Nitrate regulation of anaerobic respiratory gene expression in narX deletion mutants of Escherichia coli K-12.

Previous studies have shown that narL+ is required for nitrate regulation of anaerobic respiratory enzyme synthesis, including formate dehydrogenase-N, nitrate reductase, and fumarate reductase. Insertions in the closely linked narX gene decrease, but do not abolish, nitrate regulation of anaerobic enzyme synthesis. Analysis of sequence similarities suggests that NarX and NarL comprise a two-component regulatory pair. We constructed lacZ operon and gene fusions to investigate the operon structure of narXL. We found evidence for a complex operon with at least two promoters; PXL-narX-PL-narL. We also investigated the role of NarX in nitrate regulation of anaerobic respiratory enzyme synthesis by constructing nonpolar loss of function narX alleles. These deletions were studied on narL+ lambda specialized transducing bacteriophage. The narX deletions had no effect on nitrate regulation in delta (narXL) strains. This finding suggest that the subtle effects of previously studied narX insertions are due to decreased expression of narL and that narX+ is not essential for normal nitrate regulation. The role of NarX in nitrate regulation remains to be determined.     

Identification and characterization of narQ, a second nitrate sensor for nitrate-dependent gene regulation in Escherichia coli

In response to nitrate availability, Escherichia coli regulates the synthesis of a number of enzymes involved in anaerobic respiration and fermentation. When nitrate is present, nitrate reductase (narGHJI) gene expression is induced, while expression of the DMSO/TMAO reductase (dmsABC), fumarate reductase (frdABCD) and fermentation related genes are repressed. The narL and narX gene products are required for this nitrate-dependent control, and apparently function as members of a two-component regulatory system. NarX is a presumed sensor-transmitter for nitrate and possibly molybdenum detection. The presumed response-regulator, NarL, when activated by NarX then binds at the regulatory DNA sites of genes to modulate their expression. In this study a third nitrate regulatory gene, narQ, was identified that also participates in nitrate-dependent gene regulation. Strains defective in either narQ or narX alone exhibited no nitrate-dependent phenotype whereas mutants defective in both narQ and narX were fully inactive for nitrate-dependent repression or activation. In all conditions tested, this regulation required a functional narL gene product. These findings suggest that the narX and narQ products have complementary sensor-transmitter functions for nitrate detection, and can work independently to activate NarL, for eliciting nitrate-dependent regulation of anaerobic electron transport and fermentation functions. The narQ gene was cloned, sequenced, and compared with the narX gene. Both gene products are similar in size, hydrophobicity, and sequence, and contain a highly conserved histidine residue common to sensor-transmitter proteins.     

Intracellular pH and the metabolic status of dormant and developing Artemia embryos

Encysted embryos of the brine shrimp Artemia salina have long been known to enter profound dormancy under anaerobic conditions. Utilizing in vivo³¹P nuclear magnetic resonance, we show that the reversible transitions between anaerobic dormancy and aerobic development are accompanied by large (>1 unit) intracellular pH changes, the more acid pH being associated with the dormant state. Furthermore, dormant cyst intracellular pH is independent of that of the buffer, suggesting the potential for pH-mediated regulation of enzyme activities during dormancy. An example concerning cyst nucleotide metabolism is discussed.     

Mycobacterial Stationary Phase Induced by Low Oxygen Tension: Cell Wall Thickening and Localization of the 16-Kilodalton α-Crystallin Homolog

Most cases of tuberculosis are due to reactivation of endogenous infection which may have lain quiescent or dormant for decades. How Mycobacterium tuberculosis survives for this length of time is unknown, but it is hypothesized that reduced oxygen tension may trigger the tubercle bacillus to enter a state of dormancy. Mycobacterium bovis BCG and M. tuberculosis H37Rv were cultured under aerobic, microaerobic, and anaerobic conditions. Their ultrastructural morphology was analyzed by transmission electron microscopy (TEM), and protein expression profiles were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). TEM revealed that the microaerobically and anaerobically cultured bacilli but not the aerobically cultured bacilli developed a strikingly thickened cell wall outer layer. The thickening was not observed in aerobically cultured stationary-phase bacilli or in anaerobically cultured Mycobacterium smegmatis. A highly expressed protein was detected by SDS-PAGE in microaerobic and anaerobic cultures and was identified as the 16-kDa small heat shock protein or α-crystallin homolog. Immunolocalization by colloidal gold immunoelectron microscopy identified three patterns of protein distribution in M. bovis BCG cultured under low oxygen tension. The 16-kDa protein was strongly associated with the cell envelope, fibrous peptidoglycan-like structures, and intracellular and peripheral clusters. These results suggest that tubercle bacilli may adapt to low-oxygen conditions by developing a thickened cell wall and that the 16-kDa protein may play a role in stabilizing cell structures during long-term survival, thus helping the bacilli survive the low oxygen tension in granulomas. As such, the cell wall thickening and the 16-kDa protein may be markers for the dormant state of M. tuberculosis.     

A simple whole cell based high throughput screening protocol using Mycobacterium bovis BCG for inhibitors against dormant and active tubercle bacilli

This study aimed at developing a whole cell based high throughput screening protocol to identify inhibitors against both active and dormant tubercle bacilli. A respiratory type of nitrate reductase (NarGHJI), which was induced during dormancy, could reflect the viability of dormant bacilli of Mycobacterium bovis BCG in microplate adopted model of in vitro dormancy. Correlation between reduction in viability and nitrate reductase activity was seen clearly when dormant stage inhibitor metronidazole and itaconic anhydride were applied in this in vitro microplate model. Active replicating stage could also be monitored in the same assay by measuring the A(620) of the culture. MIC values of 0.08, 0.075, 0.3 and 3.0 microg/ml, determined through monitoring A(620) in this assay for rifampin, isoniazid, streptomycin and ethambutol respectively, were well in agreement with previously reported by BACTEC and Bio-Siv assays. S/N ratio and Z' factor for the assay were 8.5 and 0.81 respectively which indicated the robustness of the protocol. Altogether the assay provides an easy, inexpensive, rapid, robust and high content screening tool to search novel antitubercular molecules against both active and dormant bacilli.     

Expression of beta-tubulin during dormancy induction and release in apical and axillary buds of five woody species

Cell cycle activity was studied in apical and axillary buds of Norway maple ( Acer platanoides L.), apple ( Malus ' M9 ') , pedunculate oak ( Quercus robur L.), Scots pine ( Pinus sylvestris L.) and rose ( Rosa corymbifera 'Laxa') during dormancy induction and release. Flow cytometric analyses revealed that in dormant buds, cells mainly were quiescent at the G₀/G₁ phase, while in non-dormant buds, a significantly higher frequency of G₂ cells was found in all species. In western blots accumulation of 55 kDa beta -tubulin was found in active growing plant material, whereas in dormant buds the accumulation was much lower or below detection level. It was observed for all species that during dormancy induction the amount of beta -tubulin decreased, while during dormancy release a fast accumulation of beta -tubulin occurred. The dynamics of the beta -tubulin accumulation reflected the dormancy status of tree buds of the five species studied suggesting that the beta -tubulin level might be useful as a marker for the dormancy status in buds of temperate woody species.     

A QTL located on chromosome 4A associated with dormancy in white- and red-grained wheats of diverse origin

Improved resistance to preharvest sprouting in modern bread wheat (Triticum aestivum. L.) can be achieved via the introgression of grain dormancy and would reduce both the incidence and severity of damage due to unfavourable weather at harvest. The dormancy phenotype is strongly influenced by environmental factors making selection difficult and time consuming and this trait an obvious candidate for marker assisted selection. A highly significant Quantitative Trait Locus (QTL) associated with grain dormancy and located on chromosome 4A was identified in three bread wheat genotypes, two white- and one red-grained, of diverse origin. Flanking SSR markers on either side of the putative dormancy gene were identified and validated in an additional population involving one of the dormant genotypes. Genotypes containing the 4A QTL varied in dormancy phenotype from dormant to intermediate dormant. Based on a comparison between dormant red- and white-grained genotypes, together with a white-grained mutant derived from the red-grained genotype, it is concluded that the 4A QTL is a critical component of dormancy; associated with at least an intermediate dormancy on its own and a dormant phenotype when combined with the R gene in the red-grained genotype and as yet unidentified gene(s) in the white-grained genotypes. These additional genes appeared to be different in AUS1408 and SW95-50213.     

Proteins of Mycobacterium bovis BCG induced in the Wayne dormancy model

Oxygen starvation triggers the shiftdown of the obligate aerobe Mycobacterium bovis BCG to a state of dormancy. Two-dimensional electrophoresis showed a drastic up-regulation of the alpha-crystallin homolog, the putative response regulator Rv3133c, and the two conserved hypothetical proteins Rv2623 and Rv2626c in dormant bacilli.     

Differentially expressed genes associated with dormancy or germination of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> seeds

Differential display analysis using dormant and non-dormant Arabidopsis thaliana (L.) Heynh seeds resulted in a set of genes that were associated with either dormancy or germination. Expression of the germination-associated genes AtRPL36B and AtRPL27B, encoding two ribosomal proteins, was undetectable in the dry seed, low in dormant seed, and high under conditions that allowed completion of germination. Expression of these genes was also found to be light-regulated and to correlate with germination speed. Expression of the dormancy-associated genes ATS2 and ATS4, encoding a caleosin-like protein and a protein similar to a low-temperature-induced protein respectively, was high in the dry seed and decreased during germination. Expression of ATS2 and ATS4 was high in primary and secondary dormant seed but low in after-ripened or chilled seed. The expression of both genes was also light-regulated, but no relationship with temperature-dependent germination speed was found.