Further Analysis of Nitrogen Fixation (nif) Genes in Azotobacter chroococcum: Identification and Expression in Klebsiella pneumoniae of nifS, nifV, nifM and nifB Genes and Localization of nifE/N-, nifU-, nifA- and fix ABC-like Genes

Abstract

The results presented extend previous investigations on the genetics of nitrogen fixation in Azotobacter chroococcum and indicate that nif and$x-like DNA is located in at least five different regions of the genome. Region I contains functional copies of n i p , Vand M , as well as nzfH, D and K, all of which complemented mutants of Klebsiella pneumoniae. In addition, n$Eand/or nzfN-like and nzfLi-like DNA is located in this region. The organization of the nifcluster in region I closely resembles that of K. pneumoniae, though spread over 22 kb as compared with 14 kb. Region I1 contains a functional nzjB gene, which complemented a K. pneumoniae nzfB mutant, and seems to be adjacent to a n$A-like gene. Region I11 harbours nzJH*, encoding a second nitrogenase Fe-protein. Region IV contains a reiteration of nzJE-and/or nzfl-like sequences, and DNA homologous to Rhizobium meliloti$xABC is present in region V. The apparent complexity of nifDNA in A. chroococcum is probably related to the two systems for NZfixation present in this organism.

Nucleotide sequence and genetic analysis of the Azotobacter chroococcum nifUSVWZM gene cluster, including a new gene (nifP) which encodes a serine acetyltransferase

“…nifU, nifS, nifV, and nifM are clustered in a region of the A. chroococcum genome spanning about 7 kb (19). In our previous study, the approximate position of each gene was shown by (i) hybridization studies using various K. pneumoniae nifgene probes and (ii) the ability of plasmids pLC11, pSAR2a, and pSAR3a containing overlapping subfragments of this region (Fig.…”

“…Cultures were capped with Suba-Seals, and acetylene was added to 10o of the gas headspace. Acetylene reduction activities of K. pneumoniae strains were not determined as we have previously shown that strains complemented for growth on N2 display near-wild-type acetylene reduction levels, whereas strains not complemented for anaerobic growth show levels of acetylene reduction similar to the activity of the mutant strain alone (19). Protein in whole cells or crude extracts was measured by using the Folin-Ciocalteau reagent (42) with bovine serum albumin (fraction V; Sigma Chemical Co.) as a standard.…”

“…Histidine was added at 20 ,g/ml to the medium for UNF2050. Plasmids pDE15a and pDEl5b contain the 5.1-kb KpnI fragment, previously used to complement K. pneumoniae niJV and nifM mutants (19), cloned in either orientation in the vector pEMBL18+ (13). pSAR4, pSAR5, and pSAR6 contain Sau3A-KpnI fragments of pDEl5b cloned in the EcoRI site of pBR325 and are described in the text.…”

“…A decrease in A230 due to cleavage of the thioester bond of acetyl coenzyme A (acetyl-CoA) was monitored by using a Cary 2200 dual-beam spectrophotometer. The assay was carried out at 37°C in 1-cm-light-path cuvettes in a reaction volume of 1 ml which, when complete, contained the following: Tris-HCl (pH 7.6), 50 chromosome cloned in two primary plasmids pDE14 and pDE15 which were described previously (19). Restriction sites: K, KpnI; S, SaII; X, XhoI.…”

Azorhizobium caulinodans electron-transferring flavoprotein N electrochemically couples pyruvate dehydrogenase complex activity to N2 fixation

“…Rather, thermolabile nifE null mutants were obtained. Yet, while nifE null mutants have been identified in various diazotrophic bacteria Evans et al, 1988), a thermolabile phenotype was not noted. NifE is purported to be important for biogenesis of the iron-molybdenum cofactor, itself required for dinitrogenase complex activity Imperial et al, 1987).…”