Roles for enteric d-type cytochrome oxidase in N2 fixation and microaerobiosis.

Escherichia coli strains that lacked the d-type cytochrome oxidase, the terminal oxidase with a high affinity for O2, grew anaerobically as well as the wild type did and were not impaired in the ability to evolve H2 from either glucose or formate. The anaerobic synthesis and activity of nitrogenase in transconjugants of these strains carrying Klebsiella pneumoniae nif genes were also normal. However, the behavior towards O2 of anaerobically grown bacteria lacking the d-type oxidase differed from that of the wild type in the following ways: the potential O2 uptake was lower, H2 evolution and nitrogenase activity supported by fermentation were more strongly inhibited by O2, and microaerobic O2-dependent nitrogenase activity in the absence of a fermentable carbon source did not occur. These results show that the d-type oxidase serves two functions in enteric bacteria--to conserve energy under microaerobic conditions and to protect anaerobic processes from inhibition by O2.     

A haemoprotein is not involved in the control by oxygen of enteric nitrogenase synthesis

Strains of Escherichia coli containing the Nif+ plasmid pRD1 were used to investigate the possibility that haem proteins are involved in the regulation by O2 of nif expression. Strains lacking 5-aminolaevulinate synthase (HemA-), and hence normally unable to synthesize haem proteins, showed an identical response to O2 in the presence or absence of added aminolaevulinate (and hence of haem proteins). It was concluded that the regulatory protein NifL is not a haem protein.     

Phylogeny and characterization of three nifH-homologous genes from Paenibacillus azotofixans

In this paper, we report the cloning and characterization of three Paenibacillus azotofixans DNA regions containing genes involved in nitrogen fixation. Sequencing analysis revealed the presence of nifB1H1D1K1 gene organization in the 4,607-bp SacI DNA fragment. This is the first report of linkage of a nifB open reading frame upstream of the structural nif genes. The second (nifB2H2) and third (nifH3) nif homologues are confined within the 6,350-bp HindIII and 2,840-bp EcoRI DNA fragments, respectively. Phylogenetic analysis demonstrated that NifH1 and NifH2 form a monophyletic group among cyanobacterial NifH proteins. NifH3, on the other hand, clusters among NifH proteins of the highly divergent methanogenic archaea.     

A cyanobacterial recombination study, involving an efficient N2-fixing non-heterocystous partner

Many filamentous cyanobacteria fix atmospheric nitrogen under natural conditions in specialized anaerobic compartments, heterocysts, interspersed between vegetative cells, which provide protection to the O2-sensitive nitrogenase. A few unicellular cyanobacterial strains are also known to fix nitrogen aerobically at a slower rate. Filamentous cyanobacteria lacking heterocysts are not known so far to fix nitrogen. We describe the isolation and purification of a non-heterocystous filamentous cyanobacterium from the fronds of the water-fern Azolla, fixing nitrogen at 18.7+/-0.2 n moles ethylene microg Chl. a(-1) h(-1) when grown in nitrogen-free medium at a low level of oxygen between two layers of agar. This strain of Anabaena azollae has been designated as het- nif+ (non-heterocystous and nitrogen-fixing), and is found to be easily and effectively preserved in nitrogen-free medium in standard synthetic cyanobacterial nutrient medium (pH 8.5) at a continuous light intensity of 2800 lx at 25+/-1 degrees C. This het- nif+ strain is an effective donor of the nif+ marker to a het+ nif- strain of another cyanobacterium, Nostoc muscorum, when both are grown together in a recombination study.     

The roles of the nifW, nifZ and nifM genes of Klebsiella pneumoniae in nitrogenase biosynthesis

Active Fe protein of nitrogenase was synthesised in a non-nitrogen fixing organism when Escherichia coli was transformed with a plasmid encoding only two nif-specific genes, nifH and nifM of Klebsiella pneumoniae. Hence proteins NifH and NifM are sufficient to produce active Fe protein in E. coli. K. pneumoniae strains carrying chromosomal nifW- and nifZ- mutations were constructed and shown to be significant C2H2-reducing activity and to grow on N-free plates. Nevertheless, derepressing cultures of the mutant strains had reduced levels of MoFe protein activity, and consequently significantly lower levels of nitrogenase activity, than the nif+ parent strain. NifW and NifZ therefore appear to be involved in the formation or accumulation of active MoFe protein, but are not essential for nitrogen fixation in K. pneumoniae under the conditions tested.     

Isolation of nifH and part of nifD by modified capture polymerase chain reaction from a natural population of the marine cyanobacterium Trichodesmium sp.

Abstract A modified capture polymerase chain reaction (CPCR) technique was used to isolate the entire sequence of the nifH gene and its flanking regions from a natural population of Trichodesmium sp. A set of specific CPCR primers derived from a known 72-bp DNA segment of the nifH sequence permitted isolation of both the upstream and the downstream region of Trichodesmium sp. nifH . The 882-bp nifH gene presented here is the first full-length gene isolated from Trichodesmium sp. A sequence similar to a nif -like promoter was found in front of nifH . The nifH open reading frame of Trichodesmium sp. encoded 294 amino acids. Comparative analysis of the Trichodesmium sp. NifH sequence revealed strong similarity with 23 known NifH proteins. Amino acids postulated to be involved in binding of the 4Fe:4S cluster and those subjected to ADP-ribosylation were present. An open reading frame for the nifD gene was identified 189 bp downstream of nifH . A sequence similar to the consensus of the nif -like promoter was also found in front of nifD .     

Effect of Cd2+ on growth of the cadmium-resistant and -sensitive Staphylococcus aureus

The effect of Cd2+ on aerobic and anaerobic growth was studied in the Cd2+-resistant Staphylococcus aureus 17810R which harbours the cadA and cadB markers on a penicillinase plasmid pII17810. Also the effect of Cd2+ on growth of the plasmidless strain 17810S, sensitive to Cd2+ was investigated. The results indicate that under all growth conditions the Cd2+-resistant S. aureus 17810R is protected against Cd2+ toxicity up to 100 microM Cd2+ by the 2H+/Cd2+ antiporter, the product of the cadA gene. Energetics of growth of both strains under various conditions is also discussed.     

Localization and physical mapping of a plasmid-borne 23-kb nif gene cluster from Enterobacter agglomerans showing homology to the entire nif gene cluster of Klebsiella pneumoniae M5a1

A physical and genetical map of the plasmid pEA3 indigenous to Enterobacter agglomerans is presented. pEA3 is a 111-kb large plasmid containing a 23-kb large cluster of nif genes which shows extensive homology (Southern hybridization and heteroduplex analysis) to the entire nif gene cluster of Klebsiella pneumoniae (Kp) M5a1. All the nif genes on pEA3 are organized in the same manner as in K. pneumoniae, except nifJ, which is located on the left end of pEA3 nif gene cluster (near nifQB). A BamHI restriction map of pEA3 and a detailed restriction map of the 23-kb nif region on pEA3 is also presented. The nif genes of pEA3 showed a low level of acetylene reduction in Escherichia coli, demonstrating that these genes are functional and contain the whole genetic information required to fix nitrogen. The origin of vegetative replication (OriV) of pEA3 was localized about 5.5 kb from the right end of the nif gene cluster. In addition to pEA3, large plasmids from four other strains of E. agglomerans showed homology to all the Kp nif genes tested, indicating that in diazotrophic strains of E. agglomerans nif genes are usually located on plasmids. In contrast, in most of the free-living, nitrogen-fixing bacteria the nif genes are on chromosome.