Transcriptomics of Listeria monocytogenes Treated With Olive Leaf Extract

Listeria monocytogenes is a regulated foodborne pathogen that is known to cause listeriosis, a disease associated with high mortality rates in humans. Olive leaf extract (OLE) has been shown to act as a plant antimicrobial and inhibit the growth of pathogens, such as L. monocytogenes, although its mode of action has not been defined. To help identify the cellular mechanisms important for conveying these beneficial traits, RNA-Seq was used to study the transcriptome of L. monocytogenes upon exposure to a sublethal level of OLE. Results obtained from cells cultured both with and without OLE at two different time points (3.5-h and 24-h) revealed 661 genes that were differentially expressed. Of the differentially expressed genes (DEGs) identified, transcription was altered for 171 genes in response to the 3.5-h OLE treatment while 490 genes were altered in response to the 24-h OLE treatment. These DEGs included but were not limited to genes encoding for signal transduction, ATP-binding cassette (ABC) transporters, and the phosphotransferase system. Interestingly, several virulence-related genes were downregulated including an ABC transporter permease previously shown to negatively regulate biofilm formation, genes involved in flagella assembly and binding/entry into host cells as well as those regulating acid resistance suggesting that OLE may decrease the virulence potential of L. monocytogenes. Furthermore, quantitative reverse-transcription PCR was used to validate the data obtained via RNA-Seq. Our study provides insight into the mode of action of OLE treatment against L. monocytogenes and may aid in identifying synergetic strategies to inhibit L. monocytogenes in food.

The transcriptome analysis of Escherichia coli responding to tellurite

Tellurite is a strong antimicrobial agent highly toxic to many microorganisms, while its toxicity mechanism is still unclear. In this study, the comparative transcriptome analysis of E. coli MG1655 responding to the stress of tellurite was performed and the differentially transcribed genes were analyzed, to understand toxicity mechanisms of tellurite preliminaryly and uncover metabolism processes changes resulted from tellurite globally. After treated with 10 µg/mL tellurite for 1 h, high concentration and long time, the cells exhibited an obvious adaptive reaction and many metabolic processes were influenced. The transcription of the genes involved in the ribosome metabolism and the flagella assembly were changed significantly, implying they might be the major pathway affected by tellurite. The transcription of the genes encoding the transcriptional factors and small RNAs, and the genes functioned in the cell motility, metal ion metabolism and membrane function were also varied, which may participate in the metabolism adjustment and damage repair to resist the toxicity of tellurite. This work can facilitate the study of the toxicity mechanism of tellurite and promote the clinical application of this chemical.

Metagenome-wide association study revealed disease-specific landscape of the gut microbiome of systemic lupus erythematosus in Japanese

ObjectiveAlteration of the gut microbiome has been linked to the pathogenesis of systemic lupus erythematosus (SLE). However, a comprehensive view of the gut microbiome in SLE and its interaction with the host remains to be revealed. This study aimed to reveal SLE-associated changes in the gut microbiome and its interaction with the host by a comprehensive metagenome-wide association study (MWAS) followed by integrative analysis.MethodsWe performed a MWAS of SLE based on shotgun sequencing of the gut microbial DNA from Japanese individuals (Ncase=47, Ncontrol=203). We integrated the result of the MWAS with the genome-wide association study (GWAS) data and plasma metabolite data.ResultsVia species level phylogenetic analysis, we identified and validated increases of Streptococcus intermedius and Streptococcus anginosus in the patients with SLE. Microbial gene analysis revealed increases of Streptococcus-derived genes including one involved in redox reaction. Additionally, microbial pathways related to sulfur metabolism and flagella assembly were altered in the patients with SLE. We identified an overlap in the enriched biological pathways between the metagenome and the germline genome by comparing the result of the MWAS and the GWAS of SLE (ie, MWAS-GWAS interaction). α-diversity and β-diversity analyses provided evidence of dysbiosis in the metagenome of the patients with SLE. Microbiome-metabolome association analysis identified positive dosage correlation of acylcarnitine with Streptococcus intermedius, an SLE-associated taxon.ConclusionOur MWAS followed by integrative analysis revealed SLE-associated changes in the gut microbiome and its interaction with the host, which contribute to our understanding of the relationship between the microbiome and SLE.

Molecular Mechanism of Engineered Zymomonas Mobilis Response to Furfural and Acetic acid Stress

Backgroud: Acetic acid and furfural are two major inhibitors during lignocellulosic ethanol production. In our previous study, we successfully constructed an engineered Zymomonas mobilis ZM532 strain tolerant these double inhibitors by genome shuffling, but the molecular mechanisms of tolerance to these inhibitors are still unknown. This study investigated the responses of ZM532 and wild-type ZM4 to acetic acid and furfural using genomics, transcriptomics and label free quantitative proteomics. Results: By Sanger sequencing technology we re-verified of previously identified 19 mutations in ZM532, but we found a total of 23 single nucleotide polymorphisms (SNPs) in the coding sequence (CDS; 4) and intergenic region (19) in ZM532. Six SNPs were novel in this study. We also identified a total of 1865 and 14 novel differentially expressed genes (DEGs) in ZM532 and wild-type ZM4. Among these, 352 DEGs were up-regulated; while and 393 were down-regulated in AF_ZM532 vs RM_532, respectively. However, 442 DEGs were up while 463 were down-regulated in AF_ZM4 vs RM_ZM4. Moreover, 2 up and 8 down-regulated genes were identified in AF_ZM532 vs AF_ZM4; while 7 up and 1 down-regulated genes were found in RM_ZM532 vs RM_ZM. We also identified 1,532 proteins among 107 up and 204 down-regulated proteins detected in ZM4_AF vs ZM4_RM, 123 up and 205 down regulated proteins were identified in ZM532_AF vs ZM532_RM, respectively. In addition, a total of 16 up and 5 down-regulated proteins were identified out of 1462 in ZM4_AF vs ZM532_AF, while 8 up and 5 down-regulated proteins were observed out of 1491 in ZM4_RM vs ZM532_RM. These proteins and genes are involved in amino acid biosynthesis, macromolecules repair, glycolysis, flagella assembly, ABC transporter, fermentation, and ATP synthesis pathways and stress response. These mentioned genes and proteins confirmed and help to unravel the acetic acid and furfural tolerance mechanism between ZM532 and wild-type ZM4. May be these proteins and genes play key roles in ZM532 regulation with strong expressions under acids stress conditions. Furthermore, we knocked-out and overexpressed two differentially expressed genes (DEGs), ZMO_RS02740 up-regulated and ZMO_RS06525 down-regulated to investigate their roles in acetic acid and furfural tolerance. Our knockout and complementary experiments revealed that up-regulated expression gene ZMO_RS02740 and the down-regulated expression gene ZMO_RS06525 play important roles in dealing with furfural and acetic acid stress. Conclusion: ZM532 can be used to substitute ZM4 as a biocatalyst for bioethanol under acetic acid and furfural condition, with a shorter fermentation time and higher productivity. Further studies may be required to clarify the relationship between the acid resistance and the genetic disparity of mutant strains.

Improved Yield of Recombinant Protein via Flagella Regulator Deletion in Escherichia coli

Protein production requires a significant amount of intracellular energy. Eliminating the flagella has been proposed to help Escherichia coli improve protein production by reducing energy consumption. In this study, the gene encoding a subunit of FlhC, a master regulator of flagella assembly, was deleted to reduce the expression of flagella-related genes. FlhC knockout in the ptsG-deleted strain triggered significant growth retardation with increased ATP levels and a higher NADPH/NADP+ ratio. Metabolic flux analysis using a 13C-labeled carbon substrate showed increased fluxes toward the pentose phosphate and tricarboxylic acid cycle pathways in the flhC- and ptsG-deleted strains. Introduction of a high copy number plasmid or overexpression of the recombinant protein in this strain restored growth rate without increasing glucose consumption. These results suggest that the metabolic burden caused by flhC deletion was resolved by recombinant protein production. The recombinant enhanced green fluorescent protein yield per glucose consumption increased 1.81-fold in the flhC mutant strain. Thus, our study demonstrates that high-yield production of the recombinant protein was achieved with reduced flagella formation.

Reconstitution and optimisation of the biosynthesis of bacterial sugar pseudaminic acid (Pse5Ac7Ac) enables preparative enzymatic synthesis of CMP-Pse5Ac7Ac

Pseudaminic acids present on the surface of pathogenic bacteria, including gut pathogens Campylobacter jejuni and Helicobacter pylori, are postulated to play influential roles in the etiology of associated infectious diseases through modulating flagella assembly and recognition of bacteria by the human immune system. Yet they are underexplored compared to other areas of glycoscience, in particular enzymes responsible for the glycosyltransfer of these sugars in bacteria are still to be unambiguously characterised. This can be largely attributed to a lack of access to nucleotide-activated pseudaminic acid glycosyl donors, such as CMP-Pse5Ac7Ac. Herein we reconstitute the biosynthesis of Pse5Ac7Ac in vitro using enzymes from C. jejuni (PseBCHGI) in the process optimising coupled turnover with PseBC using deuterium wash in experiments, and establishing a method for co-factor regeneration in PseH tunover. Furthermore we establish conditions for purification of a soluble CMP-Pse5Ac7Ac synthetase enzyme PseF from Aeromonas caviae and utilise it in combination with the C. jejuni enzymes to achieve practical preparative synthesis of CMP-Pse5Ac7Ac in vitro, facilitating future biological studies.

Bioengineering of archaeal flagella

It was shown that the Haloferax volcanii flagella assembly system can accept alien flagellins and build functional recombinant flagella. The results can be used for targeted flagella modification to create multifunctional nanomaterials.

The fliK Gene Is Required for the Resistance of Bacillus thuringiensis to Antimicrobial Peptides and Virulence in Drosophila melanogaster

Antimicrobial peptides (AMPs) are essential effectors of the host innate immune system and they represent promising molecules for the treatment of multidrug resistant microbes. A better understanding of microbial resistance to these defense peptides is thus prerequisite for the control of infectious diseases. Here, using a random mutagenesis approach, we identify the fliK gene, encoding an internal molecular ruler that controls flagella hook length, as an essential element for Bacillus thuringiensis resistance to AMPs in Drosophila. Unlike its parental strain, that is highly virulent to both wild-type and AMPs deficient mutant flies, the fliK deletion mutant is only lethal to the latter’s. In agreement with its conserved function, the fliK mutant is non-flagellated and exhibits highly compromised motility. However, comparative analysis of the fliK mutant phenotype to that of a fla mutant, in which the genes encoding flagella proteins are interrupted, indicate that B. thuringiensis FliK-dependent resistance to AMPs is independent of flagella assembly. As a whole, our results identify FliK as an essential determinant for B. thuringiensis virulence in Drosophila and provide new insights on the mechanisms underlying bacteria resistance to AMPs.

The transcription regulator and c-di-GMP phosphodiesterase PdeL represses motility in Escherichia coli

PdeL is a transcription regulator and catalytically active c-di-GMP phosphodiesterases (PDE) in Escherichia coli. PdeL has been shown to be a transcription autoregulator, while no other target genes have been identified so far. Here, we show that PdeL represses transcription of the flagella class II operon, fliFGHIJK, and activates sslE encoding an extracellular anchored metalloprotease, among additional loci. DNA-binding studies and expression analyses using plasmidic reporters suggest that regulation of the fliF and sslE promoters by PdeL is direct. Transcription repression of the fliFGHIJK operon, encoding protein required for assembly of the flagellar basal body, results in inhibition of motility on soft agar plates and reduction of flagella assembly, as shown by fluorescence staining of the flagella hook protein FlgE. PdeL-mediated repression of motility is independent of its phosphodiesterase activity. Thus, in motility control the transcription regulator function of PdeL reducing the number of assembled flagella is apparently epistatic to its phosphodiesterase function, which can indirectly promote the activity of the flagellar motor by lowering the c-di-GMP concentration.Bacteria adopt different lifestyles depending on their environment and physiological condition. In Escherichia coli and other enteric bacteria the transition between the motile and the sessile state is controlled at multiple levels from the regulation of gene expression to the modulation of various processes by the second messenger c-di-GMP as signaling molecule. The significance of our research is in identifying PdeL, a protein of dual function that hydrolyzes c-di-GMP and that regulates transcription of genes, as a repressor of Flagella gene expression and an inhibitor of motility, which adds an additional regulatory switch to the control of motility.