Engineering of bacteria towards programmed autolysis: why, how, and when?

Programmed autolytic bacteria, also termed controlled self-disruptive or self-destructive bacteria, are bacterial systems that express certain lytic genes and undergo cell lysis at a predetermined time point to release the intracellular contents or to commit suicide. Such systems have wide applications in high-throughput screening of protein libraries, synthesis and recovery of bio-products, population control of heterogeneous cultures or synthetic co-cultures, drug delivery, and food fermentation. Recently, great achievements have been reported regarding on-demand control of cell autolysis for different purposes, highlighting the potential of autolytic strains in biomanufacturing and biomedicine. In this review article, we first introduce the various applications of such bacteria, followed by a summarization of the approaches used in the establishment of autolytic bacterial systems, including cell autolysis mediated by cell wall hydrolases with or without facilitating proteins and by membrane-disturbing proteins. Next, we describe in detail the methodologies adopted to control and initiate cell lysis, including induction by chemical inducers, stimulation by physical signals, auto-induction by metabolic status or nutrient limitation, and constitutive expression of the lytic genes. This article is ended with discussions on the remaining problems and possible future directions. This review provides comprehensive information on autolytic bacteria and insightful guidance to the development of highly efficient, robust, and smart autolytic bacterial platforms.

Transcriptome analysis of Micrococcus luteus in response to treatment with protocatechuic acid.

AIM: To reveal the antibacterial mechanism of protocatechuic acid (PCA) against Micrococcus luteus. METHODS AND RESULTS: M. luteus was exposed to PCA, and the antibacterial mechanism was revealed by measuring membrane potential, intracellular ATP and pH levels and transcriptome analysis. PCA induced the membrane potential depolarization of M. luteus, significantly decreased the intracellular ATP and pH levels of M. luteus and disrupted the integrity of the M. luteus cell membrane. Transcriptome analysis showed that PCA induced 782 differentially expressed genes (DEGs) of M. luteus. GO enrichment analysis revealed that the majority of DEGs are involved in pathways of metabolic process, cellular process, biological regulation and transport activity. In addition, PCA inhibited the growth of M. luteus in skimmed milk and extended the shelf life of skimmed milk. CONCLUSION: PCA had good bactericidal activity against M. luteus through the mechanism of cell membrane disruption and metabolic process disorder. SIGNIFICANCE AND IMPACT OF THE STUDY: PCA inhibits the growth of M. luteus in skimmed milk, suggesting that PCA is promising to be used as a novel preservative in food storage.

Evaluation of the Membrane Damage Mechanism of Chlorogenic Acid against Yersinia enterocolitica and Enterobacter sakazakii and Its Application in the Preservation of Raw Pork and Skim Milk.

Plant-derived antimicrobial agents have adequate antimicrobial effects on food-borne pathogens, which can be used as food preservatives. The purpose of this study was to evaluate the antibacterial mechanism of chlorogenic acid (CA) against Yersinia enterocolitica and Enterobacter sakazakii. The minimum inhibitory concentration (MIC) of CA was determined by employing the broth microdilution method. Then, the cell function and morphological changes of Y. enterocolitica and E. sakazakii treated with CA were characterized. Finally, the growth inhibition models of Y. enterocolitica in raw pork and E. sakazakii in skim milk were constructed through the response surface methodology. The results demonstrated that CA has a satisfactory inhibitory effect against Y. enterocolitica and E. sakazakii with a MIC of 2.5 mg/mL. In addition, CA inhibited the growth of Y. enterocolitica and E. sakazakii via cell membrane damage, such as depolarization of the cell membrane, reduction in intracellular adenosine triphosphate (ATP) and pH levels, and destruction of cell morphology. Moreover, CA reduced two log cycles of Y. enterocolitica in raw pork and E. sakazakii in skim milk at a certain temperature. According to the corresponding findings, CA has the potential to be developed as an effective preservative to control Y. enterocolitica and E. sakazakii-associated foodborne diseases.

Volatile organic compounds in the salt-lake sediments of the Tibet Plateau influence prokaryotic diversity and community assembly.

Volatile organic compounds (VOCs) are important environmental factors because they supply nutrients for microbial cells and mediate intercellular interactions. However, few studies have focused on the effects of VOCs on prokaryotic diversity and community composition. In this study, we examined the relationship between prokaryotic diversity and community composition and the content of VOCs in salt-lake sediments from the Tibet Plateau using amplicon sequencing of the 16S rRNA gene. Results showed that the alpha-diversity indices (Chao1, Shannon, and Simpson) were generally negatively correlated with the content of 36 VOCs (P < 0.05). The prokaryotic communities were significantly driven by multiple VOCs at the lineage-dependent pattern (P < 0.05). Further analysis indicated that VOCs, including 3-methylpyruvate, biuret, isocitric acid, and stearic acid, jointly explained 37.3% of the variations in prokaryotic communities. Supplemental VOCs-pyruvate, biuret, alanine, and aspartic acid-notably decreased the Chao1 and Shannon indices and significantly assembled co-occurrence networks for the bacterial communities in the saline sediments. Together, these results demonstrated that VOCs play a critical role in the regulation of the diversity, compositions, and network structures of prokaryotic communities in saline sediments.

Morphological, transcriptional, and metabolic analyses of osmotic-adapted mechanisms of the halophilic Aspergillus montevidensis ZYD4 under hypersaline conditions.

Halophilic fungi in hypersaline habitats require multiple cellular responses for high-salinity adaptation. However, the exact mechanisms behind these adaptation processes remain to be slightly known. The current study is aimed at elucidating the morphological, transcriptomic, and metabolomic changes of the halophilic fungus Aspergillus montevidensis ZYD4 under hypersaline conditions. Under these conditions, the fungus promoted conidia formation and suppressed cleistothecium development. Furthermore, the fungus differentially expressed genes (P < 0.0001) that controlled ion transport, amino acid transport and metabolism, soluble sugar accumulation, fatty acid ß-oxidation, saturated fatty acid synthesis, electron transfer, and oxidative stress tolerance. Additionally, the hypersalinized mycelia widely accumulated metabolites, including amino acids, soluble sugars, saturated fatty acids, and other carbon- and nitrogen-containing compounds. The addition of metabolites-such as neohesperidin, biuret, aspartic acid, alanine, proline, and ornithine-significantly promoted the growth (P ≤ 0.05) and the morphological adaptations of A. montevidensis ZYD4 grown in hypersaline environments. Our study demonstrated that morphological shifts, ion equilibrium, carbon and nitrogen metabolism for solute accumulation, and energy production are vital to halophilic fungi so that they can build tolerance to high-salinity environments.

Functional expression, purification, and antimicrobial activity of a novel antimicrobial peptide MLH in Escherichia coli.

In this study, a novel heterozygous antimicrobial peptide MLH was synthesized, expressed, purified, and characterized. The peptide Md-cec-LL-37_Hp (MLH) was selected through bioinformatic analysis using musca domestica antimicrobial peptide (Cec-Med), human antimicrobial peptide LL-37, and helicobacter pylori antimicrobial peptide (Hp) as parent peptides. The target gene was synthesized by overlap extension PCR (SOE-PCR) and connected to the expression vector pET-32a (+), and the recombinant plasmid pET-32a-MLH was transformed to Escherichia coli for constructing pET-32a-MLH/BL21 (DE3). Isopropyl ß-D-thiogalactoside (IPTG) was used to induce protein expression, and SDS-PAGE and western blot were adopted to test the target protein. And fermentation condition was optimized to get the mass expression of the fusion protein. The Ni2+ affinity chromatographic column was used to purify. Active heterozygous peptide was obtained after renaturation. Finally, the activity of the heterozygous antimicrobial peptide was identified. The fusion peptide showed significant antimicrobial effect on both E. coli and Staphylococcus aureus.

Key biological processes and essential genes for Proteus mirabilis biofilm development inhibition by protocatechuic acid.

Proteus mirabilis is an opportunistic pathogen linked to human urinary tract infections, and is potentially present as a foodborne pathogen within poultry products, including broiler chickens. This report outlines the inhibitory impacts of protocatechuic acid (PCA) on P. mirabilis isolated from a broiler slaughterhouse in China as well as its biofilm. This investigation encompasses assays related to motility and adhesion, bacterial metabolic activity, extracellular polymer (EPS) production, and scavenging capacity. The findings demonstrated that PCA reduced biofilm formation by 61 %. Transcriptomics findings identified that PCA limited the expression of genes like PstS that promote adhesin formation, rbsA and RcsB that alter bacterial chemotaxis, lipopolysaccharide synthesis genes LpxA and EptB, and cell wall synthesis genes MurF and MrdA, and affects the Regulator of Capsule Synthesis (RCS) two-component modulation system. Weighted gene co-expression network analysis (WGCNA) was conducted to identify the core genes. Furthermore, the binding sites of PCA to cytochrome oxidases cydA and cydB, two subunits of ATP synthase atpI and atpH, and ftsZ, which regulate bacterial division, were predicted via molecular docking. Metabolome analysis determined that PCA critically influenced coenzyme A biosynthesis, nucleotide metabolism, alanine, aspartic acid, and glutamate metabolic pathways of P. mirabilis. Therefore, PCA impacts metabolism within bacteria via various pathways, limiting the levels of extracellular polymer and bacterial viability to hinder biofilm formation. Additionally, we prepared an antibacterial plastic film containing protocatechuic acid using PVA as the monomer and CNC as the reinforcing agent. We examined the mechanical and antibacterial properties of this film. When used to wrap chicken, it reduced the total number of colonies, slowed the deterioration of chicken, and maintained the freshness of chicken. In conclusion, the information outlined in this study complements our comprehension of P. mirabilis inhibition by PCA and provides clues for the reduction of foodborne infections associated with P. mirabilis.

L-arginine alleviates heat stress-induced mammary gland injury through modulating CASTOR1-mTORC1 axis mediated mitochondrial homeostasis.

As global warming intensifies, extreme heat is becoming increasingly frequent. These extreme heatwaves have decreased the milk production of dairy animals such as cows and goats and have caused significant damage to the entire dairy industry. It is known that heat stress (HS) can induce the apoptosis and autophagy of mammary epithelial cells (MECs), leading to a decrease in lactating MECs. L-arginine can effectively attenuate HS-induced decreases in milk yield, but the exact mechanisms are not fully understood. In this study, we found that HS upregulated the arginine sensor CASTOR1 in mouse MECs. Arginine activated mTORC1 activity through CASTOR1 and promoted mitochondrial biogenesis through the mTORC1/PGC-1α/NRF1 pathway. Moreover, arginine inhibited mitophagy through the CASTOR1/PINK1/Parkin pathway. Mitochondrial homeostasis ensures ATP synthesis and a stable cellular redox state for MECs under HS, further alleviating HS-induced damage and improving the lactation performance of MECs. In conclusion, these findings reveal the molecular mechanisms by which L-arginine relieves HS-induced mammary gland injury, and suggest that the intake of arginine-based feeds or feed additives is a promising method to increase the milk yield of dairy animals in extreme heat conditions.

Peptidoglycan-Targeting Staphylolytic Enzyme Lysostaphin as a Novel and Efficient Protease toward Glycine-Rich Flexible Peptide Linkers.

Glycine-rich flexible peptide linkers have been widely adopted in fusion protein engineering; however, they can hardly be cleaved for the separation of fusion partners unless specific protease recognition sites are introduced. Herein, we report the use of the peptidoglycan-targeting staphylolytic enzyme lysostaphin to directly digest the glycine-rich flexible linkers of various lengths including oligoglycine linkers and (G4S)x linkers, without the incorporation of extra amino acids. Using His-MBP-linker-LbCpf1 as a model substrate, we show that both types of linkers could be digested by lysostaphin, and the digestion efficiency improved with increasing linker length. The enzyme LbCpf1 retained full activity after tag removal. We further demonstrated that the proteolytic activity of lysostaphin could be well maintained under different environmental conditions and in the presence of a series of chemical reagents at various concentrations that are frequently used in protein purification and stabilization. In addition, such a digestion strategy could also be applied to remove the SUMO domain linked to LwCas13a via an octaglycine linker. This study extends the applications of lysostaphin beyond an antimicrobial reagent and demonstrates its potential as a novel, efficient, and robust protease for protein engineering.

Transcriptome Analysis of Protocatechualdehyde against Listeria monocytogenes and Its Effect on Chicken Quality Characteristics.

The development of natural antimicrobial agents offers new strategies for food preservation due to the health hazards associated with the spoilage of meat products caused by microbial contamination. In this paper, the inhibitory mechanism of protocatechualdehyde (PCA) on Listeria monocytogenes was described, and its effect on the preservation of cooked chicken breast was evaluated. The results showed that the minimal inhibitory concentration (MIC) of PCA on L. monocytogenes was 0.625 mg/mL. Secondly, PCA destroyed the integrity of the L. monocytogenes cell membrane, which was manifested as a decrease in membrane hyperpolarization, intracellular ATP level, and intracellular pH value. Field emission gun scanning electron microscopy (FEG-SEM) observed a cell membrane rupture. Transcriptome analysis showed that PCA may inhibit cell growth by affecting amino acid, nucleotide metabolism, energy metabolism, and the cell membrane of L. monocytogenes. Additionally, it was discovered that PCA enhanced the color and texture of cooked chicken breast meat while decreasing the level of thiobarbituric acid active substance (TBARS). In conclusion, PCA as a natural antibacterial agent has a certain reference value in extending the shelf life of cooked chicken breast.

Membrane damage mechanism of protocatechualdehyde against Micrococcus luteus and its effect on pork quality characteristics.

This study investigated the mechanism of membrane damage by protocatechualdehyde (PCA) against Micrococcus luteus and assessed effects of PCA on the sensory and physicochemical properties of pork. The mechanism of PCA inhibition on M. luteus was studied by determining the minimum inhibitory concentration (MIC) based on membrane potential, intracellular ATP concentration, intracellular pH, confocal laser scanning microscopy (CLSM), and field emission gun scanning electron microscopy (FEG-SEM). The results showed that the MIC of PCA against M. luteus was 1.25 mg/mL. Hyperpolarization of the bacterial cell membrane, a decrease in the intracellular ATP concentration, and intracellular pH indicated that PCA damaged the cell membrane of M. luteus. FEG-SEM observation revealed that PCA could cause surface collapse, cell membrane rupture, and content outflow of M. luteus. Additionally, PCA was found to inhibit increases in the total number of colonies, the thiobarbituric acid reactive substances (TBARS) value growth rate, and moisture mobility in raw pork. Additionally, it improved the color and texture of raw pork, all of which effectively prolonged its shelf life. This study will encourage the application of PCA as a natural antibacterial agent in the food industry.

Microbial cell surface engineering for high-level synthesis of bio-products.

Microbial cell surface layers, which mainly include the cell membrane, cell wall, periplasmic space, outer membrane, capsules, S-layers, pili, and flagella, control material exchange between the cell and the extracellular environment, and have great impact on production titers and yields of various bio-products synthesized by microbes. Recent research work has made exciting achievements in metabolic engineering using microbial cell surface components as novel regulation targets without direct modifications of the metabolic pathways of the desired products. This review article will summarize the accomplishments obtained in this emerging field, and will describe various engineering strategies that have been adopted in bacteria and yeasts for the enhancement of mass transfer across the cell surface, improvement of protein expression and folding, modulation of cell size and shape, and re-direction of cellular resources, all of which contribute to the construction of more efficient microbial cell factories toward the synthesis of a variety of bio-products. The existing problems and possible future directions will also be discussed.

Metabolomics analysis and membrane damage measurement reveal the antibacterial mechanism of lipoic acid against Yersinia enterocolitica.

Yersinia enterocolitica is a pathogenic microorganism that can cause food-borne diseases. Lipoic acid (LA) has been used as an antioxidant against bacteria, but its antibacterial mechanism is rarely reported. This study aims to explore the antibacterial mechanism of LA and its effect on the metabolites of Y. enterocolitica through membrane damage and metabolomics analysis. The results showed that the minimum inhibitory concentration (MIC) of LA against Y. enterocolitica was 2.5 mg mL-1. The membrane potential was depolarized, and intracellular pH (pHin) and ATP were significantly reduced, indicating that LA destroys the cell membrane structure. Confocal laser scanning microscopy (CLSM) and field emission scanning electron microscopy (FESEM) further confirmed LA-induced cell membrane damage. The metabolic profile of Y. enterocolitica following LA treatment was analyzed by liquid chromatography-mass spectrometry (LC-MS). In the metabolome evaluation, 6209 differential metabolites were screened, including 3394 up-regulated and 2815 down-regulated metabolites. Fifteen metabolic pathways of Y. enterocolitica exhibited significant changes after LA treatment, including the pathways important for amino acid and nucleotide metabolism. The results show that LA is a bacteriostatic substance with potential application value in the food industry.

Downregulation of CASTOR1 Inhibits Heat-Stress-Induced Apoptosis and Promotes Casein and Lipid Synthesis in Mammary Epithelial Cells.

Heat stress is one of the most important factors limiting the milk yields of dairy animals. This decline can be attributed to the heat-stress-induced apoptosis of mammary epithelial cells (MECs). The cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1) is a crucial upstream regulator of the mechanistic target of rapamycin complex 1 (mTORC1) signaling, which has close connections with apoptosis. However, the specific roles of CASTOR1 in regulating the apoptosis and lactation of MECs are still obscure. In the present study, we found that heat stress promotes apoptosis and CASTOR1's expression in HC11 cells. Downregulation of CASTOR1 inhibits heat-stress-induced apoptosis through a ROS-independent pathway. In addition, silencing of CASTOR1 promotes cell proliferation, cell cycle progression, and milk component synthesis, and overexpressing of CASTOR1 reverses these observations. Furthermore, we found that silencing of CASTOR1 contributes to the nuclear transport of SREBP1 and promotes lipid synthesis. This study demonstrates the pivotal roles of CASTOR1 in heat-stress-induced apoptosis and milk component synthesis in MECs.

Evaluation of gallic acid on membrane damage of Yersinia enterocolitica and its application as a food preservative in pork.

This study was aimed to examine the membrane damage mechanism of gallic acid (GA) on Yersinia enterocolitica BNCC 108930, and to explore whether GA can prolong the shelf life of pork. The minimal inhibitory concentration (MIC) of GA against Y. enterocolitica was determined by adopting the broth microdilution method. Second, an investigation was conducted on the morphological and physiological variations of Y. enterocolitica after the GA treatment. Finally, a response surface approach was used to establish the growth inhibition model of GA against Y. enterocolitica in pork. The MIC of GA against Yersinia enterocolitica BNCC 108930 was 2.5 mg/mL. GA affects the membrane integrity of Y. enterocolitica, as demonstrated by a significant decrease in intracellular ATP and pH. The results showed that the number of Y. enterocolitica in pork meat containing 5 mg/g of GA decreased by 2 logarithmic cycles during storage at 4 °C for 3 days. According to the obtained findings, GA could be used as a food preservative to prolong the shelf life of pork.

Whole Genome Level Analysis of the Wnt and DIX Gene Families in Mice and Their Coordination Relationship in Regulating Cardiac Hypertrophy.

The Wnt signaling pathway is an evolutionarily conserved signaling pathway that plays essential roles in embryonic development, organogenesis, and many other biological activities. Both Wnt proteins and DIX proteins are important components of Wnt signaling. Systematic studies of Wnt and DIX families at the genome-wide level may provide a comprehensive landscape to elucidate their functions and demonstrate their relationships, but they are currently lacking. In this report, we describe the correlations between mouse Wnt and DIX genes in family expansion, molecular evolution, and expression levels in cardiac hypertrophy at the genome-wide scale. We observed that both the Wnt and DIX families underwent more expansion than the overall average in the evolutionarily early stage. In addition, mirrortree analyses suggested that Wnt and DIX were co-evolved protein families. Collectively, these results would help to elucidate the evolutionary characters of Wnt and DIX families and demonstrate their correlations in mediating cardiac hypertrophy.

Endolysin-Based Autolytic E. coli System for Facile Recovery of Recombinant Proteins.

Recovery of recombinant proteins from the Escherichia coli cytoplasm depends on cell disruption by mechanical, chemical, and/or enzymatic methods, which usually cause incomplete cell breakage or protein denaturation. Controllable autolytic E. coli strains have been designed to facilitate the purification of recombinant proteins; however, these strains suffer from low recovery yield, slow cell lysis, or extensive strain engineering. Herein, we report an improved, highly efficient programmable autolytic E. coli platform, in which cell lysis is initiated upon the induced expression of T4 lysozyme with N-terminal fusion of a cell-penetrating peptide. Through the engineering of the peptide sequence and copy number, and by incorporating the fusion lytic gene into the E. coli genome, more than 99.97% of cells could be lysed within 30 min of induction regardless of cell age. We further tested the expression and release of a recombinant enzyme lysostaphin (Lst) and demonstrated that 4 h induction of the lytic gene after 3 h of Lst expression resulted in 98.97% cell lysis. Lst obtained from this system had the same yield, yet 1.63-fold higher activity, compared with that obtained from cells lysed by freeze-thawing and sonication. This autolytic platform shows potential for use in large-scale microbial production of proteins and other biopolymers.

Wall teichoic acids: physiology and applications.

Wall teichoic acids (WTAs) are charged glycopolymers containing phosphodiester-linked polyol units and represent one of the major components of Gram-positive cell envelope. WTAs have important physiological functions in cell division, gene transfer, surface adhesion, drug resistance and biofilm formation, and are critical virulence factors and vital determinants in mediating cell interaction with and tolerance to environmental factors. Here, we first briefly introduce WTA structure, biosynthesis and its regulation, and then summarize in detail four major physiological roles played by WTAs, i.e. WTA-mediated resistance to antimicrobials, virulence to mammalian cells, interaction with bacteriolytic enzymes and regulation of cell metabolism. We also review the applications of WTAs in these fields that are closely related to the human society, including antibacterial drug discovery targeting WTA biosynthesis, development of vaccines and antibodies regarding WTA-mediated pathogenicity, specific and sensitive detection of pathogens in food using WTAs as a surface epitope and regulation of WTA-related pathways for efficient microbial production of useful compounds. We also point out major problems remaining in these fields, and discuss some possible directions in the future exploration of WTA physiology and applications.

Evaluation of the membrane damage mechanism of protocatechualdehyde against Yersinia enterocolitica and simulation of growth inhibition in pork.

This study sought to explore the antibacterial mechanism associated with membrane damage in Yersinia enterocolitica by protocatechualdehyde (PCA), thus providing improved knowledge of whether PCA is suitable for pork preservation. The minimum inhibitory concentration (MIC) of PCA was determined by micro-broth dilution. We then characterized functional and morphological changes of Y. enterocolitica treated with PCA. Finally, the growth inhibition model of PCA against Y. enterocolitica in pork was established using the response surface method. Accordingly, the MIC of PCA against Y. enterocolitica was found to be 0.3125 mg/mL. Significant observations incorporated membrane depolarization, a markedly decreased intracellular ATP and pH, and morphological changes induced by PCA treatment. After PCA treatment under low temperatures, the average Y. enterocolitica count in pork decreased by two log cycles. According to the obtained findings, PCA exhibited satisfactory performances as a food preservative to control the growth and reproduction of Y. enterocolitica in pork.

Ecosystem services changes between 2000 and 2015 in the Loess Plateau, China: A response to ecological restoration.

The Loess Plateau of China is one of the most severe soil and water loss areas in the world. Since 1999, the Grain to Green Program (GTGP) has been implemented in the region. This study aimed to analyze spatial and temporal variations of ecosystem services from 2000 to 2015 to assess the effects of the GTGP, including carbon sequestration, water regulation, soil conservation and sand fixation. During the study period, the area of forest land and grassland significantly expanded, while the area of farmland decreased sharply. Ecosystem services showed an overall improvement with localized deterioration. Carbon sequestration, water regulation and soil conservation increased substantially. Sand fixation showed a decreasing trend mainly because of decreased wind speeds. There were synergies between carbon sequestration and water regulation, and tradeoffs between soil conservation and sand fixation. It was concluded that ecological projects have contributed significantly to the rehabilitation of the fragile ecosystems of this region. To make these projects more successful and sustainable, long-term management procedures are necessary to maintain and consolidate the improvements.