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A system is described for the single-copy, stable insertion of cloned DNA sequences into the chromosomes of Gram- bacteria. Two narrow-host-range plasmids form the basis of this system: the 'carrier' plasmid contains the mini Tn7-Km transposon, into which foreign DNA can be cloned; the 'helper' plasmid provides the Tn7 transposition functions in trans. Both plasmids are readily transferred into Gram- bacteria by conjugation. The functionality of this system has been demonstrated in Rhodospirillum rubrum.

Dinitrogenase reductase (the nifH product) from Rhodospirillum rubrum is regulated by a post-translational modification system encoded by draTG. As demonstrated in this report, the cloning, sequencing, and functional characterization of the nifH gene provides a basis for further analysis as well as revealing interesting features of gene organization. The coding regions of nifH and draT are separated by only 400 bp, though the genes are divergently transcribed and differentially regulated. The construction of a nifH insertion caused a Nif- phenotype and destroyed the mutant's ability to synthesize both dinitrogenase and dinitrogenase reductase, verifying functionality and transcriptional organization of the nifHDK genes.

A genomic library of Azospirillum lipoferum was constructed with phage lambda EMBL4 as vector. From this library, the genes encoding dinitrogenase reductase ADP-ribosyltransferase (DRAT), draT, and dinitrogenase reductase-activating glycohydrolase (DRAG), draG, were cloned by hybridization with the heterologous probes of Rhodospirillum rubrum. As in R. rubrum, draT is located between draG and nifH, the gene encoding dinitrogenase reductase (a substrate for the DRAG/DRAT system). In the crude extract of Escherichia coli harboring the expression vector for this region, DRAT and DRAG enzyme activities were detected, confirming the identity of the cloned genes. Southern hybridization with genomic DNA from different Azospirillum spp., demonstrated a correlation between observable draTG hybridization and the biochemical demonstration of this covalent modification system.

A temperate actinophage, TG1, was isolated from soil by growth on Streptomyces cattleya and has been shown to be potentially useful for the cloning of DNA in this organism and other streptomycetes. It forms stable lysogens by integration at a unique site on the chromosome. The phage genome consists of 41 kb of double-stranded DNA with cohesive ends. It has unique sites for ClaI, NdeI, PstI, SmaI, and XbaI. The PstI site has been shown to be in a dispensable region of the phage genome. Deletions (2 kb in length) were obtained which retain this site and should be useful for the cloning of DNA.

The function of the cloned draT gene of Rhodospirillum rubrum was studied by placing it under the control of the tac promoter in the vector, pKK223-3. After induction with isopropyl-beta-D-thiogalactopyranoside, dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was detected in crude extracts of the heterologous hosts Escherichia coli and Klebsiella pneumoniae. In addition, the expression of draT produced a Nif- phenotype in the otherwise wild-type K. pneumoniae strains, the result of the ADP-ribosylation of accumulated dinitrogenase reductase (DR). DR from a nifF- background was also susceptible to ADP-ribosylation, indicating that the oxidized form of DR will serve as a substrate for DRAT in vivo. A mutation that changes the Arg-101 residue of DR, the ADP-ribose attaching site, eliminates the ADP-ribosylation of DR in vivo, confirming the necessity of this residue for modification.