNA-segments at these positions which represent the structural preconditions for the genetic properties of Tn1771. The role of a cryptic plasmid involved in the generation of the endogenous R-plasmids pFS401 and pFS402 is discussed.

SUMMARY: 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. aeruginosa chromosome. Immunological and enzymic analysis showed the expression of functional primase polypeptides in the constructed P. aeruginosa strain. This strain also restored wild-type conjugational transfer proficiency, by complementation, to mutants of the IncP-1 plasmid R18 affected in transfer from P. aeruginosa to P. stutzeri or to Acinetobacter calcoaceticus due to transposon Tn7 insertion mutations in the primase gene. This strategy of cloning into a transposon and integration into the bacterial chromosome should facilitate genetic manipulation and studies of gene expression in a range of Gram-negative bacteria.

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. Since the positions of these restriction sites on the genetic map of RP4 are known, the maps could be compared. They revealed a high AT content for the Tn1 transposon and the kanamycin resistance gene. The tra-1 region is also distinguished by a sharply defined AT-rich region, whereas tra-2 and the tetracycline resistance gene have an AT content which is not distinctly different from the average AT content of RP4.

Phosphinothricin-tripeptide (Ptt), also known as bialaphos, contains phosphinothricin (Pt), a potent inhibitor of glutamine synthetase. A 4.0-kb BamHI fragment coding for Ptt resistance was cloned in Streptomyces lividans TK23. The fragment was isolated from a Ptt-resistant mutant of Streptomyces viridochromogenes Tu494. Subcloning experiments revealed that Ptt resistance can be assigned to a 0.8-kb BglII fragment. This fragment was shown to include the Ptt-resistance promoter. Subcloning this fragment downstream from the lacZ promoter conferred Ptt resistance to Escherichia coli JM83 in one of the two possible orientations. Biochemical investigations revealed that the BglII fragment codes for a Pt N-acetyltransferase.

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. They offer the selection markers and cloning sites characteristic of the basic E. coli vectors. Therefore, they can be applied and adapted to a variety of cloning strategies. However, the cloning of very large fragments (e.g., in cosmid hybrids of pSUP106) was found to affect the stability of the recombinant molecules in a Rec+ background. This instability was not observed with smaller inserts of about 5 kilobases.

A plasmid-cloning system was developed for Streptomyces viridochromogenes Tu494, a producer of the tripeptide antibiotic phosphinothricyl-alanyl-alanine (PTT). Parameters affecting protoplast formation and transformability of S. viridochromogenes were investigated in detail. A procedure giving rise to transformation efficiencies of 10(4)-10(5) transformants per microgram DNA was worked out. Several Streptomyces plasmid vectors such as pIJ350, pIJ61, pEB2, pGM4 and pSW2 were tested in S. viridochromogenes. Some of these vectors (pGM4, pEB2) showed a high copy number, whereas the copy number of others (pIJ350, pIJ61 and pSW2) was markedly lower. Under non-selective conditions some of the vectors (pIJ350, pEB2) were not stably maintained, in contrast to the vectors pGM4 and pSW2 which did not require any selection pressure for stable maintenance. Therefore, the plasmid vectors pGM4 and pSW2 derived from endogenous replicons of Streptomyces ghanaensis strains represent appropriate plasmid vehicles for cloning experiments in S. viridochromogenes.

Publisher Summary This chapter describes the experimental application of a vector system for Rhizobium that enables efficient transposon mutagenesis, site-specific insertion of selectable markers, creation of deletions in predetermined DNA regions, complementation studies, and high-frequency transfer of any DNA molecule between different strains. The essential ingredients of the system are the broad host range transfer and mobilization functions of plasmid RP4, transposable elements, mainly Tn5, and usual Escherichia coli specific vector plasmids. The major area of research in Rhizobium genetics is the identification, characterization, and manipulation of symbiotic genes. Modern recombinant DNA techniques have been applied to the analysis of cloned rhizobial genes in their parental environment by the construction of broad host range vector plasmids. The antibiotic resistance plasmid RP4 is known to be self-transmissible to any Gram-negative bacterium. The advantages of the use of antibiotic resistance transposons as mutagenic agents are insertion of a transposon into a gene leads to a complete loss of its function, transposon insertion mutation can be directly selected so that the yield of a transposon mutagenesis experiment is 100%, and the antibiotic resistance marker carried by the transposon acts as a probe for the genetic and physical location of the affected gene.

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. The loss of the cryptic plasmid pAtC58 has no effect on the tumor-forming ability of the Agrobacterium strains; when the Ti plasmid is present, normal tumors are formed on Kalanchoe daigremontiana.

SUMMARYStrains ofEscherichia coliharbouring a conjugative plasmid that carries a transposon for tetracycline resistance (Tn1771) were found to be adaptable to very high tetracycline concentrations. The molecular analysis of plasmids isolated from strains with enhanced levels of tetracycline resistance revealed an intramolecular amplification of the resistance determinant of the tetracycline transposon.A model for the molecular structure of the transposon is presented, which suggests that there are three repetitive DNA segments on Tn1771. This accounts for the properties both of transposition and of gene amplification.

Intact and osmotically sensitive cells of Corynebacterium glutamicum can be efficiently transformed by electroporation. This was shown by using the plasmid vector pUL-330 (5.2 kb), containing the kanamycin resistance gene of transposon Tn5. The following electric parameters yielded efficient transformation. For intact cells: one exponentially decaying field pulse \(E = E_0 exp( - t/\tau _E )\) with time constants \(\tau _E = 450 - 500\) and with initial field intensities of E0=35–40 kV cm-1; prepulse temperature 20°C. Cell regeneration (survival) was 100%–80%. Transformation efficiency can be increased by an additional freeze and thaw cycle of the cells, prior to electroporation. Lysozyme treated cells (osmotically sensitive) were transformed with three successive pulses of E0=25–30 kV cm-1. Cell regeneration under these conditions was found to be 20–30%. The optimum yield of transformants/μg plasmid-DNA was 3×103 for intact cells, 2×104 for intact cells which were frozen and thawed twice and 7×104 for osmotically sensitive cells if the cell suspension was pulsed at a cell density of 1–3×108/ml and at a DNA concentration of 0.2 μg/ml up to ≤2 μg/ml. The data obtained for osmotically sensitive cells suggest that the temperature increase accompanying the electric field pulse enhances colony formation and transformation efficiency if the initial prepulse temperature is ≥20°C, although regeneration of electroporated C. glutamicum cells starts to decrease at temperatures≥20°C.

Rhodobacter capsulatus mutants unable to fix nitrogen were isolated by random transposon Tn5 mutagenesis. The Tn5 insertion sites of 30 Nif- mutants were mapped within three unlinked chromosomal regions designated A, B, and C. The majority of Tn5 insertions (21 mutants) map within nif region A, characterized by two ClaI fragments of 2.5 and 25 kilobases (kb). The 17-kb ClaI fragment of nif region B contains six nif::Tn5 insertions, and the three remaining mutations are located on a 32-kb ClaI fragment of nif region C. Hybridization experiments using all 17 Klebsiella pneumoniae nif genes individually as probes revealed homology to nifE, nifS, nifA, and nifB in nif region A. The nifHDK genes were localized in nif region B. About 2 kb away from this operon, a second copy of the DNA fragments homologous to nifA and nifB, originally found in nif region A, was identified.

Sequencing of the Rhizobium meliloti DNA region downstream of nifA revealed the existence of nifB, fdxN and ORF3. The molecular weight of the fdxN protein (Mr 6830) and the distribution of cysteine residues in its deduced amino acid sequence is typical for low molecular weight bacterial ferredoxins. Interposon insertion and plasmid integration mutagenesis demonstrated that FdxN is essential for nitrogen fixation in R. meliloti, whereas the predicted translation product of ORF3 (Mr 8708) is not necessary for this process. In contrast, ferredoxin-like proteins, which are encoded by nifB-associated genes, are not required for nitrogen fixation in all other organisms analysed so far. Plasmid integration mutagenesis additionally revealed that nifA, nifB and fdxN form one transcriptional unit. This result was confirmed by complementation analysis of polar interposon insertion mutants of nifA, nifB and fdxN and by complementation of a non-polar nifA deletion mutant. A DNA sequence resembling a typical nif consensus promoter, which is preceded by two putative NifA-binding sites, is located in front of nifB. This nifB promoter can be activated in Escherichia coli by the nifA gene product of Klebsiella pneumoniae to the same level as that of the R. meliloti nifH promoter. In contrast, R. meliloti NifA stimulates the nifH promoter more efficiently than the nifB promoter. This low-level activation of the nifB promoter may be the reason why transcription of nifB and fdxN is initiated primarily at a promoter in front of nifA.

Spheroplasts were prepared from Corynebacterium glutamicum ATCC13032 by growing cells in the presence of glycine followed by digestion with lysozyme. Using pUL330 a spheroplast transformation system was established routinely yielding 103 to 104 transformants per μg of plasmid DNA. Spheroplasts were converted into protoplasts after incubation with the lytic enzyme achromopeptidase in the presence of additional lysozyme. Protoplasts prepared by this method regenerated at efficiencies of 10 to 30%. A protoplast transformation system was established routinely yielding 105 to 106 transformants per μg of plasmid DNA. The Escherichia coli Brevibacterium lactofermentum shuttle vector pUL62 prepared from E. coli could be introduced into spheroplasts of C. glutamicum after heat treatment at 48 to 49°C for 10 min.

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. DNA sequence analysis revealed an open reading frame (ORF) of 469 codons specifying a polypeptide of 52,040 daltons. Its deduced amino acid sequence was found to be highly homologous to other glutamine synthetase sequences. This ORF was expressed in Escherichia coli minicells and the corresponding polypeptide reacted with an antiserum raised against GSI. Upstream of glnA we found an ORF of 111 codons (ORF111) preceded by the consensus sequence for an ntrA-dependent promoter. Minicells experiments showed a protein band, with a molecular weight in good agreement with that (10,469) deduced from the nucleotide sequence. On the basis of homology studies we discuss the possibility that the product of ORF111 is equivalent to the P_II protein of E. coli and plays a similar role in regulation of nitrogen metabolism.

The cryptic plasmid pSG5 of Streptomyces ghanaensis 5/1B (DSM 2932) was characterized to have a molecular size of 12.7 kb and an approximate copy number of 20–50 per chromosome. A bifunctional derivative, designated pSW344E, consisting of pSG5 and an Escherichia coli vector plasmid was constructed. Following Tn5 mutagenesis in E. coli, the replication functions of the mutagenized pSW344E plasmids were analysed in S. lividans. A 2 kb DNA fragment of the pSG5 replicon was found to carry replication functions. Subcloning of pSG5 DNA into various replication probe vectors resulted in the identification of the pSG5 minimal replicon, identical to the above mentioned 2 kb DNA region. Several small bifunctional plasmids, able to replicate in E. coli as well as in Streptomyces, were generated during subcloning. Some of these plasmids were found to be useful shuttle vectors.

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. pneumoniae nifN and was therefore renamed as the R. meliloti nifN gene. Upstream of the nifN coding region a second open reading frame was identified coding for a putative polypeptide of 110 amino acids (ORF110). By fragment-specific Tn5 mutagenesis it was shown that the nifN gene and ORF110 form an operon. The control region of this operon contains a nif promoter and also the putative nifA-binding sequence. For the deduced amino acid sequence of the nifN gene product a striking homology to the R. meliloti nifK protein was found. One cysteine residue and its adjacent amino acid sequence, which are highly conserved in the R. meliloti nifK, R. meliloti nifN, and K. pneumoniae nifN proteins, may play a role in binding the FeMo cofactor.

We describe the isolation and characterization of alfalfa-nodulating rhizobia from acid soils of different locations in Central Argentina and Uruguay. A collection of 465 isolates was assembled, and the rhizobia were characterized for acid tolerance. Growth tests revealed the existence of 15 acid-tolerant (AT) isolates which were able to grow at pH 5.0 and formed nodules in alfalfa with a low rate of nitrogen fixation. Analysis of those isolates, including partial sequencing of the genes encoding 16S rRNA and genomic PCR-fingerprinting with MBOREP1 and BOXC1 primers, demonstrated that the new isolates share a genetic background closely related to that of the previously reported Rhizobium sp. Or191 recovered from an acid soil in Oregon (B. D. Eardly, J. P. Young, and R. K. Selander, Appl. Environ. Microbiol. 58:1809–1815, 1992). Growth curves, melanin production, temperature tolerance, and megaplasmid profiles of the AT isolates were all coincident with these characteristics in strain Or191. In addition to the ability of all of these strains to nodulate alfalfa (Medicago sativa) inefficiently, the AT isolates also nodulated the common bean and Leucaena leucocephala, showing an extended host range for nodulation of legumes. In alfalfa, the time course of nodule formation by the AT isolate LPU 83 showed a continued nodulation restricted to the emerging secondary roots, which was probably related to the low rate of nitrogen fixation by the largely ineffective nodules. Results demonstrate the complexity of the rhizobial populations present in the acidic soils represented by a main group of N2-fixing rhizobia and a second group of ineffective and less-predominant isolates related to the AT strain Or191., Instituto de Biotecnologia y Biologia Molecular