Analysis of the Plasmid-Based ts-Mutant ΔfabA/pTS-fabA Reveals Its Lethality under Aerobic Growth Conditions That Is Suppressed by Mild Overexpression of desA at a Restrictive Temperature in Pseudomonas aeruginosa.

It is uncertain whether PA1610|fabA is essential or dispensable for growth on LB-agar plates under aerobic conditions in Pseudomonas aeruginosa PAO1. To examine its essentiality, we disrupted fabA in the presence of a native promoter-controlled complementary copy on ts-plasmid. In this analysis, we showed that the plasmid-based ts-mutant ΔfabA/pTS-fabA failed to grow at a restrictive temperature, consistent with the observation by Hoang and Schweizer (T. T. Hoang, H. P. Schweizer, J Bacteriol 179:5326-5332, 1997, https://doi.org/10.1128/jb.179.17.5326-5332.1997), and expanded on this by showing that ΔfabA exhibited curved cell morphology. On the other hand, strong induction of fabA-OE or PA3645|fabZ-OE impeded the growth of cells displaying oval morphology. Suppressor analysis revealed a mutant sup gene that suppressed a growth defect but not cell morphology of ΔfabA. Genome resequencing and transcriptomic profiling of sup identified PA0286|desA, whose promoter carried a single-nucleotide polymorphism (SNP), and transcription was significantly upregulated (level increase of >2-fold, P < 0.05). By integration of the SNP-bearing promoter-controlled desA gene into the chromosome of ΔfabA/pTS-fabA, we showed that the SNP is sufficient for ΔfabA to phenocopy the sup mutant. Furthermore, mild induction of the araC-PBAD-controlled desA gene but not desB rescued ΔfabA. These results validated that mild overexpression of desA fully suppressed the lethality but not the curved cell morphology of ΔfabA. Similarly, Zhu et al. (Zhu K, Choi K-H, Schweizer HP, Rock CO, Zhang Y-M, Mol Microbiol 60:260-273, 2006, https://doi.org/10.1111/j.1365-2958.2006.05088.x) showed that multicopy desA partially alleviated the slow growth phenotype of ΔfabA, the difference in which was that ΔfabA was viable. Taken together, our results demonstrate that fabA is essential for aerobic growth. We propose that the plasmid-based ts-allele is useful for exploring the genetic suppression interaction of essential genes of interest in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen whose multidrug resistance demands new drug development. Fatty acids are essential for viability, and essential genes are ideal drug targets. However, the growth defect of essential gene mutants can be suppressed. Suppressors tend to be accumulated during the construction of essential gene deletion mutants, hampering the genetic analysis. To circumvent this issue, we constructed a deletion allele of fabA in the presence of a native promoter-controlled complementary copy in the ts-plasmid. In this analysis, we showed that ΔfabA/pTS-fabA failed to grow at a restrictive temperature, supporting its essentiality. Suppressor analysis revealed desA, whose promoter carried a SNP and whose transcription was upregulated. We validated that both the SNP-bearing promoter-controlled and regulable PBAD promoter-controlled desA suppressed the lethality of ΔfabA. Together, our results demonstrate that fabA is essential for aerobic growth. We propose that plasmid-based ts-alleles are suitable for genetic analysis of essential genes of interest.

Tracing phosphorus cycle in global watershed using phosphate oxygen isotopes.

The Phosphorus (P) cycle is a crucial biochemical process in the earth system. However, an extensive increase of P input into watersheds destroyed the ecosystem. To explore the effects of internal P loading and external P input in global watersheds, we reviewed the research progress and synthesized the isotope data of experimental results from literatures. An integrated result of the observational and experimental studies revealed that both internal P and external P largely contribute to watershed P loadings in watersheds. Internal P can be released to the overlying water during sediment resuspension process and change of redox conditions near the sediment-water interface. Growing fertilizer application on farmlands to meet food demand with population rise and diet improvement contributed to an huge increase of external P input to watersheds. Therefore, water quality cannot be improved by only reducing internal P or external P loadings. In addition, we found that phosphate oxygen isotope technology is an effectively way to trace the P biogeochemical cycle in watersheds. To better predict the dynamic of P in watersheds, future research integrating oxygen isotope fractionation mechanisms and phosphate oxygen isotope technology would be more effective.

Phosphorus fractions and oxygen isotope composition of inorganic phosphate in typical agricultural soils.

Phosphorus (P), despite being an essential nutrient element for plants growth in agricultural ecosystem, the low utilization rate of soil P and the environmental problems caused by soil P losses are serious. Therefore, scoping knowledge of the possible sources and utilization extent of soil P by microorganisms is very helpful for better understanding of promoting P utilization for sustainable agriculture. Oxygen isotope of phosphate technology is an effective tool to trace the sources of P. In this study, P contents and oxygen isotope composition of inorganic phosphate (δ18OP) of different pools (H2O-P, NaHCO3-P, NaOH-P, and HCl-P) in typical agricultural soil from Northeast China and Central China were analyzed and quantified. The results showed that fertilizer and land use were important factors influencing the contents of H2O-Pt and NaHCO3-Pt and the soil TP contents from different types of soils were greatly affected by soil weathering degree. The δ18OP of different P pools indicated that the difference in utilization extent of different P fractions by microorganisms and the δ18OP values of different P fractions could be due to accumulation of multiple factors. The results will provide effective information for further study on sources and effective utilization of different P fractions in soil.

Impact of Fish Farming on Phosphorus in Reservoir Sediments.

Fish farming has seriously influenced the aquatic environment in Sancha reservoir in SW China since 1985 and has been strongly restricted since 2005. Thus, phosphorus speciation in a sediment core dated between 1945 and 2010 at cm-resolution and in surface sediments from Sancha reservoir may allow us track how fish farming impacts phosphorus dynamics in lake sediments. Fish farming shifts the major binding forms of phosphorus in sediments from organic to residual phosphorus, which mostly originated from fish feed. Sorption to metal oxides and association with organic matters are important mechanisms for phosphorus immobilisation with low fish farming activities, whereas calcium-bound phosphorous had an essential contribution to sediment phosphorus increases under intensive fish framing. Notwithstanding the shifting, the aforementioned phosphorus fractions are usually inert in the lake environment, therefore changing phosphorus mobility little. The use of fish feed and water-purification reagents, the most important additives for fish farming, introduce not only phosphorus but also large amounts of sand-sized minerals such as quartz into the lake, to which phosphorus weakly sorbs. The sand-sized minerals as additional sorbents increase the pool of easily mobilisable phosphorus in sediments, which will slow down the recovery of reservoir water due to its rapid re-mobilisation.

Uptake and speciation of vanadium in the rhizosphere soils of rape (Brassica juncea L.).

The response of rape (Brassica juncea L.) to different vanadium (V) speciation in rhizosphere soils was investigated in pot experiments using an agricultural soil containing 147 mg V kg(-1) supplemented with 0-500 mg V kg(-1) of pentavalent V [V(V)] and a mining soil containing 774 mg V kg(-1). Tetravalent V [V(IV)] accounted for 76.1 and 85.9 % of total V in the untreated agricultural soil and mining soil, respectively. The proportion of both V(V) and water-extractable V increased with increasing concentrations of V(V) in the agricultural soil. The growth of rape substantially reduced the concentrations of V(V) but not V(IV) in the rhizosphere soil, suggesting that V(V) was actively involved in the soil-rape interaction of V. Both soil V(V) and water-extractable V were negatively related to the total rape biomass, but were positively correlated with the concentration of root V. No such relationships were found for total V and soil V(IV). Together, these results indicate that soil V(V) and water-extractable V might better reflect the toxicity of V in soils than total V and soil V(IV). Rape accumulated V in the sequence: roots > > stem > leaf > seed. As indicated by the remarkably low root bioconcentration factor of V(V) (0.41-7.24 %), rape had a lower ability to accumulate V than other plants reported in the literature (14.6-298 %). Only a small fraction of V in rape roots was translocated to the aboveground organs (the translocation factor was 3.57-46.9 %). No V was detectable in seeds in the soils at 147 and 197 mg V kg(-1), and no seed was produced in the soils at higher V concentrations. Thus, the risk of V intake by humans via the consumption of rapeseed-based foods under normal conditions is considered to be lower than that of other plants.