An RND (resistance-nodulation-division)-type transporter having the capacity to export free fatty acids (FFAs) was identified in the cyanobacterium Synechococcus elongatus strain PCC 7942 during characterization of a mutant strain engineered to produce FFAs. The basic strategy for construction of the FFA-producing mutant was a commonly used one, involving inactivation of the endogenous acyl-acyl carrier protein synthetase gene (aas) and introduction of a foreign thioesterase gene ('tesA), but a nitrate transport mutant NA3 was used as the parental strain to achieve slow, nitrate-limited growth in batch cultures. Also, a nitrogen-regulated promoter PnirA was used to drive 'tesA to maximize thioesterase expression during the nitrate-limited growth. The resulting mutant (dAS2T) was, however, incapable of growth under the conditions of nitrate limitation, presumably due to toxicity associated with FFA overproduction. Incubation of the mutant culture under the non-permissive conditions allowed for isolation of a pseudorevertant (dAS2T-pr1) capable of growth on nitrate. Genome sequence and gene expression analyses of this strain suggested that expression of an RND-type efflux system had rescued growth on nitrate. Targeted inactivation of the RND-type transporter genes in the wild-type strain resulted in loss of tolerance to exogenously added FFAs including capric, lauric, myristic, oleic and linolenic acids. Overexpression of the genes in dAS2T, on the other hand, enhanced FFA excretion and cell growth in nitrate-containing medium, verifying that the genes encode an efflux pump for FFAs. These results demonstrate the importance of the efflux system in efficient FFA production using genetically engineered cyanobacteria.
Among the known functions of the P(II) protein (the glnB gene product) in the cyanobacterium Synechococcus elongatus, negative regulation of the activity of PipX, a transcriptional co-activator of the NtcA regulon, has been thought to be essential for cell viability, because all the P(II)-less mutants thus far constructed carry spontaneous mutations in pipX. PipX is thus deduced to be a toxic protein, but its toxicity has not been clearly defined because of the lack of P(II)-deficient mutants carrying wild-type pipX. In this study, we developed a method to construct a targeted P(II)-less mutant of S. elongatus without the pipX mutation and determined the contribution of PipX to the detrimental effects of P(II) deficiency. Growth defects of the mutant were severe under nitrogen-replete conditions, i.e. in the presence of ammonium, but were also apparent under nitrogen-limited conditions. Genetic analyses indicated that the growth impairment observed under the nitrogen-limited conditions is largely due to the toxicity of PipX. Some of the phenotypes observed under the nitrogen-replete conditions, including reduced pigmentation and death of most of the cells after transfer from nitrogen-limited conditions to nitrogen-replete conditions, were ascribed to the toxicity of PipX, but inactivation of pipX only partially rescued the growth defect observed in the presence of ammonium, indicating the presence of an as yet unknown P(II) function(s) required for normal growth. Effects of ammonium addition on the nitrite uptake activity of the glnB mutant revealed a new function for P(II) in regulation of the activity of the ABC-type cyanate/nitrite transporter.
Serotonin (5-hydroxytryptamine, 5-HT) is an important biogenic amine that acts as a neural circuit modulator. It is widespread in the central nervous system of insects. However, little is known about the distribution of serotonin in the nervous system of the cotton bollworm Helicoverpa armigera . In the present study, we performed immunohistochemical experiments with anti-serotonin serum to examine the distribution of serotonin in the central nervous system of H. armigera larvae. We found about 40 serotonin-immunoreactive neurons in the brain and about 20 in the gnathal ganglion. Most of these neurons are wide-field neurons giving rise to processes throughout the neuropils of the brain and the gnathal ganglion. In the central brain, serotonin-immunoreactive processes are present bilaterally in the tritocerebrum, the deutocerebrum, and major regions of the protocerebrum, including the central body (CB), lateral accessory lobes (LALs), clamps, crepine, superior protocerebrum, and lateral protocerebrum. The CB, anterior ventrolateral protocerebrum (AVLP), and posterior optic tubercle (POTU) contain extensive serotonin-immunoreactive process terminals. However, the regions of mushroom bodies, the lateral horn, and protocerebral bridges (PBs) are devoid of serotonin-immunoreactivity. In the gnathal ganglion, the serotonin-immunoreactive processes are also widespread throughout the neuropil, and some process projections extend to the tritocerebrum. Our results provide the first comprehensive description of the serotonergic neuronal network in H. armigera larvae, and they reveal the neural architecture and the distribution of neural substances, allowing us to explore the neural mechanisms of behaviors by using electrophysiological and pharmacological approaches on the target regions.
Elucidating the stoichiometry and resorption patterns of multiple nutrients is of essential importance to holistically understanding plant nutrition and biogeochemical cycling. Although many studies on ecological stoichiometry have been carried out, surprisingly few of them were simultaneously done on the investigation of both nutrient resorption efficiency and stoichiometry for different-aged plantations of a perennial tree. Here, both green and senesced leaf samples were collected from four Larix kaempfer plantations aged of 8, 15, 22, and 32 years in the Qinling Mountains to examine nutrients resorption efficiency and stoichiometry characteristics. The results suggested that the nutrient concentrations of N, P, K, Ca, Mg, Al, and Fe in both green and senesced leaves of L. kaempferi (a deciduous conifer tree) did not show a regular change trends along the plantation ages in the Qinling Mountains. The concentrations of the most nutrients examined, except for Fe, in the green leaves were relatively lower than or close to the required physiological concentrations, suggesting a relative limitation of multiple nutrients exists in L. kaempferi for its above-ground biomass growth. The rank order of resorption efficiencies of four key nutrients (N, P, K, and Mg) was K (80.89%) > N (67.42%) > P (65.34%) > Mg (41.16%), whereas the nutrient Ca and Fe tended to accumulate in senesced leaves. Overall, the nutrient resorption efficiency of all examined elements did not exhibit a regular trend corresponding to the change of the plantation ages in L. kaempferi, but it was positively related to the nutrient concentrations in green leaves. The mean C:N and C:P ratios in the green and senesced leaves were significantly higher than those reported globally (on average). By contrast, the N:P ratio, at <14, was not only much lower than that reported for both China’s flora and globally (on average), but it did suggest that the N nutrient limits growth of L. kaempferi in these plantations. Taken together, the results of this study are of substantial interest and value to forest managers and for the sustainable development of the Qinling forest ecosystems.
Attractants and repellents are considered to be an environment-friendly approach for pest management. Odorant receptors (ORs), which are located on the dendritic membranes of olfactory sensory neurons in insects, are essential genes for recognizing attractants and repellents. In the Asian corn borer, Ostrinia furnacalis, ORs that respond to sex pheromones have been characterized, but general ORs for plant odorants, especially for repellents, have not been identified. Nonanal is a plant volatile of maize that could result in avoidance of the oviposition process for female adults in O. furnacalis. In this study, we identified a female-biased OR that responds to nonanal using a Xenopus oocyte expression system. In addition, we found that OfurOR27 was also sensitive to two other compounds, octanal and 1-octanol. Behavioral analysis showed that octanal and 1-octanol also caused female avoidance of oviposition. Our results indicated that OfurOR27 is an OR that is sensitive to repellents. Moreover, the two newly identified repellents may help to develop a chemical ecology approach for pest control in O. furnacalis.
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