Inactivation of Carotenogenic-Biosynthesizing Genes Altered Lipids Composition and Intensity in Cronobacter sakazakii.

Cronobacter sakazakii, an opportunistic milk-borne pathogen responsible for severe neonatal meningitis and bacteremia, can synthesize yellow pigment (various carotenoids) benefiting for bacterial survival, while little literature was available about the influence of various carotenoids on bacterial resistance to a series of stresses and the characteristics of cell membrane, obstructing the development of novel bactericidal strategies overcoming the strong tolerance of C. sakazakii. Thus in this study, for the first time, five carotenogenic genes of C. sakazakii BAA-894 were inactivated, respectively, to construct a series of mutants producing various carotenoids and their effects on the cell membrane properties, and resistances to food- and host-related stresses, were investigated systematically. Furthermore, to explore its possible mode of action, comparative lipidomics analysis was performed to reveal the change of lipids that were mainly located at cell membranes. The results showed that five mutants (ΔcrtB, ΔcrtI, ΔcrtY, ΔcrtZ, and ΔcrtX) displayed negligible change in growth rate but higher permeability of the outer membrane and lower fluidity of cell membrane compared to the wild type. Besides, these mutants exhibited poorer ability of biofilm formation and lower resistances to acid, oxidative, osmotic, and desiccation stresses, indicating that different carotenoid composition significantly affected environmental tolerance of C. sakazakii. To discover the possible causes, lipidomics analysis of C. sakazakii was conducted and more than 500 lipid species belonging to 27 classes had been identified at first. Compared to that of BAA-894, the composition and relative intensity of lipid species in five mutants varied significantly, especially the monounsaturated and biunsaturated phosphatidylethanolamine. The evidence presented in this study demonstrated that the varied composition of carotenoids in C. sakazakii significantly altered the lipid profile and intensity, which maybe a crucial means to influencing the characteristics of cell membranes and resistance to environmental stresses.

Comparative lipidomics of Pichia pastoris using constitutive promoter reveals lipid diversity and variability at different growth phases.

Pichia pastoris is an expression platform widely used for foreign protein expression, while it is unknown how the global lipid profiles changed during the cultivation process, which is crucial for fermentation optimization and chassis design. Therefore, this study aimed to reveal the diverse lipid profiles of P. pastoris controlled by constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase gene and to unravel their change in the lag, logarithmic, stationary, and death phases, using ultra-performance liquid chromatography/nano-electrospray ionization-tandem mass spectrometry. Two hundred forty lipid species across 11 lipid classes were detected, including various glycerolipids, glycerophospholipids, and sphingolipids. Pichia cells displayed high diversity and variability of lipids in lipid profile, relative intensity, phosphatidylinositol/phosphatidylserine ratio, fatty acid chain length, and unsaturation degree. Notably, increase of unsaturated triacylglycerol level was accompanied by rise of malondialdehyde level under oxidative stress. The increased ceramide with long fatty acid chain could be a key feature at death phase. This work deepened our understanding of the physiology of P. pastoris during cultivation and provided valuable information for further improvement of the P. pastoris expression system.

Enhanced Sensitivity of Salmonella to Antimicrobial Blue Light Caused by Inactivating rfaC Gene Involved in Lipopolysaccharide Biosynthesis.

Salmonella is a global foodborne pathogen that causes human diseases ranging from mild gastroenteritis to severe systemic infections. Recently, antimicrobial blue light (aBL) showed effective bactericidal activity against a variety of bacteria (e.g., Salmonella) with varying efficiency. However, the antimicrobial mechanism of aBL has not been fully elucidated. Our previous report showed that the outer membrane (OM) is a key target of aBL. The major component of the OM, lipopolysaccharide (LPS), may play a role in aBL bactericidal effect. Therefore, the influence of LPS truncation on the sensitivity of Salmonella Typhimurium SL1344 to aBL was investigated for the first time. First, the rfaC gene in the SL1344 strain likely involved in linking lipid A to the core region of LPS was inactivated and the influence on LPS structure was verified in the mutant strain SL1344ΔrfaC. SL1344ΔrfaC showed a significant increase in sensitivity to aBL, and the bactericidal efficiency exceeded 8 log CFU at an aBL dose of 383 J/cm2, while that of its parental SL1344 strain approached 4 log CFU. To discover the possible mechanism of higher sensitivity, the permeability of OM was determined. Compared to SL1344, SL1344ΔrfaC showed 2.7-fold higher permeability of the OM at 20 J/cm2, this may explain the higher vulnerability of the OM to aBL. Furthermore, the fatty acid profile was analyzed to reveal the detailed changes in the OM and inner membrane of the mutant. Results showed that the membrane lipids of SL1344ΔrfaC were markedly different to SL1344, indicating that change in fatty acid profile might mediate the enhancement of OM permeability and the increased sensitivity to aBL in SL1344ΔrfaC. Hence, we concluded that disruption of rfaC in Salmonella Typhimurium led to the formation of truncated LPS and thus enhanced the permeability of the OM, which contributed to the increased sensitivity to aBL.

Comparative lipidomics of methanol induced Pichia pastoris cells at different culture phases uncovers the diversity and variability of lipids.

Pichia pastoris is an attractive eukaryotic host widely employed in industrial biotechnology for protein production and biocatalysis, and oxidative stress and other harsh conditions were frequently encountered during the cultivation cycle, however, the global lipidomic profile change needed to be revealed. The present study aimed to discover the variation in P. pastoris lipids at different stages (lag, logarithmic, stationary, induction and decline phases) by a sensitive ultra-performance liquid chromatography/nanoelectrospray-mass/mass spectrometry. We identified 253 lipid species across 11 lipids classes, including glycerophospholipids, glycerolipids and sphingolipids. High diversity and flexibility of lipids (including the composition and relative intensity) were observed during different phases, especially when glycerol was shifted to methanol. Especially, the unsaturated-double-bonds containing lipids showed a close relationship with the change of carbon source, which also led to increase of oxidative stress. Additionally, the relative intensity of sphingolipids was increased obviously in decline phase, likely associated with cell apoptosis. The current study expanded our understanding of the cell physiology of P. pastoris through the lipid profile change and lay the foundation for chassis design strains in the future by engineering microbial membrane.

Blue Light for Inactivation of Meatborne Pathogens and Maintaining the Freshness of Beef.

ABSTRACT: Beef is rich in various nutrients but easily spoils due to bacterial contamination; thus, a bactericidal method is needed to inactivate meatborne pathogens while maintaining the freshness of beef. The present study was conducted to investigate for the first time the bactericidal effect of blue light (BL) at 415 nm against four meatborne pathogens (methicillin-resistant Staphylococcus aureus, Escherichia coli, Salmonella Typhimurium, and Listeria monocytogenes) both in vitro and inoculated onto the surface of fresh beef. The populations of the four pathogens on the nonirradiated control beef did not change significantly (P > 0.05), whereas a dose-dependent inactivation effect was found for BL-treated beef both in vitro and in vivo. On the beef cuts, BL at 109.44 J/cm2 inactivated 90% of inoculated cells of the tested strains (P < 0.05), and this inactivation effect was sustained during 7 days of cold storage. Insignificant changes in lipid oxidation rate, water holding capacity, and cooking loss were found during storage between the control beef and the beef irradiated at 109.44 J/cm2 at the same time. BL had a minor and nonsignificant effect on surface color and free amino acid concentrations. The pH of the treated beef increased more slowly (P < 0.05) than did that of untreated beef. These results suggest that BL could be a novel bactericide and could help maintain the freshness of beef.

Understanding a defensive response of methicillin-resistant Staphylococcus aureus after exposure to multiple cycles of sub-lethal blue light.

Blue light (BL) has shown bactericidal effectiveness against methicillin-resistant Staphylococcus aureus (MRSA), one of the major clinical pathogens with antibiotic resistance. Bacteria likely respond to the oxidative stress induced by BL; however, the defensive response is still unclear. This study aimed to reveal the phenotypic change in MRSA after being exposed to 15 cycles of sub-lethal BL illumination. The comparative transcriptomic results showed that the expression of peptidoglycan (PG) synthesis gene glmS was significantly upregulated in the cells after the multiple cycle light treatment, and the biochemical analysis determined that the content of PG synthesized was increased by 25.86% when compared with that in control cells. Furthermore, significant thickening of the cell wall was observed under a transmission electron microscope (P < .05). The light sensitivity of the tested MRSA strain was reduced after the multiple cycle light treatment, indicating the possibility of MRSA being more adaptive to the BL stress. The present study suggested that multiple cycles of sub-lethal BL could change the light susceptibility of MRSA through thickening the cell wall.

Testing for heteroskedasticity in two-way fixed effects panel data models.

In this paper, we propose a new method for testing heteroskedasticity in two-way fixed effects panel data models under two important scenarios where the cross-sectional dimension is large and the temporal dimension is either large or fixed. Specifically, we will develop test statistics for both cases under the conditional moment framework, and derive their asymptotic distributions under both the null and alternative hypotheses. The proposed tests are distribution free and can easily be implemented using the simple auxiliary regressions. Simulation studies and two real data analyses demonstrate that our proposed tests perform well in practice, and may have the potential for wide application in econometric models with panel data.

The disc separation and the eigenvalue distribution of the Schur complement of nonstrictly diagonally dominant matrices.

The result on the Gersgorin disc separation from the origin for strictly diagonally dominant matrices and their Schur complements in (Liu and Zhang in SIAM J. Matrix Anal. Appl. 27(3):665-674, 2005) is extended to nonstrictly diagonally dominant matrices and their Schur complements, showing that under some conditions the separation of the Schur complement of a nonstrictly diagonally dominant matrix is greater than that of the original grand matrix. As an application, the eigenvalue distribution of the Schur complement is discussed for nonstrictly diagonally dominant matrices to derive some significant conclusions. Finally, some examples are provided to show the effectiveness of theoretical results.