Distinct Interaction Mechanism of RNAP and ResD and Distal Subsites for Transcription Activation of Nitrite Reductase in Bacillus subtilis ψ

The ResD-ResE signal transduction system plays a pivotal role in anaerobic nitrate respiration in Bacillus subtilis . The nasD operon encoding nitrite reductase is essential for nitrate respiration and is tightly controlled by the ResD response regulator. To understand the mechanism of ResD-dependent transcription activation of the nasD operon, we explored ResD-RNA polymerase (RNAP), ResD-DNA, and RNAP-DNA interactions required for nasD transcription. Full transcriptional activation requires the upstream promoter region where five molecules of ResD bind. The distal ResD-binding subsite at −87 to −84 partially overlaps a sequence similar to the consensus distal subsite of the upstream (UP) element with which the Escherichia coli C-terminal domain of the α subunit (αCTD) of RNAP interacts to stimulate transcription. We propose that interaction between αCTD and ResD at the promoter-distal site is essential for stimulating nasD transcription. Although nasD has an extended −10 promoter, it lacks a reasonable −35 element. Genetic analysis and structural simulations predicted that the absence of the −35 element might be compensated by interactions between σ A and αCTD, and between αCTD and ResD at the promoter-proximal ResD-binding subsite. Thus, our work suggested that ResD likely participates in nasD transcription activation by binding to two αCTD subunits at the proximal and distal promoter sites, representing a unique configuration for transcription activation. IMPORTANCE A significant number of ResD-controlled genes have been identified and transcription regulatory pathways in which ResD participates have emerged. Nevertheless, the mechanism of how ResD activates transcription of different genes in a nucleotide sequence-specific manner has been less explored. This study suggested that among the five ResD-binding subsites in the promoter of the nasD operon, the promoter-proximal and -distal ResD-binding subsites play important roles in nasD activation by adapting different modes of protein-protein and protein-DNA interactions. The finding of a new-type of protein-promoter architecture provides insight into the understanding of transcription activation mechanisms controlled by transcription factors including the ubiquitous response regulators of two-component regulatory systems particularly in Gram-positive bacteria.

Nature's nitrite-to-ammonia expressway, with no stop at dinitrogen

Since the characterization of cytochrome c552 as a multiheme nitrite reductase, research on this enzyme has gained major interest. Today, it is known as pentaheme cytochrome c nitrite reductase (NrfA). Part of the NH4+ produced from NO2− is released as NH3 leading to nitrogen loss, similar to denitrification which generates NO, N2O, and N2. NH4+ can also be used for assimilatory purposes, thus NrfA contributes to nitrogen retention. It catalyses the six-electron reduction of NO2− to NH4+, hosting four His/His ligated c-type hemes for electron transfer and one structurally differentiated active site heme. Catalysis occurs at the distal side of a Fe(III) heme c proximally coordinated by lysine of a unique CXXCK motif (Sulfurospirillum deleyianum, Wolinella succinogenes) or, presumably, by the canonical histidine in Campylobacter jejeuni. Replacement of Lys by His in NrfA of W. succinogenes led to a significant loss of enzyme activity. NrfA forms homodimers as shown by high resolution X-ray crystallography, and there exist at least two distinct electron transfer systems to the enzyme. In γ-proteobacteria (Escherichia coli) NrfA is linked to the menaquinol pool in the cytoplasmic membrane through a pentaheme electron carrier (NrfB), in δ- and ε-proteobacteria (S. deleyianum, W. succinogenes), the NrfA dimer interacts with a tetraheme cytochrome c (NrfH). Both form a membrane-associated respiratory complex on the extracellular side of the cytoplasmic membrane to optimize electron transfer efficiency. This minireview traces important steps in understanding the nature of pentaheme cytochrome c nitrite reductases, and discusses their structural and functional features. Graphical abstract

Distribution of Denitrification among Haloarchaea: A Comprehensive Study

Microorganisms from the Halobacteria class, also known as haloarchaea, inhabit a wide range of ecosystems of which the main characteristic is the presence of high salt concentration. These environments together with their microbial communities are not well characterized, but some of the common features that they share are high sun radiation and low availability of oxygen. To overcome these stressful conditions, and more particularly to deal with oxygen limitation, some microorganisms drive alternative respiratory pathways such as denitrification. In this paper, denitrification in haloarchaea has been studied from a phylogenetic point of view. It has been demonstrated that the presence of denitrification enzymes is a quite common characteristic in Halobacteria class, being nitrite reductase and nitric oxide reductase the enzymes with higher co-occurrence, maybe due to their possible role not only in denitrification, but also in detoxification. Moreover, copper-nitrite reductase (NirK) is the only class of respiratory nitrite reductase detected in these microorganisms up to date. The distribution of this alternative respiratory pathway and their enzymes among the families of haloarchaea has also been discussed and related with the environment in which they constitute the major populations. Complete denitrification phenotype is more common in some families like Haloarculaceae and Haloferacaceae, whilst less common in families such as Natrialbaceae and Halorubraceae.

Resequencing and transcriptomic analysis reveal differences in nitrite reductase in jujube fruit (Ziziphus jujuba Mill.)

Background Jujube is a typical fruit tree species from China. ‘Muzao’, a cracking-susceptible cultivar, and ‘Linhuang No. 1’, a cracking-resistant cultivar, were selected in a previous study as contrasting research materials. Whole-genome resequencing and transcriptomic analysis of ‘Linhuang No. 1’ and ‘Muzao’ allowed the identification of differentially expressed genes with different gene structures between the two cultivars and could be helpful in explaining the differences and similarities between the two cultivars. Results Resequencing identified 664,129 polymorphic variable sites between ‘Linhuang No. 1’ and ‘Muzao’. To determine the genetic relationship among ‘Linhuang No. 1’, ‘Muzao’ and the jujube genome reference cultivar ‘Dongzao’, the characteristic polymorphic variable sites were analysed by principal component analysis. The genetic relationship between ‘Linhuang No. 1’ and ‘Muzao’ was closer than that of either variety and ‘Dongzao’. Nineteen differentially expressed genes were identified by combining transcriptomic analysis with resequencing analysis. LOC107427052 (encoding a nitrite reductase) was identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for further study. The identified insertion was not in the domain region of the LOC107427052 gene coding sequence (CDS) region and was verified by the finding that the insertion did not affect translation of the protein. The LOC107427052 gene expression levels, nitrite reductase activities and nitrite contents of ‘Muzao’ were significantly higher than the corresponding values of ‘Linhuang No. 1’ at the young fruit stage. There was no significant difference in the quantity of the product of nitrite reductase, namely, ammonia, between the two cultivars. Conclusions The present study was the first to explore the differences between different jujube cultivars (‘Linhuang No. 1’ and ‘Muzao’) by combining genome resequencing and transcriptomics. LOC107427052 (encoding a nitrite reductase) was characterized by KEGG enrichment analysis. The insertion in the CDS region of the LOC107427052 gene provides a new direction for the study of nitrogen metabolism in jujube. Our study has laid a foundation for the comparative analysis of nitrite metabolism between the jujube cultivars ‘Linhuang No. 1’ and ‘Muzao’.