Zhou Wang

Abstract Soil bacterial communities are pivotal in regulating terrestrial biogeochemical cycles and ecosystem functions. The increase in global nitrogen (N) deposition has impacted various aspects of terrestrial ecosystems, but we still have a rudimentary understanding of whether there is a threshold for N input level beyond which soil bacterial communities will experience critical transitions. Using high‐throughput sequencing of the 16S rRNA gene, we examined soil bacterial responses to a long‐term (13 yr), multi‐level, N addition experiment in a temperate steppe of northern China. We found that plant diversity decreased in a linear fashion with increasing N addition. However, bacterial diversity responded nonlinearly to N addition, such that it was unaffected by N input below 16 g N·m −2 ·yr −1 , but decreased substantially when N input exceeded 32 g N·m −2 ·yr −1 . A meta‐analysis across four N addition experiments in the same study region further confirmed this nonlinear response of bacterial diversity to N inputs. Substantial changes in soil bacterial community structure also occurred between N input levels of 16 to 32 g N·m −2 ·yr −1 . Further analysis revealed that the loss of soil bacterial diversity was primarily attributed to the reduction in soil pH, whereas changes in soil bacterial community were driven by the combination of increased N availability, reduced soil pH, and changes in plant community structure. In addition, we found that N addition shifted bacterial communities toward more putatively copiotrophic taxa. Overall, our study identified a threshold of N input level for bacterial diversity and community composition. The nonlinear response of bacterial diversity to N input observed in our study indicates that although bacterial communities are resistant to low levels of N input, further increase in N input could trigger a critical transition, shifting bacterial communities to a low‐diversity state.

Anthropogenic nitrogen (N) input is known to alter plant and microbial α-diversity, but how N enrichment influences β-diversity of plant and microbial communities remains poorly understood. Using a long-term multilevel N addition experiment in a temperate steppe, we show that plant, soil bacterial and fungal communities exhibited different responses in their β-diversity to N input. Plant β-diversity decreased linearly as N addition increased, as a result of increased directional environmental filtering, where soil environmental properties largely explained variation in plant β-diversity. Soil bacterial β-diversity first increased then decreased with increasing N input, which was best explained by corresponding changes in soil environmental heterogeneity. Soil fungal β-diversity, however, remained largely unchanged across the N gradient, with plant β-diversity, soil environmental properties, and heterogeneity together explaining an insignificant fraction of variation in fungal β-diversity, reflecting the importance of stochastic community assembly. Our study demonstrates the divergent effect of N enrichment on the assembly of plant, soil bacterial and fungal communities, emphasizing the need to examine closely associated fundamental components (i.e., plants and microorganisms) of ecosystems to gain a more complete understanding of ecological consequences of anthropogenic N enrichment.

Lipopolysaccharides, major molecules in the outer membrane of Gram-negative bacteria, play important roles on membrane integrity of the cell. However, how the core oligosaccharide of lipopolysaccharide affect the membrane behavior is not well understood. In this study, the relationship between the core oligosaccharide of lipopolysaccharide and the membrane behavior was investigated using a series of Escherichia coli mutants defective in genes to affect the biosynthesis of core oligosaccharide of lipopolysaccharide. Cell surface hydrophobicity, outer membrane permeability, biofilm formation and auto-aggregation of these mutant cells were compared. Compared to the wild type W3110, cell surface hydrophobicities of mutant ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaP, ΔwaaY and ΔwaaB were enhanced, outer membrane permeabilities of ΔwaaC, ΔwaaF, ΔwaaG and ΔwaaP were significantly increased, abilities of biofilm formation by ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaR, ΔwaaP, ΔwaaQ and ΔwaaY decreased, and auto-aggregation abilities of ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaO, ΔwaaR, ΔwaaU, ΔwaaP and ΔwaaY were strongly enhanced. These results give new insight into the influence of core oligosaccharide of lipopolysaccharide on bacterial cell membrane behavior.

UNLABELLED: When 10 Escherichia coli mutant strains with defects in lipopolysaccharide (LPS) core biosynthesis were grown on agar medium at 30°C, four of them, the ΔwaaF, ΔwaaG, ΔwaaP, and ΔwaaB strains, formed mucoid colonies, while the other six, the ΔwaaU, ΔwaaR, ΔwaaO, ΔwaaC, ΔwaaQ, and ΔwaaY strains, did not. Using light microscopy with tannin mordant staining, the presence of exopolysaccharide around the cells of the mutants that formed mucoid colonies could be discerned. The ΔwaaF mutant produced the largest amounts of exopolysaccharide, regardless of whether it was grown on agar or in liquid medium. The exopolysaccharide was isolated from the liquid growth medium of ΔwaaF cells, hydrolyzed, and analyzed by high-performance liquid chromatography with an ion-exchange column, and the results indicated that the exopolysaccharide was consistent with colanic acid. When the key genes related to the biosynthesis of colanic acid, i.e., wza, wzb, wzc, and wcaA, were deleted in the ΔwaaF background, the exopolysaccharide could not be produced any more, further confirming that it was colanic acid. Colanic acid could not be produced in strains in which rcsA, rcsB, rcsD, or rcsF was deleted in the ΔwaaF background, but a reduced level of colanic acid production was detected when the rcsC gene was deleted, suggesting that a change of lipopolysaccharide structure in ΔwaaF cells might be sensed by the RcsCDB phosphorelay system, leading to the production of colanic acid. The results demonstrate that E. coli cells can activate colanic acid production through the RcsCDB phosphorelay system in response to a structural deficiency of lipopolysaccharide. IMPORTANCE: Lipopolysaccharide and colanic acid are important forms of exopolysaccharide for Escherichia coli cells. Their metabolism and biological significance have been investigated, but their interrelation with the cell stress response process is not understood. This study demonstrates, for the first time, that E. coli cells can activate colanic acid production through the RcsCDB phosphorelay system in response to a structural change of lipopolysaccharide, suggesting that bacterial cells can monitor the outer membrane integrity, which is essential for cell survival and damage repair.

Flagella assembly was investigated in the Escherichia coli W3110 wild-type strain and ΔwaaF, ΔwaaC, and ΔwaaG mutant strains that only synthesize lipopolysaccharide with different lengths, using transmission electron microscopy and whole genome transcriptome profiling. Under the electron microscope, the flagella were observed on the cell surface of the W3110 strain but not the ΔwaaC, ΔwaaF, or ΔwaaG strains. Transcriptional profiling showed that 1382 genes in ΔwaaC, 526 genes in ΔwaaF, and 965genes in ΔwaaG were significantly regulated compared to the control W3110 strain. These genes were further analyzed by gene ontology and KEGG pathway. Although there were significant transcriptional differences among the ΔwaaC, ΔwaaF, and ΔwaaG strains, genes related to flagella assembly and bacterial chemotaxis (the linkage between the flagella and the environment) were significantly down-regulated in all three strains. The data demonstrated that flagella assembly in E. coli depends on the length of lipopolysaccharide.