Cyclopropane fatty acid (CFA) formation is a post‐synthetic modification of the lipid bilayer that occurs as cultures of Escherichia coli and many other bacteria enter stationary phase. We report the first distinct phenotype for this membrane modification; early stationary phase cultures of strains lacking CFA (as a result of a null mutation in the cfa gene) are abnormally sensitive to killing by a rapid shift from neutral pH to pH 3. This sensitivity to acid shock is dependent on CFA itself because resistance to acid shock is restored to cfa mutant strains by incorporation of CFAs from the growth medium or by introduction of a functional cfa gene on a plasmid. The synthesis of CFA depends in part on the RpoS sigma factor, but the role of RpoS in resistance to acid shock involves additional factors because strains with null mutations in both cfa and rpoS are more sensitive to acid shock than either single mutant strain. Exponential phase cultures of E. coli are much more sensitive to acid shock than stationary phase cultures, but survival is greatly increased if the exponential phase cultures are exposed to moderately acid conditions (pH 5) before shift to pH 3. We show that exposure to moderately acid conditions gives a marked increase in cfa transcription. The efficiency of the survival of acid shock is extremely strain dependent, even among putative wild‐type strains. Much, but not all, of this variability can be explained by the partially or totally defective RpoS alleles carried by many strains.

As decomposers, fungi are key players in recycling plant material in global carbon cycles. We hypothesized that genomes of early diverging fungi may have inherited pectinases from an ancestral species that had been able to extract nutrients from pectin-containing land plants and their algal allies (Streptophytes). We aimed to infer, based on pectinase gene expansions and on the organismal phylogeny, the geological timing of the plant–fungus association. We analyzed 40 fungal genomes, three of which, including Gonapodya prolifera, were sequenced for this study. In the organismal phylogeny from 136 housekeeping loci, Rozella diverged first from all other fungi. Gonapodya prolifera was included among the flagellated, predominantly aquatic fungal species in Chytridiomycota. Sister to Chytridiomycota were the predominantly terrestrial fungi including zygomycota I and zygomycota II, along with the ascomycetes and basidiomycetes that comprise Dikarya. The Gonapodya genome has 27 genes representing five of the seven classes of pectin-specific enzymes known from fungi. Most of these share a common ancestry with pectinases from Dikarya. Indicating functional and sequence similarity, Gonapodya, like many Dikarya, can use pectin as a carbon source for growth in pure culture. Shared pectinases of Dikarya and Gonapodya provide evidence that even ancient aquatic fungi had adapted to extract nutrients from the plants in the green lineage. This implies that 750 million years, the estimated maximum age of origin of the pectin-containing streptophytes represents a maximum age for the divergence of Chytridiomycota from the lineage including Dikarya.

The activity of Escherichia coli pyruvate oxidase (PoxB) was shown to be growth-phase dependent; the enzyme activity reaches a maximum at early stationary phase. We report that PoxB activity is dependent on a functional rpoS(katF) gene which encodes a sigma factor required to transcribe a number of stationary-phase-induced genes. PoxB activity as well as the beta-galactosidase encoded by a poxB::lacZ protein fusion was completely abolished in a strain containing a defective rpoS gene. Northern and primer extension analyses showed that poxB expression was regulated at the transcriptional level and was transcribed from a single promoter; the 5' end of the mRNA being located 27 bp upstream of the translational initiation codon of poxB. The poxB gene was expressed at decreased levels under anaerobiosis; however, the anaerobic regulatory genes arcA, arcB or fnr were not involved in anaerobic poxB gene expression. Expression of the rpoS(katF) gene has been reported to be affected by acetate, the product of PoxB reaction. However, we found that poxB null mutations had no effect on rpoS(katF) expression. Inactivation of two genes involved in acetate metabolism, ackA and pta, had no effect on either poxB or rpoS(katF) expression.

With the advent of the genomics era, heterologous gene expression has been used extensively as a means of accessing natural products (NPs) from environmental DNA samples. However, the heterologous production of NPs often has very low efficiency or is unable to produce targeted NPs. Moreover, due to the complicated transcriptional and metabolic regulation of NP biosynthesis in native producers, especially in the cases of genome mining, it is also difficult to rationally and systematically engineer synthetic pathways to improved NPs biosynthetic efficiency. In this study, various strategies ranging from heterologous production of a NP to subsequent application of omics-guided synthetic modules optimization for efficient biosynthesis of NPs with complex structure have been developed. Heterologous production of spinosyn in Streptomyces spp. has been demonstrated as an example of the application of these approaches. Combined with the targeted omics approach, several rate-limiting steps of spinosyn heterologous production in Streptomyces spp. have been revealed. Subsequent engineering work overcame three of selected rate-limiting steps, and the production of spinosad was increased step by step and finally reached 1460 μg/L, which is about 1000-fold higher than the original strain S. albus J1074 (C4I6-M). These results indicated that the omics platform developed in this work was a powerful tool for guiding the rational refactoring of heterologous biosynthetic pathway in Streptomyces host. Additionally, this work lays the foundation for further studies aimed at the more efficient production of spinosyn in a heterologous host. And the strategy developed in this study is expected to become readily adaptable to highly efficient heterologous production of other NPs with complex structure.