Phosphoenolpyruvate-dependent Phosphotransferase System Research Articles

Structure and mechanism of a phosphotransferase system glucose transporter

Glucose is the primary source of energy for many organisms and is efficiently taken up by bacteria through a dedicated transport system that exhibits high specificity. In Escherichia coli, the glucose-specific transporter IICBGlc serves as the major glucose transporter and functions as a component of the phosphoenolpyruvate-dependent phosphotransferase system. Here, we report cryo-electron microscopy (cryo-EM) structures of the glucose-bound IICBGlc protein. The dimeric transporter embedded in lipid nanodiscs was captured in the occluded, inward- and occluded, outward-facing conformations. Together with biochemical and biophysical analyses, and molecular dynamics (MD) simulations, we provide insights into the molecular basis and dynamics for substrate recognition and binding, including the gates regulating the binding sites and their accessibility. By combination of these findings, we present a mechanism for glucose transport across the plasma membrane. Overall, this work provides molecular insights into the structure, dynamics, and mechanism of the IICBGlc transporter in a native-like lipid environment.

Antibacterial characteristics and mechanistic insights of combined tea polyphenols, Nisin, and epsilon-polylysine against feline oral pathogens: a comprehensive transcriptomic and metabolomic analysis.

This study evaluates the antibacterial characteristics and mechanisms of combined tea polyphenols (TPs), Nisin, and ε-polylysine (PL) against Streptococcus canis, Streptococcus minor, Streptococcus mutans, and Actinomyces oris, common zoonotic pathogens in companion animals. Pathogenic strains were isolated from feline oral cavities and assessed using minimum inhibitory concentration (MIC) tests, inhibition zone assays, growth kinetics, and biofilm inhibition studies. Among single agents, PL exhibited the lowest MIC values against all four pathogens. TP showed significant resistance against S. minor, and Nisin against S. mutans. The combination treatment (Comb) of TP, Nisin, and PL in a ratio of 13:5:1 demonstrated broad-spectrum antibacterial activity, maintaining low MIC values, forming large inhibition zones, prolonging the bacterial lag phase, reducing growth rates, and inhibiting biofilm formation. RNA sequencing and metabolomic analysis indicated that TP, Nisin, and PL inhibited various membrane-bound carbohydrate-specific transferases through the phosphoenolpyruvate-dependent phosphotransferase system in S. canis, disrupting carbohydrate uptake. They also downregulated glycolysis and the citric acid cycle, inhibiting cellular energy metabolism. Additionally, they modulated the activities of peptidoglycan glycosyltransferases and d-alanyl-d-alanine carboxypeptidase, interfering with peptidoglycan cross-linking and bacterial cell wall stability. The Comb therapy significantly enhances antibacterial efficacy by targeting multiple bacterial pathways, offering potential applications in food and pharmaceutical antimicrobials.

Engineering PTS-based glucose metabolism for efficient biosynthesis of bacterial cellulose by Komagataeibacter xylinus

Bacterial cellulose (BC) is a renewable biomaterial that has attracted significant attention due to its excellent properties and wide applications. Komagataeibacter xylinus CGMCC 2955 is an important BC-producing strain. It primarily produces BC from glucose while simultaneously generating gluconic acid as a by-product, which acidifies the medium and inhibits BC synthesis. To enhance glucose uptake and BC synthesis, we reconstructed the phosphoenolpyruvate-dependent glucose phosphotransferase system (PTSGlc) and strengthened glycolysis by introducing heterologous genes, resulting in a recombinant strain (GX08PTS03; Δgcd::ptsHIcrrE. coli::ptsGE. coli::pfkAE. coli). Strain GX08PTS03 efficiently utilized glucose for BC production without accumulating gluconic acid. Subsequently, the fermentation process was systematically optimized. Under optimal conditions, strain GX08PTS03 produced 7.74 g/L of BC after 6 days of static fermentation, with a BC yield of 0.39 g/g glucose, which were 87.41 % and 77.27 % higher than those of the wild-type strain, respectively. The BC produced by strain GX08PTS03 exhibited a longer fiber diameter along with a lower porosity, significantly higher solid content, crystallinity, tensile strength, and Young's modulus. This study is novel in reporting that the engineered PTSGlc-based glucose metabolism could effectively enhance the production and properties of BC, providing a future outlook for the biopolymer industry.

Maribacter halichondriae sp. nov., isolated from the marine sponge Halichondria panicea, displays features of a sponge-associated life style

A new member of the family Flavobacteriaceae (termed Hal144T) was isolated from the marine breadcrumb sponge Halichondria panicea. Sponge material was collected in 2018 at Schilksee which is located in the Kiel Fjord (Baltic Sea, Germany). Phylogenetic analysis of the full-length Hal144T 16S rRNA gene sequence revealed similarities from 94.3 to 96.6% to the nearest type strains of the genus Maribacter. The phylogenetic tree of the 16S rRNA gene sequences depicted a cluster of strain Hal144T with its closest relatives Maribacter aestuarii GY20T (96.6%) and Maribacter thermophilus HT7-2T (96.3%). Genome phylogeny showed that Maribacter halichondriae Hal144T branched from a cluster consisting of Maribacter arenosus, Maribacter luteus, and Maribacter polysiphoniae. Genome comparisons of strain Maribacter halichondriae Hal144T with Maribacter sp. type strains exhibited average nucleotide identities in the range of 75–76% and digital DNA-DNA hybridisation values in the range of 13.1–13.4%. Compared to the next related type strains, strain Hal144T revealed unique genomic features such as phosphoenolpyruvate-dependent phosphotransferase system pathway, serine-glyoxylate cycle, lipid A 3-O-deacylase, 3-hexulose-6-phosphate synthase, enrichment of pseudogenes and of genes involved in cell wall and envelope biogenesis, indicating an adaptation to the host. Strain Hal144T was determined to be Gram-negative, mesophilic, strictly aerobic, flexirubin positive, resistant to aminoglycoside antibiotics, and able to utilize N-acetyl-β-D-glucosamine. Optimal growth occurred at 25–30 °C, within a salinity range of 2–6% sea salt, and a pH range between 5 and 8. The major fatty acids identified were C17:0 3-OH, iso-C15:0, and iso-C15:1 G. The DNA G + C content of strain Hal144T was 41.4 mol%. Based on the polyphasic approach, strain Hal144T represents a novel species of the genus Maribacter, and we propose the name Maribacter halichondriae sp. nov. The type strain is Hal144T (= DSM 114563T = LMG 32744T).

Green Tea-Derived Catechins Suppress the Acid Productions of Streptococcus mutans and Enhance the Efficiency of Fluoride

Green tea-derived catechins, which can be divided into galloylated (epicatechin gallate: ECG, epigallocatechin gallate: EGCG) and non-galloylated (catechin: C, epicatechin: EC, epigallocatechin: EGC) catechins, are considered to be the main contributors to the caries control potential of green tea. In this study, we intended to compare the antimicrobial effects of these representative green tea-derived catechins and their combined effects with fluoride on the acid production and aggregation of Streptococcus mutans. The effects of different catechins on the growth, aggregation and acid production of S. mutans, and the combined effect of catechins and potassium fluoride (2 m<sc>m</sc> at pH 7.0, 0.3 m<sc>m</sc> at pH 5.5) on S. mutans acid production were measured by anaerobic culture, turbidity changes due to aggregation, and pH-stat methods. Molecular docking simulations were also performed to investigate the interactions between catechins and membrane-embedded enzyme II complex (EIIC), a component of the phosphoenolpyruvate-dependent phosphotransferase system (sugar uptake-related enzyme). ECG or EGCG at 1 mg/mL significantly inhibited the growth of S. mutans, induced bacterial aggregation, and decreased glucose-induced acid production (p < 0.05). All catechins were able to bind to EIIC in silico, in the following order of affinity: EGCG, ECG, EGC, EC, and C. Furthermore, they enhanced the inhibitory effects of fluoride at pH 5.5 and significantly inhibited S. mutans acid production by 47.5–86.6% (p < 0.05). These results suggest that both galloylated and non-galloylated catechins exhibit antimicrobial activity, although the former type demonstrates stronger activity, and that the caries control effects of green tea may be due to the combined effects of multiple components, such as catechins and fluoride. The detailed mechanisms underlying these phenomena and the in vivo effect need to be explored further.

Erythritol alters gene transcriptome signatures, cell growth, and biofilm formation in Staphylococcus pseudintermedius

BackgroundErythritol was found to inhibit the growth of microorganisms. The present study aimed to demonstrate the growth inhibition of Staphylococcus pseudintermedius by erythritol and to define the changes in gene transcription signatures induced by erythritol. Changes in the gene transcription profiles were analysed by RNA sequencing and quantitative reverse transcription PCR. Gene ontology analysis was performed to assign functional descriptions to the genes.ResultsErythritol inhibited S. pseudintermedius growth in a dose-dependent manner. We then performed a transcriptome analysis of S. pseudintermedius with and without 5% (w/w) erythritol exposure to validate the mechanism of growth inhibition. We revealed that erythritol induced up-regulation of three genes (ptsG, ppdK, and ppdkR) that are related to the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS). Glucose supplementation restored the up-regulation of the PTS-related genes in response to erythritol. In addition, erythritol down-regulated eleven genes that are located in a single pur-operon and inhibited biofilm formation of S. pseudintermedius.ConclusionsThese findings indicated that erythritol antagonistically inhibits PTS-mediated glucose uptake, thereby exerting a growth inhibitory effect on S. pseudintermedius. Moreover, erythritol inhibits the ‘de novo’ IMP biosynthetic pathway that may contribute to biofilm synthesis in S. pseudintermedius.

Growth response of Bacillus coagulans on TiO2 nanoparticles by RNA-Seq analysis of the customary probiotic strain

The phosphoenolpyruvate sugar phosphotransferase systems in bacteria are complex enzyme systems carrying out various important functions like detection, phosphorylation and transport of several sugar substrates including mono and disaccharides, amino sugars, etc. This key enzyme complex prevents the efflux of sugar substrates by phosphorylation and prepares them for energy production and metabolism. In this study, a high throughput RNA sequencing method was used to compare the gene expression in Bacillus coagulans treated with nano titanium dioxide (TiO2) rutile. Out of 3175 genes, 499 were differentially expressed. Gene Ontology analysis revealed 239 significant genes. In the biological process category, we found the phosphoenolpyruvate-dependent sugar phosphotransferase systems are the dominant ones and are involved in the uptake of specific carbohydrate sources. In our previous study, we showed increased growth of bacteria in presence of nano TiO2 anatase with a sharp rise in ATP concentrations. We know that, among metal oxide-based nanoparticles, TiO2 is placed at the topmost position in the agri-food sector based on its use and their reactivity with human beneficial gut flora, commonly known as probiotics, becoming a major concern. Hence, the interaction of TiO2 rutile nanoparticles on Bacillus coagulans was studied thoroughly at the transcriptomics level; with depth.

Glucose Transport in Escherichia coli: From Basics to Transport Engineering.

Escherichia coli is the best-known model for the biotechnological production of many biotechnological products, including housekeeping and heterologous primary and secondary metabolites and recombinant proteins, and is an efficient biofactory model to produce biofuels to nanomaterials. Glucose is the primary substrate used as the carbon source for laboratory and industrial cultivation of E. coli for production purposes. Efficient growth and associated production and yield of desired products depend on the efficient sugar transport capabilities, sugar catabolism through the central carbon catabolism, and the efficient carbon flux through specific biosynthetic pathways. The genome of E. coli MG1655 is 4,641,642 bp, corresponding to 4702 genes encoding 4328 proteins. The EcoCyc database describes 532 transport reactions, 480 transporters, and 97 proteins involved in sugar transport. Nevertheless, due to the high number of sugar transporters, E. coli uses preferentially few systems to grow in glucose as the sole carbon source. E. coli nonspecifically transports glucose from the extracellular medium into the periplasmic space through the outer membrane porins. Once in periplasmic space, glucose is transported into the cytoplasm by several systems, including the phosphoenolpyruvate-dependent phosphotransferase system (PTS), the ATP-dependent cassette (ABC) transporters, and the major facilitator (MFS) superfamily proton symporters. In this contribution, we review the structures and mechanisms of the E. coli central glucose transport systems, including the regulatory circuits recruiting the specific use of these transport systems under specific growing conditions. Finally, we describe several successful examples of transport engineering, including introducing heterologous and non-sugar transport systems for producing several valuable metabolites.

The molecular mechanism for carbon catabolite repression of the chitin response in Vibrio cholerae.

Vibrio cholerae is a facultative pathogen that primarily occupies marine environments. In this niche, V. cholerae commonly interacts with the chitinous shells of crustacean zooplankton. As a chitinolytic microbe, V. cholerae degrades insoluble chitin into soluble oligosaccharides. Chitin oligosaccharides serve as both a nutrient source and an environmental cue that induces a strong transcriptional response in V. cholerae. Namely, these oligosaccharides induce the chitin sensor, ChiS, to activate the genes required for chitin utilization and horizontal gene transfer by natural transformation. Thus, interactions with chitin impact the survival of V. cholerae in marine environments. Chitin is a complex carbon source for V. cholerae to degrade and consume, and the presence of more energetically favorable carbon sources can inhibit chitin utilization. This phenomenon, known as carbon catabolite repression (CCR), is mediated by the glucose-specific Enzyme IIA (EIIAGlc) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). In the presence of glucose, EIIAGlc becomes dephosphorylated, which inhibits ChiS transcriptional activity by an unknown mechanism. Here, we show that dephosphorylated EIIAGlc interacts with ChiS. We also isolate ChiS suppressor mutants that evade EIIAGlc-dependent repression and demonstrate that these alleles no longer interact with EIIAGlc. These findings suggest that EIIAGlc must interact with ChiS to exert its repressive effect. Importantly, the ChiS suppressor mutations we isolated also relieve repression of chitin utilization and natural transformation by EIIAGlc, suggesting that CCR of these behaviors is primarily regulated through ChiS. Together, our results reveal how nutrient conditions impact the fitness of an important human pathogen in its environmental reservoir.

Non-redundant functionality of Lactiplantibacillus plantarum phospho-β-glucosidases revealed by carbohydrate utilization signatures associated to pbg2 and pbg4 gene mutants.

To increase our knowledge on the functionality of 6-phospho-β-glucosidases linked to phosphoenolpyruvate-dependent phosphotransferase systems (PTS) that are encountered in high redundancy in the Lactiplantibacillus plantarum WCFS1 genome. Two L. plantarum WCFS1 gene mutants that lacked one of the 6-phospho-β-glucosidases, ∆pbg2 (or ∆lp_0906) or ∆pbg4 (or ∆lp_2777) were constructed and the metabolic impact of these mutations assessed by high-throughput phenotyping (Omnilog). The ∆pbg2 mutant displayed a reduced metabolic performance, having lost the capacity to utilize 20 out of 57 carbon (C)-sources used by the wild-type strain. Conversely, the ∆pbg4 mutant conserved the capacity to metabolize most of the C-sources preferred by the wild type strain. This mutant utilized 56 C-sources albeit the range of substrates used and hence its metabolic profiling differed from that of the WCFS1 strain. The ∆pbg2 mutant notably reduced or abolished the capacity to metabolize substrates related to pentose and glucoronate interconversions and was unable to assimilate fatty acids or nucleosides as sole C-sources for growth. The ∆pbg4 mutant acquired the capacity to utilize efficiently glycogen, indicating an efficient supply of glucose from this source. Lactiplantibacillus plantarum gene mutants that lack individual 6-phospho-β-glucosidases display very different carbohydrate utilization signatures showing that these enzymes can be crucial to determine the capacity of L. plantarum to consume different C-sources and hence for the nutrition and physiology of this microorganism.

Confirmation of Glucose Transporters through Targeted Mutagenesis and Transcriptional Analysis in Clostridium acetobutylicum

The solvent-producing bacterium Clostridium acetobutylicum is able to grow on a variety of carbohydrates. The main hexose transport system is the phosphoenolpyruvate-dependent phosphotransferase system (PTS). When the gene glcG that encodes the glucose transporter was inactivated, the resulting mutant glcG::int(1224) grew as well as the wild type, yet its glucose consumption was reduced by 17% in a batch fermentation. Transcriptomics analysis of the phosphate-limited continuous cultures showed that the cellobiose transporter GlcCE was highly up-regulated in the mutant glcG::int(1224). The glcCE mutation did not affect growth and even consumed slightly more glucose during solventogenesis growth compared to wild type, indicating that GlcG is the primary glucose-specific PTS. Poor growth of the double mutant glcG::int(1224)-glcCE::int(193) further revealed that GlcCE was the secondary glucose PTS and that there must be other PTSs capable of glucose uptake. The observations obtained in this study provided a promising foundation to understand glucose transport in C. acetobutylicum.

Stably transmitted defined microbial community in honeybees preserves Hafnia alvei inhibition by regulating the immune system.

The gut microbiota of honeybees is highly diverse at the strain level and essential to the proper function and development of the host. Interactions between the host and its gut microbiota, such as specific microbes regulating the innate immune system, protect the host against pathogen infections. However, little is known about the capacity of these strains deposited in one colony to inhibit pathogens. In this study, we assembled a defined microbial community based on phylogeny analysis, the 'Core-20' community, consisting of 20 strains isolated from the honeybee intestine. The Core-20 community could trigger the upregulation of immune gene expressions and reduce Hafnia alvei prevalence, indicating immune priming underlies the microbial protective effect. Functions related to carbohydrate utilization and the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS systems) are represented in genomic analysis of the defined community, which might be involved in manipulating immune responses. Additionally, we found that the defined Core-20 community is able to colonize the honeybee gut stably through passages. In conclusion, our findings highlight that the synthetic gut microbiota could offer protection by regulating the host immune system, suggesting that the strain collection can yield insights into host-microbiota interactions and provide solutions to protect honeybees from pathogen infections.

Residual Helicity at the Active Site of the Histidine Phosphocarrier, HPr, Modulates Binding Affinity to Its Natural Partners.

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr9–30), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr9–30 is mainly disordered. We attempted to improve the affinity of HPr9–30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr9–30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. Therefore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.

Capacity of soybean carbohydrate metabolism in Leuconostoc mesenteroides, Lactococcus lactis and Streptococcus thermophilus

Few studies have focused on the capacity of lactic acid bacteria in utilizing soybean carbohydrates by genetic and phenotypic approaches. In the current study, genetic and phenotypic characteristics of soybean carbohydrate metabolism in Leuconostoc mesenteroides, Lactococcus lactis and Streptococcus thermophilus were investigated. The results indicated that all the 3 species have the potential capacity to utilize sucrose with a sucrose phosphoenolpyruvate-dependent phosphotransferase system (PTS) pathway mainly consisted of scrA, sacA, scrK and scrR. Among the 15 strains, only 1 L. lactis strain possessed both sucrose permease and PTS pathway. On the other hand, only Leu. mesenteroides among the 3 species have the genes related to α-galactosidases for potential capacity to metabolize raffinose-family oligosaccharides (RFO) in soybean. Four Leu. mesenteroides strains, DQHXN_Q03M16, FSDLZ60M2, DSCAB2M6 and DQHXN_Q38M5, possessed the complete α-galactosidases gene clusters of lacS-galA-galK-galT and high α-galactosidases activity, which contributed to the capacity of these strains to utilize RFO. These findings provide an approach to investigate the genetic and the phenotypic characteristics of Leu. mesenteroides, L. lactis and S. thermophilus in soybean carbohydrate metabolism for suitable strain selection in soybean fermentation.

Real-time PCR method for qualitative and quantitative detection of Lactobacillus sakei group species targeting novel markers based on bioinformatics analysis

Latilactobacillus sakei group comprises four closely related species, making it difficult to accurately distinguish them with standard markers such as the 16S rRNA gene. The objective of our study was to mine novel markers for PCR detection and discrimination of L. sakei group species and L. sakei subspecies by comparative pan-genomic analysis. A total of 63 genome sequences of L. sakei group species consisted of 119,899 coding genes, yielding 5741 pan-genomes, 831 core-genomes, 3347 accessory-genomes, and 1563 unique-genomes. The accessory-genome was compared to extract unique candidate genes common only to genomes of the same species. The candidate genes were then aligned with the other bacterial genomes to select marker genes present in all genomes of a given species, but not in the genomes of other species. We identified the arginine/ornithine antiporter, putative cell surface protein precursor, sodium:solute symporter, PRD domain protein, PTS sugar transporter subunit IIC, and phosphoenolpyruvate-dependent sugar phosphotransferase system EIIC as marker genes for L. sakei, L. sakei subsp. sakei, L. sakei subsp. carnosus, L. curvatus, L. graminis, and L. fuchuensis, respectively. Primer pairs were designed for each marker and showed 100% specificity for 48 lactic acid bacterial reference strains. The PCR method developed in this study was used to evaluate 106 strains isolated from fermented foods to demonstrate that the marker genes provided a viable alternative to the 16S rRNA gene. We also applied the method to the monitoring of kimchi samples to quantify L. sakei group species or subspecies. Our PCR method based on novel markers can rapidly identify L. sakei group with high accuracy and high throughput.

Green Tea-Derived Epigallocatechin Gallate Inhibits Acid Production and Promotes the Aggregation of Streptococcus mutans and Non-Mutans Streptococci

It has been suggested that green tea-derived epigallocatechin gallate (EGCG), which has antimicrobial properties, might help prevent dental caries. However, the detailed properties of EGCG remain unclear. In this study, the antimicrobial properties of EGCG were evaluated by examining its bactericidal activity, its inhibitory effects against bacterial growth, acid production, acidic end-product formation, and sugar uptake (phosphoenolpyruvate-dependent phosphotransferase system, PEP-PTS activity), and its effects on bacterial aggregation, using monocultured planktonic cells of Streptococcus mutans and non-mutans streptococci. Coincubating S. mutans with EGCG (1 mg/mL) for 4 h had no bactericidal effects, while it decreased the growth and acid production of S. mutans by inhibiting the activity of the PEP-PTS. EGCG (2 mg/mL) caused rapid bacterial cell aggregation and had reduced the optical density of S. mutans cell suspension by 86.7% at pH 7.0 and 90.7% at pH 5.5 after 2 h. EGCG also reduced the acid production of non-mutans streptococci, including S. sanguinis, S. gordonii, and S. salivarius, and promoted the aggregation of these non-mutans streptococci. Furthermore, these antimicrobial effects of short-term EGCG treatment persisted in the presence of saliva. These results suggest that EGCG might have short-term antibacterial effects on caries-associated streptococci in the oral cavity.

Genetic Mutations That Confer Fluoride Resistance Modify Gene Expression and Virulence Traits of Streptococcus mutans.

Fluoride is an inorganic monatomic anion that is widely used as an anti-cariogenic agent for the control of caries development. The aims of this study were to identify the mutated genes that give rise to fluoride-resistant (FR) strains of the cariogenic pathogen Streptococcus mutans and explore how genetic alterations in the genome of an S. mutans FR strain optimize the metabolism(s) implicated in the expression of virulence-associated traits. Here, we derived an S. mutans FR strain from a wild-type UA159 strain by continuous shifts to a medium supplemented with increasing concentrations of fluoride. The FR strain exhibited a slow growth rate and low yield under aerobic and oxidative stress conditions and was highly sensitive to acid stress. Notably, microscopy observation displayed morphological changes in which the FR strain had a slightly shorter cell length. Next, using the sequencing analyses, we found six mutations in the FR genome, which decreased the gene expression of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Indeed, the ability to intake carbohydrates was relatively reduced in the FR strain. Collectively, our results provide evidence that the genetic mutations in the genome of the FR strain modulate the expression of gene(s) for carbon metabolism(s) and cellular processes, leading to diminished fitness with respect to virulence and persistence.

Composition and potential functions of the dominant microbiota in deep-sea hagfish gut from the South China Sea

Hagfishes, the most ancient jawless fishes, have a pivotal biological status. Gut bacterial communities are considered to be another organ that supports host survival and reproduction, while most gut microbiota cannot be cultured in vitro. In this study, four deep-sea hagfish individuals were collected from the South China Sea (>900 m). 16S rRNA and metagenomics approaches were used to analyze the gut microbiota assembly and the functions of the dominant microbes. The results revealed 16 phyla and 21 genera (relative abundance ≥ 0.5%) of microbes in total. Ureaplasma and Arcobacter were the dominant genera across all the gut samples, and the microbiota diversity showed no significant differences among the different gut regions. To investigate the potential functions of the dominant microbial taxa, the draft genomes of Ureaplasma (Hg1 and Hg2) and Arcobacter (LC1) from the midgut and hindgut were successfully recovered. The Hg1 and Hg2 genomes were enriched in genes encoding phosphoenolpyruvate-dependent phosphotransferase systems (PTSs) and sodium-driven symporters responsible for the uptake of sugars and other carbohydrates. The functional repertoire of Ureaplasma was likely to help the host in the metabolism of multiple carbohydrates, especially chitin degradation. The LC1 genome harbored genes encoding a restriction-methylation system, two CRISPR-Cas systems and multiple toxin-antitoxin systems. The enrichment of genes related to bacterial defense indicated that the functions of LC1 may involve protection against harmful or foreign microorganisms and balancing the host intestinal flora. These results hint that genetic and functional diversification might be present among Ureaplasma and Arcobacter. Though the exact functions of the dominant microbes remain to be further identified, our results allow us a glimpse into hagfish-microbe alliances in deep-sea ecosystems.

Effect of carbon catabolite repression on lactose and galactose catabolism in Lacticaseibacillus paracasei

Previous research has identified phosphoenolpyruvate-dependent phosphotransferase system and tagatose-6-phosphate (T6P) pathway of lactose/galactose metabolism in Lacticaseibacillus casei groups, which led to no galactose accumulation in fermented milk produced by Lb. paracasei. However, Lb. paracasei preferred lactose to galactose when grown in a mixture of the two sugars, indicating possibility of carbon catabolite repression of galactose catabolism in the fermented milk. Therefore, galactose accumulation was still observed during co-culturing of Lb. paracasei with Streptococcus thermophilus in fermented milk. We demonstrated that the transcriptional regulator lacR acts as a transcriptional repressor of the T6P pathway genes (lacA, lacC and lacD) and lacT acts as a transcriptional activator for genes (lacE) encoding the lactose-transporting PTS in the mixed lactose/galactose CDM medium, indicating possible dual repressive effects of global or local regulation on the carbon catabolism of galactose in Lb. paracasei.

Deciphering the Aldolase Function of STM3780 from a Bovine Enteric Infection-Related Gene Cluster in Salmonella enterica Serotype Typhimurium.

Non-typhoidal Salmonella are capable of colonizing livestock and humans, where they can progressively cause disease. Previously, a library of targeted single-gene deletion mutants of Salmonella enterica serotype Typhimurium was inoculated to ligated ileal loops in calves to identify genes under selection. Of those genes identified, a cluster of genes is related to carbohydrate metabolism and transportation. It is proposed that an incoming carbohydrate is first phosphorylated by a phosphoenolpyruvate-dependent phosphotransferase system. The metabolite is further phosphorylated by the kinase STM3781 and then cleaved by the aldolase STM3780. STM3780 is functionally annotated as a class II fructose-bisphosphate aldolase. The aldolase was purified to homogeneity, and its aldol condensation activity with a range of aldehydes was determined. In the condensation reaction, STM3780 was shown to catalyze the abstraction of the pro-S hydrogen from C3 of dihydroxyacetone and subsequent formation of a carbon-carbon bond with S stereochemistry at C3 and R stereochemistry at C4. The best aldehyde substrate was identified as l-threouronate. Surprisingly, STM3780 was also shown to catalyze the condensation of two molecules of dihydroxyacetone phosphate to form the branched carbohydrate dendroketose bisphosphate.