The ntrA, ntrB and ntrC products are responsible for regulating the transcription of many genes involved in the assimilation of poor nitrogen sources in enteric bacteria. The presence of a similar system in the non‐enteric bacterium Azotobacter vinelandii is reported here. Genes analogous to ntrA and ntrC were isolated from an A. vinelandii gene library by complementation of Escherichia coli mutants. The gene encoding glutamine synthetase, glnA, was also isolated and found to be adjacent to ntrC but distant from ntrA, as it is in enteric organisms. The cloned Azotobacter genes also complemented Klebsiella pneumoniae mutants and hybridized to K. pneumoniae ntrA, ntrC and glnA gene probes. The role of ntrA and ntrC in A. vinelandii was established by using Tn5 insertions in the cloned genes to construct mutants by marker exchange. These mutants show that both ntrA and ntrC are required for the utilization of nitrate as a nitrogen source. However, ntrC is not required for nitrogen fixation by A. vinelandii, in contrast with K. pneumoniae where both ntrA and ntrC are essential.

Plasmid RK2 is unusual in its ability to replicate stably in a wide range of Gram-negative bacteria. The replication origin (oriV) and a plasmid-encoded initiation protein (TrfA; expressed as 33 and 44 kDa forms) are essential for RK2 replication. To examine initiation events in bacteria unrelated to Escherichia coli, the genes encoding the replicative helicase, DnaB, of Pseudomonas putida and Pseudomonas aeruginosa were isolated and used to construct protein expression vectors. The purified proteins were tested for activity along with E.coli DnaB at RK2 oriV. Each helicase could be recruited and activated at the RK2 origin in the presence of the host-specific DnaA protein and the TrfA protein. Escherichia coli or P.putida DnaB was active with either TrfA-33 or TrfA-44, while P.aeruginosa DnaB required TrfA-44 for activation. Moreover, unlike the E.coli DnaB helicase, both Pseudomonas helicases could be delivered and activated at oriV in the absence of an ATPase accessory protein. Thus, a DnaC-like accessory ATPase is not universally required for loading the essential replicative helicase at a replication origin.

The broad host range plasmid RK2 replicates and regulates its copy number in a wide range of Gram-negative bacteria. The plasmid-encoded trans-acting replication protein TrfA

No abstract

The TrfA protein encoded by the broad host range bacterial plasmid RK2 specifically binds to eight direct repeats (iterons) present at the plasmid replication origin to initiate DNA replication. Purified TrfA protein is largely in the form of a dimer, and using a dimerization test system that involves the fusion of the amino-terminal domain of the cI repressor protein to TrfA, we show that the TrfA protein forms dimers in vivo. Because of the high stability of the dimer form of TrfA, the formation of heterodimers between the wild-type and different sized TrfA proteins requires in vivo de novo folding of the primary protein sequence or in vitro denaturation and renaturation. The results of gel mobility shift assays using in vitro or in vivo formed heterodimers indicated that the TrfA protein binds to the iteron DNA as a monomer. Furthermore, when the monomeric and dimeric forms of TrfA are separated by gel filtration chromatography, only the protein in the chromatographic position of the monomeric form demonstrated significant DNA binding activity. These results indicate that only the monomer form of the TrfA protein is active for binding to the iterons at the RK2 replication origin.