Moderate salinity improves the availability of soil P by regulating P-cycling microbial communities in coastal wetlands

Accelerated sea-level rise is expected to cause the salinization of freshwater wetlands, but the responses to salinity of the availability of soil phosphorus (P) and of microbial genes involved in the cycling of P remain unexplored. We conducted a field experiment to investigate the effects of salinity on P cycling by soil microbial communities and their regulatory roles on P availability in coastal freshwater and brackish wetlands. Salinity was positively correlated with P availability, with higher concentrations of labile P but lower concentrations of moderately labile P in the brackish wetland. The diversity and richness of microbial communities involved in P cycling were higher in the brackish wetland than the freshwater wetland. Salinity substantially altered the composition of the P-cycling microbial community, in which those of the brackish wetland were separated from those of the freshwater wetland. Metagenomic sequence analysis indicated that functional genes involved in the solubilization of inorganic P and the subsequent transport and regulation of P were more abundant in coastal soils. The relative abundances of most of the target genes differed between the wetlands, with higher abundances of P-solubilization (gcd and ppa) and -mineralization (phoD, phy, and ugpQ) genes and lower abundances of P-transport genes (pstB, ugpA, ugpB, ugpE, and pit) in the brackish wetland. A significant positive correlation between the concentration of labile P and the abundances of the target genes suggested that salinity may, at least in part, improve P availability by regulating the P-cycling microbial community. Our results suggest that the P-cycling microbial community abundance and P availability respond positively to moderate increases in salinity by promoting the microbial solubilization and mineralization of soil P. Changes in microbial communities and microbially mediated P cycling may represent microbial strategies to adapt to moderate salinity levels, which in turn control soil function and nutrient balance.     

Faster accumulation and greater contribution of glomalin to the soil organic carbon pool than amino sugars do under tropical coastal forest restoration

Microbial metabolic products play a vital role in maintaining ecosystem multifunctionality, such as soil physical structure and soil organic carbon (SOC) preservation. Afforestation is an effective strategy to restore degraded land. Glomalin-related soil proteins (GRSP) and amino sugars are regarded as stable microbial-derived C, and their distribution within soil aggregates affects soil structure stability and SOC sequestration. However, the information about how afforestation affects the microbial contribution to SOC pools within aggregates is poorly understood. We assessed the accumulation and contribution of GRSP and amino sugars within soil aggregates along a restoration chronosequence (Bare land, Eucalyptus exserta plantation, native species mixed forest, and native forest) in tropical coastal terraces. Amino sugars and GRSP concentrations increased, whereas their contributions to the SOC pool decreased along the restoration chronosequence. Although microaggregates harbored greater microbial abundances, amino sugars and GRSP concentrations were not significantly affected by aggregate sizes. Interestingly, the contributions of amino sugars and GRSP to SOC pools decreased with decreasing aggregate size which might be associated with increased accumulation of plant-derived C. However, the relative change rate of GRSP was consistently greater in all restoration chronosequences than that of amino sugars. The accumulation of GRSP and amino sugars in SOC pools was closely associated with the dynamics of soil fertility and the microbial community. Our findings suggest that GRSP accumulates faster and contributes more to SOC pools during restoration than amino sugars did which was greatly affected by aggregate sizes. Afforestation substantially enhanced soil quality with native forest comprising species sequestering more SOC than the monoculture plantation did. Such information is invaluable for improving our mechanistic understanding of microbial control over SOC preservation during degraded ecosystem restoration. Our findings also show that plantations using arbuscular mycorrhizal plants can be an effective practice to sequester more soil carbon during restoration.     

The start of frozen dates over northern permafrost regions with the changing climate

The soil freeze–thaw cycle in the permafrost regions has a significant impact on regional surface energy and water balance. Although increasing efforts have been made to understand the responses of spring thawing to climate change, the mechanisms controlling the global interannual variability of the start date of permafrost frozen (SOF) remain unclear. Using long-term SOF from the combinations of multiple satellite microwave sensors between 1979 and 2020, and analytical techniques, including partial correlation, ridge regression, path analysis, and machine learning, we explored the responses of SOF to multiple climate change factors, including warming (surface and air temperature), start date of permafrost thawing (SOT), soil properties (soil temperature and volume of water), and the snow depth water equivalent (SDWE). Overall, climate warming exhibited the maximum control on SOF, but SOT in spring was also an important driver of SOF variability; among the 65.9% significant SOT and SOF correlations, 79.3% were positive, indicating an overall earlier thawing would contribute to an earlier frozen in winter. The machine learning analysis also suggested that apart from warming, SOT ranked as the second most important determinant of SOF. Therefore, we identified the mechanism responsible for the SOT–SOF relationship using the SEM analysis, which revealed that soil temperature change exhibited the maximum effect on this relationship, irrespective of the permafrost type. Finally, we analyzed the temporal changes in these responses using the moving window approach and found increased effect of soil warming on SOF. In conclusion, these results provide important insights into understanding and predicting SOF variations with future climate change.     

Lipopolysaccharide (LPS) increases thein vivo oxidation of branched-chain amino acids in the rat: A cytokine-mediated effect

Septic rats (as induced by cecal puncture and ligation) showed an increased rate ofin vivo leucine oxidation as measured from the formation of¹⁴CO₂ from an intravenously injected [1-¹⁴C]leucine tracer dose. Acute lipopolysaccharide (LPS) administration (1 mg/kg) to rats caused a similar effect on the rate ofin vivo leucine oxidation. Additionally, both tumour necrosis factor- (TNF) and interleukin-1- (IL-1), in an acute dose of 100 g/kg, also increased the rate of the oxidation of the amino acid, although only IL-1 caused a similar increase to that observed following LPS. The observed increased leucine oxidation was related to lower leucine concentrations both in LPS- and cytokine-treated rats. Important decreases were also observed in the other branched-chain amino acids (valine and isoleucine) in the LPS- and IL-1-treated animals. Isolated incubated muscles from TNF- and IL-1-treated rats did not show any changes in the rate of leucine utilization, thus suggesting that the mechanism by which the cytokines stimulate whole-body leucine oxidation is not based on an increase in the activity of the enzymatic machinery responsible for leucine oxidation. Additionally, glucocorticoids do not seem to mediate the enhancedin vivo oxidation of the amino acid since, although they are increased by both LPS and cytokines, treatment of the animals with RU486 (a glucocorticoid antagonist) was not able to suppress the effects of the cytokine onin vivo leucine oxidation.     

Differential Effects of Ipsapirone on 5-Hydroxytryptamine Release in the Dorsal and Median Raphe Neuronal Pathways

Abstract: Serotonergic neurons of the dorsal and median raphe nuclei are morphologically dissimilar. Recent results challenge previous evidence indicating a greater inhibition of dorsal raphe neurons after 5-hydroxytryptamine_1A (5-HT_1A) autoreceptor activation. As both nuclei innervate different forebrain territories, this issue is critical to understanding the changes in brain function induced by anxiolytic and antidepressant drugs. Using microdialysis, we examined the modifications of 5-HT release induced by the selective 5-HT_1A agonist ipsapirone in both neuronal pathways. Maximal and minimal basal 5-HT values (in the presence of 1 µ M citalopram) were 45.0 ± 4.8 fmol/fraction in the median raphe nucleus and 8.4 ± 0.4 fmol/fraction in the dorsal hippocampus. Ipsapirone (0.3, 3, and 10 mg/kg s.c.) reduced dose-dependently 5-HT in the two raphe nuclei and four forebrain areas. Maximal reductions (to ∼25% of predrug values) were observed in cortex and striatum and in median raphe nucleus. The effects were more moderate in dorsal and ventral hippocampus (to 66 and 50% of baseline, respectively). These results are consistent with a higher sensitivity of dorsal raphe neurons to 5-HT_1A autoreceptor activation. Yet the differential reduction of 5-HT release in the median raphe nucleus and hippocampus suggests the presence of complex mechanisms of control of 5-HT release in these neurons.     

Glucose handling by hepatocytes from obese Zucker rats

Hepatocytes isolated from obese Zucker rats showed a significantly higher rate of both [U-¹⁴C]glucose and [U-¹⁴C]lactate incorporation into [¹⁴C]lipid than those from their lean counterparts. This was associated with a marked increase in the lipogenic rate measured by the incorporation of³H₂O into the cell esterified fatty acids. Although there were no changes in the incorporation of the tracer into either [¹⁴C]glycogen or¹⁴CO₂, the [¹⁴C] total uptake was significantly higher in the obese animals. The high rate of [¹⁴C]lipid synthesis from glucose was observed both at 15 and 30 mM substrate concentrations and was linked to an enhanced uptake of the tracer into the cell as measured using the decarboxilation of [1-¹⁴C]glucose in the presence of phenazine methosulphate. The presence of insulin in the incubation medium had no effect on the uptake of glucose by the liver cells. However, the large uptake of glucose by the hepatocytes from the obese animals was not related to an enhanced rate of transport as measured using 3-O-methyl[U-¹⁴C]glucose. The activity of glucose-6-phosphate dehydrogenase together with a higher [1-¹⁴C]glucose/[U-¹⁴C]glucose descarboxylation ratio indicate a predominant very active pentose phosphate pathway which may be responsible for the enhanced glucose uptake observed in the hepatocytes from the obese animals.     

Mathematical modelling of the urea cycle. A numerical investigation into substrate channelling.

Metabolite channelling, the process in which consecutive enzymes have confined substrate transfer in metabolic pathways, has been proposed as a biochemical mechanism that has evolved because it enhances catalytic rates and protects unstable intermediates. Results from experiments on the synthesis of radioactive urea [Cheung, C., Cohen, N.S. & Raijman, L (1989) J. Biol. Chem.264, 4038-4044] have been interpreted as implying channelling of arginine between argininosuccinate lyase and arginase in permeabilized hepatocytes. To investigate this interpretation further, a mathematical model of the urea cycle was written, using Mathematica it simulates time courses of the reactions. The model includes all relevant intermediates, peripheral metabolites, and subcellular compartmentalization. Analysis of the output from the simulations supports the argument for a high degree of, but not absolute, channelling and offers insights for future experiments that could shed more light on the quantitative aspects of this phenomenon in the urea cycle and other pathways.     

In the rat, tumor necrosis factor α administration results in an increase in both UCP2 and UCP3 mRNAs in skeletal muscle: a possible mechanism for cytokine-induced thermogenesis?

Since the discovery of the new members of the UCP (uncoupling protein) family, UCP2 and UCP3, very few studies have dealt with the regulation of their expression. Bearing this in mind, administration of a single intravenous injection of TNF-α (100 μg/kg body weight) to rats resulted in a significant increase in UCP2 (242%) and UCP3 (113%) gene expression in skeletal muscle. The results suggest a possible role for UCP2 and UCP3 in the increase of energy expenditure associated with cytokine treatment.     

Cell Division Inhibition in Salmonella typhimurium Histidine-Constitutive Strains: an ftsI-Like Defect in the Presence of Wild-Type Penicillin-Binding Protein 3 Levels

Histidine-constitutive (His^c) strains of Salmonella typhimurium undergo cell division inhibition in the presence of high concentrations of a metabolizable carbon source. Filaments formed by His^c strains show constrictions and contain evenly spaced nucleoids, suggesting a defect in septum formation. Inhibitors of penicillin-binding protein 3 (PBP3) induce a filamentation pattern identical to that of His^c strains. However, the His^c septation defect is caused neither by reduced PBP3 synthesis nor by reduced PBP3 activity. Gross modifications of peptidoglycan composition are also ruled out. d-Cycloserine, an inhibitor of the soluble pathway producing peptidoglycan precursors, causes phenotypic suppression of filamentation, suggesting that the septation defect of His^c strains may be caused by scarcity of PBP3 substrate.When histidine-constitutive (His^c) mutants of Salmonella typhimurium were first isolated, the authors noted that high levels of histidine biosynthetic enzymes caused wrinkled colony morphology on 2% glucose plates (25). Wrinkledness reflects cell filamentation (12, 19), which is triggered by overproduction of hisH and hisF gene products (5, 9, 19). A similar response has been described for Escherichia coli (11). HisH and HisF are subunits of the heterodimeric imidazole-glycerol-phosphate synthase (1, 34), which catalyzes the formation of imidazole-glycerol-phosphate (IGP) with release of the purine precursor AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) (17, 26). However, division inhibition does not require metabolic flow through the histidine biosynthetic pathway, suggesting that HisH and HisF trigger filamentation through an activity unrelated to IGP synthesis (10, 19). The involvement of AICAR has been also ruled out (10, 11). In both S. typhimurium and E. coli, the cell division defect of His^c strains is unrelated to the SOS response and does not involve the cell division inhibitor SulA (11, 12). We show below that the cell division defect of S. typhimurium His^c strains is a block in septum formation, as proposed by Frandsen and D’Ari (11). We also describe the unexpected finding that strains that overproduce IGP synthase contain wild-type levels of active penicillin-binding protein 3 (PBP3). These contradictory data are tentatively reconciled by the ability of d-cycloserine to suppress filamentation in His^c mutants. The latter observation suggests that HisHF overproduction may cause a shortage in PBP3 substrate.

Filament formation by His^c strains.

Mid-exponential-phase cultures of strains LT2 (hisO⁺) and TR6753 (hisO1242 [5, 16]) were observed under the microscope by using Hiraga’s fluo-phase combined method, a procedure that permits the simultaneous observation of nucleoids and cells (15). Nucleoid staining was achieved with DAPI (4′,6-diamino-2-phenylindole). The His^c strain formed long filaments which contained evenly spaced nucleoids, indicating that their division defect is unrelated to DNA synthesis or chromosome partition (Fig. ​(Fig.1).1). Moreover, the presence of blunt constrictions indicates that the division block lies beyond the stage of FtsZ action (8). The filaments are similar in morphology and length to those formed by ftsI and ftsA mutants of E. coli (8). The ftsI gene encodes PBP3, an essential cell division protein involved in septum formation (27). FtsA is a membrane-bound protein that interacts with PBP3 (22, 31). A difference is that ftsI and ftsA mutants are conditional (thermosensitive) lethals unable to form colonies under restrictive conditions (3, 18), while the filaments produced by His^c strains of S. typhimurium in the presence of 2% glucose give rise to colonies which are distinctly wrinkled (9, 19, 25). Open in a separate windowFIG. 1Microscopic photographs of cells and filaments prepared with Hiraga’s fluo-phase combined method (15). (A) Filaments formed by the His^c strain TR6753 grown in E medium containing 2% glucose. (B) An isogenic His⁺ strain (LT2) grown under the same conditions does not form filaments. (C) Addition of aztreonam to a culture of strain LT2 in E medium induces filaments identical to those formed by a His^c strain in high-glucose medium (compare panels A and C). Bar, 5 μm.Antibiotics that inhibit PBP3, such as aztreonam and azlocillin, produce phenocopies of ftsI mutants in E. coli (29). Based on this precedent, we investigated whether PBP3 inhibitors were able to reproduce the filamentation phenotype of His^c strains. Addition of aztreonam (1 mg/liter) to a culture of strain LT2 triggered filament formation (Fig. ​(Fig.1C),1C), and the filaments were identical in morphology and length to those formed by the His^c strain TR6753 (Fig. ​(Fig.1A).1A). The same effect was induced by azlocillin (data not shown). At the concentrations used, these antibiotics inhibit specifically PBP3 (21). These observations suggest that His^c strains of S. typhimurium behave as ftsI mutants.Additional evidence against an FtsA-like defect was provided by the failure of a plasmid carrying the E. coli ftsA gene to relieve cell division inhibition when introduced in strain TR6753. If His^c strains were ftsA-like, a plasmid-borne ftsA gene should restore the FtsA/FtsZ ratio (6), thereby causing a certain degree of suppression. However, the actual result was that pMFV26, an ftsA⁺ plasmid provided by Miguel Vicente (CIB-CSIC, Madrid, Spain), failed to relieve septation inhibition. The conclusion that His^c strains behave as ftsI (rather than ftsA) mutants receives further support from the ability of d-cycloserine to suppress filamentation (see below).

HisHF overproduction does not cause reduced synthesis of PBP3.

The levels of PBP3 produced by HisO⁺ and HisO^c strains were compared by using envelope extracts from mid-exponential-phase cultures (optical density at 600 nm, 0.5 to 0.6) in E medium containing 2% glucose (33). Envelope proteins were fractionated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (8% acrylamide) and detected by immunoblotting against a polyclonal anti-PBP3 serum (21). The levels of PBP3 protein are similar in HisO⁺ and HisO^c strains (Fig. ​(Fig.2);2); densitometric analysis (not shown) confirmed the absence of differences. Thus, the cell division defect associated with IGP synthase overproduction is not caused by reduced synthesis of PBP3. An additional observation is that the electrophoretic mobilities of PBP3 are similar in HisO⁺ and HisO^c strains, thereby eliminating the occurrence of gross structural changes in the protein. Open in a separate windowFIG. 2Immunodetection of PBP3 from the wild type (lanes A and C) and the His^c strain TR6753 (lanes B and D). Lanes A and B contain membrane vesicles corresponding to 80 μg of protein in a final reaction volume of 20 μl. Lanes C and D contain membrane vesicles corresponding to 120 μg of protein in a final volume of 20 μl.

HisHF overproduction does not cause reduced activity of PBP3.

As an indicator of the functionality of PBP3 in membrane extracts from HisO⁺ and HisO^c strains, we analyzed their capacities to bind covalently ³H-labelled benzylpenicillin (2). Cultures were prepared as described above. Protein separation was performed on an SDS-polyacrylamide gel (2). Radioactivity was detected by fluorography (28, 30). Protein quantitation was performed by the method of Bradford (4). No differences in penicillin binding were found (Fig. ​(Fig.33 and densitometric data not shown). A side observation is that the remaining high-molecular-weight PBPs were also unaffected (Fig. ​(Fig.3).3). Open in a separate windowFIG. 3Binding of ³H-labelled benzylpenicillin to cell envelopes from exponentially growing cells of the His⁺ strain LT2 (lane I) and the His^c strain TR6753 (lane II). PBPs are numbered on the right by standard nomenclature (27).

Peptidoglycan composition of His^c strains.

To investigate the possibility that His^c strains might synthesize an abnormal cell wall, we prepared peptidoglycan extracts from strains LT2 and TR6753 grown in E medium containing 2% glucose. Concentrated exponential cultures containing approximately 10¹¹ bacterial cells were cooled to 4°C, centrifuged at 12,000 × g for 15 min, and resuspended in 3 ml of phosphate-buffered saline, pH 7.4. The suspension was mixed 1:1 (vol/vol) with a boiling solution of 8% SDS (24). The SDS-insoluble material was washed in distilled water (13). Peptidoglycan was digested with Cellosyl muramidase (20 μg/ml) (Hoechst, Sommerville, N.J.); this treatment yields muropeptides of low molecular weight (23). The reaction was stopped in a boiling bath for 5 min. Insoluble material was removed by centrifugation (1,000 × g, 10 min). Peptidoglycan composition was determined by high-performance liquid chromatography analysis (13, 14). The main conclusions are that (i) abnormal peptidoglycan muropeptides were not found and (ii) gross differences in peptidoglycan composition or organization were not found between HisO⁺ and HisO^c strains (Table ​(Table1).1). These experiments do not rule out the formation of an abnormal PBP3 substrate but certainly exclude the possibility that any unusual substrate is incorporated into growing peptidoglycan chains. This absence of differences does not eliminate the possibility that His^c strains may have a defect in the reaction catalyzed by PBP3; peptidoglycan from strain LT2 treated with the PBP3 inhibitor aztreonam showed also standard composition (Table ​(Table1).1). In fact, a well-known and surprising feature of cell wall synthesis is that the global peptidoglycan composition remains unaltered under conditions that cause major changes in cell shape (20).

TABLE 1

Muropeptide composition of peptidoglycan from His⁺ and His^c strains and from a His⁺ strain treated with aztreonam

The Salmonella typbimurium RecJ function permits growth of P22 abc phage on recBCD+ hosts

Summary We describe recJ mutants of Salmonella typhimurium. The recJ gene maps between sufD and serA (min 62) and is transcribed counterclockwise. Unlike recJ mutants of Escherichia coli, recJ strains of S. typhimurium are sensitive to irradiation with UV light. This sensitivity is equivalent to or greater that that displayed by recBCD mutant strains. The residual ability of phage P22 abc (anti-recBCD) mutants to form plaques on recBCD ⁺ strains is eliminated in recJ hosts. Thus host RecJ function appears to substitute for the anti-RecBCD functions of phage P22 and may serve to limit RecBCD activity.