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.

Aims To increase the l‐isoleucine production in Corynebacterium glutamicum by overexpressing the global regulator Lrp and the two‐component export system BrnFE. Methods and Results The brnFE operon and the lrp gene were cloned into the shuttle vector pDXW‐8 individually or in combination. The constructed plasmids were transformed into an l‐isoleucine‐producing strain C. glutamicum JHI3‐156, and the l‐isoleucine production in these different strains was analysed and compared. More l‐isoleucine was produced when only Lrp was expressed than when only BrnFE was expressed. Significant increase in l‐isoleucine production was observed when Lrp and BrnFE were expressed in combination. Compared to the control strain, l‐isoleucine production in JHI3‐156/pDXW‐8‐lrp‐brnFE increased 63% in flask cultivation, and the specific yield of l‐isoleucine increased 72% in fed‐batch fermentation. Conclusions Both Lrp and BrnFE are important to enhance the l‐isoleucine production in C. glutamicum. Significance and Impact of the Study The results provide useful information to enhance l‐isoleucine or other branched‐chain amino acid production in C. glutamicum.

Lipid A, the hydrophobic anchor of lipopolysaccharide (LPS), is an essential component in the outer membrane of Gram-negative bacteria. It can stimulate the innate immune system via Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD2), leading to the release of inflammatory cytokines. In this study, six Escherichia coli strains which can produce lipid A with different acylation patterns were constructed; the influence of lipid A acylation pattern on the membrane permeability and innate immune stimulation has been systematically investigated. The lipid A species were isolated and identified by matrix assisted laser ionization desorption-time of flight/tandem mass spectrometry. N-Phenyl naphthylamine uptake assay and antibiotic susceptibility test showed that membrane permeability of these strains were different. The lower the number of acyl chains in lipid A, the stronger the membrane permeability. LPS purified from these strains were used to stimulate human or mouse macrophage cells, and different levels of cytokines were induced. Compared with wild type hexa-acylated LPS, penta-acylated, tetra-acylated and tri-acylated LPS induced lower levels of cytokines. These results suggest that the lipid A acylation pattern influences both the bacterial membrane permeability and innate immune stimulation. The results would be useful for redesigning the bacterial membrane structure and for developing lipid A vaccine adjuvant.