A Systems Approach Discovers the Role and Characteristics of Seven LysR Type Transcription Factors in Escherichia Coli

Although Escherichia coli K-12 strains represent perhaps the best known model bacteria, we do not know the identity or functions of all of their transcription factors (TFs). It is now possible to systematically discover the physiological function of TFs in E. coli BW25113 using a set of synergistic methods; including ChIP-exo, growth phenotyping, conserved gene clustering, and transcriptome analysis. Among 47 LysR-type TFs (LTFs) found on the E. coli K-12 genome, many regulate nitrogen source utilization or amino acid metabolism. However, 19 LTFs remain unknown. In this study, we elucidated the regulation of seven of these 19 LTFs: YbdO, YbeF, YgfI, YiaU, YneJ, YcaN, YbhD. We show that: 1) YbdO regulation has an effect on bacterial growth at low pH with citrate supplementation. YbdO is a repressor of the ybdNM operon and is implicated in the regulation of citrate lyase genes (citCDEFG); 2) YgfI activates the dhaKLM operon that encodes the phosphotransferase system involved in glycerol and dihydroxyacetone utilization; 3) YiaU regulates the yiaT gene encoding an outer membrane protein, and waaPSBOJYZU operon is also important in determining cell density at the stationary phase; 4) YneJ, re-named here as PtrR, directly regulates the expression of the succinate-semialdehyde dehydrogenase, Sad (also known as YneI), and is a predicted regulator of fnrS (a small RNA molecule). PtrR is important for bacterial growth in the presence of L-glutamate and putrescine as nitrogen sources; and 5) YbhD and YcaN regulate adjacent y-genes on the genome and YbeF is involved in flagella gene regulation. We have thus established the functions for four LTFs and identified the target genes for three LTFs.

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.

A systems approach discovers the role and characteristics of seven LysR type transcription factors in Escherichia coli

Although Escherichia coli K-12 strains represent perhaps the best known model bacteria, we do not know the identity or functions of all of their transcription factors (TFs). It is now possible to systematically discover the physiological function of TFs in E. coli BW25113 using a set of synergistic methods; including ChIP-exo, growth phenotyping, conserved gene clustering, and transcriptome analysis. Among 47 LysR-type TFs (LTFs) found on the E. coli K-12 genome, many regulate nitrogen source utilization or amino acid metabolism. However, 19 LTFs remain unknown. In this study, we elucidated the regulation of seven of these 19 LTFs: YbdO, YbeF, YgfI, YiaU, YneJ, YcaN, YbhD. We show that: 1) YbdO regulation has an effect on bacterial growth at low pH with citrate supplementation. YbdO is a repressor of the ybdNM operon and is implicated in the regulation of citrate lyase genes (citCDEFG); 2) YgfI activates the dhaKLM operon that encodes the phosphotransferase system involved in glycerol and dihydroxyacetone utilization; 3) YiaU regulates the yiaT gene encoding an outer membrane protein, and waaPSBOJYZU operon is also important in determining cell density at the stationary phase; 4) YneJ, re-named here as PtrR, directly regulates the expression of the succinate-semialdehyde dehydrogenase, Sad (also known as YneI), and is a predicted regulator of fnrS (a small RNA molecule). PtrR is important for bacterial growth in the presence of L-glutamate and putrescine as nitrogen sources; and 5) YbhD and YcaN regulate adjacent y-genes on the genome and YbeF is involved in flagella gene regulation. We have thus established the functions for four LTFs and identified the target genes for three LTFs.

Yeast β-Glucan Altered Intestinal Microbiome and Metabolome in Older Hens

The prebiotics- and probiotics-mediated positive modulation of the gut microbiota composition is considered a useful approach to improve gut health and food safety in chickens. This study explored the effects of yeast β-glucan (YG) supplementation on intestinal microbiome and metabolites profiles as well as mucosal immunity in older hens. A total of 256 43-week-old hens were randomly assigned to two treatments, with 0 and 200 mg/kg of YG. Results revealed YG-induced downregulation of toll-like receptors (TLRs) and cytokine gene expression in the ileum without any effect on the intestinal barrier. 16S rRNA analysis claimed that YG altered α- and β-diversity and enriched the relative abundance of class Bacilli, orders Lactobacillales and Enterobacteriales, families Lactobacillaceae and Enterobacteriaceae, genera Lactobacillus and Escherichia–Shigella, and species uncultured bacterium-Lactobacillus. Significant downregulation of cutin and suberin, wax biosynthesis, atrazine degradation, vitamin B6 metabolism, phosphotransferase system (PTS), steroid degradation, biosynthesis of unsaturated fatty acids, aminobenzoate degradation and quorum sensing and upregulation of ascorbate and aldarate metabolism, C5-branched dibasic acid metabolism, glyoxylate and dicarboxylate metabolism, pentose and glucuronate interconversions, steroid biosynthesis, carotenoid biosynthesis, porphyrin and chlorophyll metabolism, sesquiterpenoid and triterpenoid biosynthesis, lysine degradation, and ubiquinone and other terpenoid-quinone biosyntheses were observed in YG-treated hens, as substantiated by the findings of untargeted metabolomics analysis. Overall, YG manifests prebiotic properties by altering gut microbiome and metabolite profiles and can downregulate the intestinal mucosal immune response of breeder hens.

Regulation of dctA and DctA by cAMP-CRP and EIIAGlc at the transcriptional and post-translational levels in E. coli: Consequences for aerobic uptake and metabolism of C4-dicarboxylates

The expression of dctA, encoding the aerobic C4-dicarboxylate (C4-DC) transporter DctA of Escherichia coli, and its use in the presence of alternative carbon sources was characterized. dctA is regulated by cAMP-CRP and substrates that control cAMP levels, either through the phosphotransferase system (PTS), or through their metabolic link to PEP synthesis. The data indicates that phosphorylation of the regulator EIIAGlc of the glucose-specific PTS represents the mediator for regulation. The dctA promotor region contains a class I CRP-binding site (position -81.5) and a DcuR-binding site (position -105.5). The response regulator DcuR of the C4-DC-activated DcuS-DcuR two-component system is known to stimulate expression of dctA, and cAMP-CRP is known to stimulate expression of dcuS-dcuR. Thus, activation of dctA expression by cAMP-CRP and DcuR is organized in a coherent feed-forward loop (FFL) where cAMP-CRP positively regulates the expression of dctA by direct stimulation and by stimulating the expression of dcuR. Stimulation by DcuR is presumed to require DNA bending by cAMP-CRP. In this way, CRP-FFL integrates carbon catabolite control and C4-DC-specific regulation. Moreover, EIIAGlc of the glucose-specific PTS strongly interacts with DctA, which could lead to substrate exclusion of C4-DCs when preferred carbon substrates such as sugars are present. Since C4-DCs are perceived in the periplasmic space by the sensor DcuS, the substrate exclusion is not linked to inducer exclusion, contrasting classical inducer exclusion known for the lactose permease LacY. Thus, aerobic C4-DC metabolism is tightly regulated at the transcriptional and post-translational levels, whereas uptake of L-aspartate by DcuA is essentially unaffected. Overall, transcriptional and post-translational regulation of dctA expression and DctA function efficiently fine-tunes C4-DC catabolism in response to other preferred carbon sources.

Enzyme systems involved in glucosinolate metabolism in Companilactobacillus farciminis KB1089

Cruciferous vegetables are rich sources of glucosinolates (GSLs). GSLs are degraded into isothiocyanates, which are potent anticarcinogens, by human gut bacteria. However, the mechanisms and enzymes involved in gut bacteria-mediated GSL metabolism are currently unclear. This study aimed to elucidate the enzymes involved in GSL metabolism in lactic acid bacteria, a type of gut bacteria. Companilactobacillus farciminis KB1089 was selected as a lactic acid bacteria strain model that metabolizes sinigrin, which is a GSL, into allylisothiocyanate. The sinigrin-metabolizing activity of this strain is induced under glucose-absent and sinigrin-present conditions. A quantitative comparative proteomic analysis was conducted and a total of 20 proteins that were specifically expressed in the induced cells were identified. Three candidate proteins, β-glucoside-specific IIB, IIC, IIA phosphotransferase system (PTS) components (CfPttS), 6-phospho-β-glucosidase (CfPbgS) and a hypothetical protein (CfNukS), were suspected to be involved in sinigrin-metabolism and were thus investigated further. We hypothesize a pathway for sinigrin degradation, wherein sinigrin is taken up and phosphorylated by CfPttS, and subsequently, the phosphorylated entity is degraded by CfPbgS. As expression of both pttS and pbgS genes clearly gave Escherichia coli host strain sinigrin converting activity, these genes were suggested to be responsible for sinigrin degradation. Furthermore, heterologous expression analysis using Lactococcus lactis suggested that CfPttS was important for sinigrin degradation and CfPbgS degraded phosphorylated sinigrin.

Structural basis of pore formation in the mannose phosphotransferase system (man-PTS) by pediocin PA-1

Bacteriocins are ribosomally synthesized bacterial antimicrobial peptides that have a narrow spectrum of antibacterial activity against species closely related to the producers. Pediocin-like (or class IIa) bacteriocins (PLBs) exhibit antibacterial activity against several Gram-positive bacterial strains by forming pores in the cytoplasmic membrane of target cells with the specific receptor, the mannose phosphotransferase system (man-PTS). In this study, we report the cryo-electron microscopy structures of man-PTS from Listeria monocytogenes alone and its complex with pediocin PA-1, the first and most extensively studied representative PLB at a resolution of 3.12 Å and 2.45 Å, respectively. The structures revealed that the binding of pediocin PA-1 opens the Core domain of man-PTS away from its Vmotif domain, creating a pore through the cytoplasmic membranes of target cells. During this process, the N-terminal β-sheet region of pediocin PA-1 can specifically attach to the extracellular surface of the man-PTS Core domain, whereas the C-terminal half penetrates the membrane and cracks the man-PTS like a wedge. Thus, our findings shed light on a design of novel PLBs that can kill target pathogenic bacteria. Importance Listeria monocytogenes is a ubiquitous microorganism responsible for listeriosis, a rare but severe disease in humans who become infected by ingesting contaminated food products ( i.e. , dairy, meat, fish, and vegetables), which have a fatality rate of 33%. Pediocin PA-1 is an important commercial additive used in food production to inhibit Listeria species. The mannose phosphotransferase system (man-PTS) is responsible for the sensitivity of Listeria monocytogenes to pediocin PA-1. In this study, we report the cryo-EM structures of man-PTS from Listeria monocytogenes alone and its complex with pediocin PA-1 at a resolution of 3.12 Å and 2.45 Å, respectively. Our results facilitate the understanding of the mode of action of class IIa bacteriocins as an alternative to antibiotics.

Proteomic analysis of the colistin-resistant E. coli clinical isolate: Explorations of the resistome

Background: Antimicrobial resistance is a worldwide problem after the emergence of colistin resistance since it was the last option left to treat carbapenemase-resistant bacterial infections. The mcr gene and its variants are one of the causes for colistin resistance. Besides mcr genes, some other intrinsic genes are also involved in colistin resistance but still need to be explored. Objective: The aim of this study was to investigate differential proteins expression of colistin-resistant E. coli clinical isolate and to understand their interactive partners as future drug targets. Methods: In this study, we have employed the whole proteome analysis through LC-MS/MS. The advance proteomics tools were used to find differentially expressed proteins in the colistin-resistant Escherichia coli clinical isolate compared to susceptible isolate. Gene ontology and STRING were used for functional annotation and protein-protein interaction networks, respectively. Results: LC-MS/MS analysis showed overexpression of 47 proteins and underexpression of 74 proteins in colistin-resistant E. coli. These proteins belong to DNA replication, transcription and translational process; defense and stress related proteins; proteins of phosphoenol pyruvate phosphotransferase system (PTS) and sugar metabolism. Functional annotation and protein-protein interaction showed translational and cellular metabolic process, sugar metabolism and metabolite interconversion. Conclusion: We conclude that these protein targets and their pathways might be used to develop novel therapeutics against colistin-resistant infections. These proteins could unveil the mechanism of colistin resistance.

Ribosome Profiling and RNA Sequencing Reveal Genome-Wide Cellular Translation and Transcription Regulation Under Osmotic Stress in Lactobacillus rhamnosus ATCC 53103

To determine whether osmotic pressure affects the translation efficiency of Lactobacillus rhamnosus, the ribosome profiling assay was performed to analyze the changes in translation efficiency in L. rhamnosus ATCC 53103. Under osmotic stress, differentially expressed genes (DEGs) involved in fatty acid biosynthesis and metabolism, ribosome, and purine metabolism pathways were co-regulated with consistent expression direction at translation and transcription levels. DEGs involved in the biosynthesis of phenylalanine, tyrosine, and tryptophan, and the phosphotransferase system pathways also were co-regulated at translation and transcription levels, while they showed opposite expression direction at two levels. Moreover, DEGs involved in the two-component system, amino acid metabolism, and pyruvate metabolism pathways were only regulated at the transcription level. And DEGs involved in fructose and mannose metabolism were only regulated at the translation level. The translation efficiency of DEGs involved in the biosynthesis of amino acids was downregulated while in quorum sensing and PTS pathways was upregulated. In addition, the ribosome footprints accumulated in open reading frame regions resulted in impaired translation initiation and elongation under osmotic stress. In summary, L. rhamnosus ATCC 53103 could respond to osmotic stress by translation regulation and control the balance between survival and growth of cells by transcription and translation.