Profiling Gene Expression to Distinguish the Likely Active Diazotrophs from a Sea of Genetic Potential in Marine Sediments

Nitrogen (N) cycling microbial communities in marine sediments are extremely diverse, and it is unknown whether this diversity reflects extensive functional redundancy. Sedimentary denitrifiers remove significant amounts of N from the coastal ocean and diazotrophs are typically regarded as inconsequential. Recently, N fixation has been shown to be a potentially important source of N in estuarine and continental shelf sediments. Analysis of expressed genes for nitrite reductase (nirS) and a nitrogenase subunit (nifH) was used to identify the likely active denitrifiers and nitrogen fixers in surface sediments from different seasons in Narragansett Bay (Rhode Island, USA). The overall diversity of diazotrophs expressing nifH decreased along the estuarine gradient from the estuarine head to an offshore continental shelf site. Two groups of sequences related to anaerobic sulphur/iron reducers and sulphate reducers dominated libraries of expressed nifH genes. Quantitative polymerase chain reaction (qPCR) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) data shows the highest abundance of both groups at a mid bay site, and the highest nifH expression at the head of the estuary, regardless of season. Several potential environmental factors, including water temperature, oxygen concentration and metal contamination, may influence the abundance and nifH expression of these two bacterial groups

The heme-copper oxidase family consists of three distinct types of terminal oxidases and is related to nitric oxide reductase

Among aerobic prokaryotes, many different terminal oxidase complexes have been described. Sequence comparison has revealed that the aa3-type cytochrome c oxidase and the bo3-type quinol oxidase are variations on the same theme: the heme-copper oxidase. A third member of this family has recently been recognized: the cbb3-type cytochrome c oxidase. Here we give an overview, and report that nitric oxide (NO) reductase, a bc-type cytochrome involved in denitrification, shares important features with these terminal oxidases as well. Tentative structural, functional and evolutionary implications are discussed

Transcription regulation of the nir gene cluster encoding nitrite reductase of Paracoccus denitrificans involves NNR and NirI, a novel type of a membrane bound regulator.

The nirIX gene cluster of Paracoccus denitrificans is located between the nir and nor gene clusters encoding nitrite and nitric oxide reductases respectively. The NirI sequence corresponds to that of a membrane-bound protein with six transmembrane helices, a large periplasmic domain and cysteine-rich cytoplasmic domains that resemble the binding sites of [4Fe-4S] clusters in many ferredoxin-like proteins. NirX is soluble and apparently located in the periplasm, as judged by the predicted signal sequence. NirI and NirX are homologues of NosR and NosX, proteins involved in regulation of the expression of the nos gene cluster encoding nitrous oxide reductase in Pseudomonas stutzeri and Sinorhizobium meliloti. Analysis of a NirI-deficient mutant strain revealed that NirI is involved in transcription activation of the nir gene cluster in response to oxygen limitation and the presence of N-oxides. The NirX-deficient mutant transiently accumulated nitrite in the growth medium, but it had a final growth yield similar to that of the wild type. Transcription of the nirIX gene cluster itself was controlled by NNR, a member of the family of FNR-like transcriptional activators. An NNR binding sequence is located in the middle of the intergenic region between the nirI and nirS genes with its centre located at position -41.5 relative to the transcription start sites of both genes. Attempts to complement the NirI mutation via cloning of the nirIX gene cluster on a broad-host-range vector were unsuccessful, the ability to express nitrite reductase being restored only when the nirIX gene cluster was reintegrated into the chromosome of the NirI-deficient mutant via homologous recombination in such a way that the wild-type nirI gene was present directly upstream of the nir operon