Activation of a silent lactose utilization pathway in an evolved Listeria monocytogenes F2365 outbreak isolate.

Listeria monocytogenes, a widespread food-borne pathogen, utilizes diverse growth substrates including mono- and di-saccharides via PEP-phosphotransferase (PTS) systems. We evaluated a collection of L. monocytogenes isolates of different origins for their ability to utilize lactose, a disaccharide composed of galactose and glucose and the main carbon source in milk and dairy products. Notably, the dairy-associated outbreak strain F2365 could not utilize lactose efficiently, conceivably due to a frameshift mutation (lacR887del) resulting in a truncated LacR. Transcriptional activator LacR is involved in the expression of two PTS systems, encoded by the lpo operon lmo1718-1720 in combination with lmo2708 and the lmo2683-2685 operon, and linked to lactose and/or cellobiose metabolism in L. monocytogenes. Via experimental evolution of the ancestral strain F2365, an evolved isolate F2365 EV was obtained which showed enhanced growth and metabolism of lactose. Using the lactose-positive model strain L. monocytogenes EGDe as a control, HPLC experiments showed that EGDe and F2365 EV could consume lactose and utilize the glucose moiety, while the galactose moiety was exported from the cells. Genome sequencing of F2365 EV found the original lacR887del mutation was still present but an additional point mutation lmo2766C415T had occurred, resulting in an amino acid substitution in the putative regulator Lmo2766. The lmo2766 gene is located next to operon lmo2761-2765 with putative PTS genes in the genome. Notably, comparative RNAseq analysis confirmed that the lmo2761-2765 operon was strongly upregulated in F2365 EV in the presence of lactose but not in EGDe and F2365. Conversely, the LacR-regulated lpo operon, lmo2708, and lmo2683-2685 operon were only upregulated in EGDe. Additional growth and HPLC experiments, using mutants constructed in lactose-positive L. monocytogenes EGDe, showed reduced growth of the EGDe lacR887del mutant with no utilization of lactose, while the double mutant EGDe lacR887dellmo2766C415T showed enhanced growth and lactose utilization. Hence, these results demonstrate that an amino acid substitution in the Lmo2766 regulator activates a previously silent lactose utilization pathway encoded by PTS operon lmo2761-2765, facilitating the growth and metabolism of L. monocytogenes with lactose as a substrate. This finding enhances our understanding of the metabolic capabilities and adaptability of L. monocytogenes, offering a broader view of the lactose utilization capacity of this pathogen.

The Streptococcus pyogenes stand-alone regulator RofA exhibits characteristics of a PRD-containing virulence regulator.

Streptococcus pyogenes [group A streptococcus (GAS)] is a human pathogen capable of infecting diverse tissues. To successfully infect these sites, GAS must detect available nutrients and adapt accordingly. The phosphoenolpyruvate transferase system (PTS) mediates carbohydrate uptake and metabolic gene regulation to adapt to the nutritional environment. Regulation by the PTS can occur through phosphorylation of transcriptional regulators at conserved PTS-regulatory domains (PRDs). GAS has several PRD-containing stand-alone regulators with regulons encoding both metabolic genes and virulence factors [PRD-containing virulence regulators (PCVRs)]. One is RofA, which regulates the expression of virulence genes in multiple GAS serotypes. It was hypothesized that RofA is phosphorylated by the PTS in response to carbohydrate levels to coordinate virulence gene expression. In this study, the RofA regulon of M1T1 strain 5448 was determined using RNA sequencing. Two operons were consistently differentially expressed across growth in the absence of RofA; the pilus operon was downregulated, and the capsule operon was upregulated. This correlated with increased capsule production and decreased adherence to keratinocytes. Purified RofA-His was phosphorylated in vitro by PTS proteins EI and HPr, and phosphorylated RofA-FLAG was detected in vivo when GAS was grown in low-glucose C medium. Phosphorylated RofA was not observed when C medium was supplemented 10-fold with glucose. Mutations of select histidine residues within the putative PRDs contributed to the in vivo phosphorylation of RofA, although phosphorylation of RofA was still observed, suggesting other phosphorylation sites exist in the protein. Together, these findings support the hypothesis that RofA is a PCVR that may couple sugar metabolism with virulence regulation.

Genetic characterization of glyoxalase pathway in oral streptococci and its contribution to interbacterial competition.

Objectives: To analyze contributions to microbial ecology of Reactive Electrophile Species (RES), including methylglyoxal, generated during glycolysis. Methods: Genetic analyses were performed on the glyoxalase pathway in Streptococcus mutans (SM) and Streptococcus sanguinis (SS), followed by phenotypic assays and transcription analysis. Results: Deleting glyoxalase I (lguL) reduced RES tolerance to a far greater extent in SM than in SS, decreasing the competitiveness of SM against SS. Although SM displays a greater RES tolerance than SS, lguL-null mutants of either species showed similar tolerance; a finding consistent with the ability of methylglyoxal to induce the expression of lguL in SM, but not in SS. A novel paralogue of lguL (named gloA2) was identified in most streptococci. SM mutant ∆gloA2SM showed little change in methylglyoxal tolerance yet a significant growth defect and increased autolysis on fructose, a phenotype reversed by the addition of glutathione, or by the deletion of a fructose: phosphotransferase system (PTS) that generates fructose-1-phosphate (F-1-P). Conclusions: Fructose contributes to RES generation in a PTS-specific manner, and GloA2 may be required to degrade certain RES derived from F-1-P. This study reveals the critical roles of RES in fitness and interbacterial competition and the effects of PTS in modulating RES metabolism.

Elucidation of the Mechanisms of Inter-domain Coupling in the Monomeric State of Enzyme I by High-pressure NMR.

The catalytic cycle of Enzyme I (EI), a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate, is characterized by a series of local and global conformational rearrangements. This multistep process includes a monomer-to-dimer transition, followed by an open-to-closed rearrangement of the dimeric complex upon PEP binding. In the present study, we investigate the thermodynamics of EI dimerization using a range of high-pressure solution NMR techniques complemented by SAXS experiments. 1H-15N TROSY and 1H-13C methyl TROSY NMR spectra combined with 15N relaxation measurements revealed that a native-like engineered variant of full-length EI fully dissociates into stable monomeric state above 1.5 kbar. Conformational ensembles of EI monomeric state were generated via a recently developed protocol combining coarse-grained molecular simulations with experimental backbone residual dipolar coupling measurements. Analysis of the structural ensembles provided detailed insights into the molecular mechanisms driving formation of the catalytically competent dimeric state, and reveals that each step of EI catalytical cycle is associated with a significant reduction in either inter- or intra-domain conformational entropy. Altogether, this study completes a large body work conducted by our group on EI and establishes a comprehensive structural and dynamical description of the catalytic cycle of this prototypical multidomain, oligomeric enzyme.

Integrated Genomics and Transcriptomics Provide Insights into Salt Stress Response in Bacillus subtilis ACP81 from Moso Bamboo Shoot (Phyllostachys praecox) Processing Waste.

Salt stress is detrimental to the survival of microorganisms, and only a few bacterial species produce hydrolytic enzymes. In this study, we investigated the expression of salt stress-related genes in the salt-tolerant bacterial strain Bacillus subtilis ACP81, isolated from bamboo shoot processing waste, at the transcription level. The results indicate that the strain could grow in 20% NaCl, and the sub-lethal concentration was 6% NaCl. Less neutral protease and higher cellulase and ß-amylase activities were observed for B. subtilis ACP81 under sub-lethal concentrations than under the control concentration (0% NaCl). Transcriptome analysis showed that the strain adapted to high-salt conditions by upregulating the expression of genes involved in cellular processes (membrane synthesis) and defense systems (flagellar assembly, compatible solute transport, glucose metabolism, and the phosphotransferase system). Interestingly, genes encoding cellulase and ß-amylase-related (malL, celB, and celC) were significantly upregulated and were involved in starch and sucrose metabolic pathways, and the accumulated glucose was effective in mitigating salt stress. RT-qPCR was performed to confirm the sequencing data. This study emphasizes that, under salt stress conditions, ACP81 exhibits enhanced cellulase and ß-amylase activities, providing an important germplasm resource for saline soil reclamation and enzyme development.

Minimizing acetate formation from overflow metabolism in Escherichia coli: comparison of genetic engineering strategies to improve robustness toward sugar gradients in large-scale fermentation processes.

Introduction: Escherichia coli, a well characterized workhorse in biotechnology, has been used to produce many recombinant proteins and metabolites, but have a major drawback in its tendency to revert to overflow metabolism. This phenomenon occurs when excess sugar triggers the production of mainly acetate under aerobic conditions, a detrimental by-product that reduces carbon efficiency, increases cell maintenance, and ultimately inhibits growth. Although this can be prevented by controlled feeding of the sugar carbon source to limit its availability, gradients in commercial-scale bioreactors can still induce it in otherwise carbon-limited cells. While the underlying mechanisms have been extensively studied, these have mostly used non-limited cultures. In contrast, industrial production typically employs carbon-limited processes, which results in a substantially different cell physiology. Objective: The objective of this study was to evaluate and compare the efficiency of different metabolic engineering strategies with the aim to reduce overflow metabolism and increase the robustness of an industrial 2'-O-fucosyllactose producing strain under industrially relevant conditions. Methods: Three distinct metabolic engineering strategies were compared: i) alterations to pathways leading to and from acetate, ii) increased flux towards the tricarboxylic acid (TCA) cycle, and iii) reduced glucose uptake rate. The engineered strains were evaluated for growth, acetate formation, and product yield under non-limiting batch conditions, carbon limited fed-batch conditions, and after a glucose pulse in fed-batch mode. Results and Discussion: The findings demonstrated that blockage of the major acetate production pathways by deletion of the pta and poxB genes or increased carbon flux into the TCA cycle by overexpression of the gltA and deletion of the iclR genes, were efficient ways to reduce acetate accumulation. Surprisingly, a reduced glucose uptake rate did not reduce acetate formation despite it having previously been shown as a very effective strategy. Interestingly, overexpression of gltA was the most efficient way to reduce acetate accumulation in non-limited cultures, whereas disruption of the poxB and pta genes was more effective for carbon-limited cultures exposed to a sudden glucose shock. Strains from both strategies showed increased tolerance towards a glucose pulse during carbon-limited growth indicating feasible ways to engineer industrial E. coli strains with enhanced robustness.

The Klebsiella mannose phosphotransferase system promotes proliferation and the production of extracellular polymeric substances from mannose, facilitating adaptation to the host environment.

OBJECTIVES: Klebsiella spp., an opportunistic infectious organism, is commensal in the nasal and oral cavities of humans. Recently, it has been reported that oral Klebsiella spp. ectopically colonize the intestinal tract and induce the accumulation of intestinal Th1 cells. For oral bacteria to colonize the intestinal tract, they need to compete for nutrients with indigenous intestinal bacteria. Therefore, we focused on mannose, a mucus-derived sugar, and the mannose phosphotransferase system (PTS) (ManXYZ), a mechanism for mannose uptake, in terms of their effects on intestinal colonization and immune responses to Klebsiella spp. METHODS: We generated a Klebsiella manXYZ-deficient strain and investigated whether the utilization of intestinal mucus-derived sugars is associated with the growth. Furthermore, we examine the virulence of this organism in the mouse intestinal tract, especially the ability to colonize the host using competition assay. RESULTS: We found that Klebsiella ManXYZ is a PTS that specifically takes up mannose and glucosamine. Through ManXYZ, mannose was used for bacterial growth and the upregulated production of extracellular polymeric substances. In vivo competition assays showed that mannose metabolism promoted intestinal colonization. However, ManXYZ was not involved in Th1 and Th17 induction in the intestinal tract. CONCLUSION: The fundamental roles of ManXYZ were to ensure that mannose, which is present in the host, is made available for bacterial growth, to promote the production of extracellular polymeric substances, thus facilitating bacterial adaptation to the host environment.

Purine and carbohydrate availability drive Enterococcus faecalis fitness during wound and urinary tract infections.

IMPORTANCE: Although E. faecalis is a common wound pathogen, its pathogenic mechanisms during wound infection are unexplored. Here, combining a mouse wound infection model with in vivo transposon and RNA sequencing approaches, we identified the E. faecalis purine biosynthetic pathway and galactose/mannose MptABCD phosphotransferase system as essential for E. faecalis acute replication and persistence during wound infection, respectively. The essentiality of purine biosynthesis and the MptABCD PTS is driven by the consumption of purine metabolites by E. faecalis during acute replication and changing carbohydrate availability during the course of wound infection. Overall, our findings reveal the importance of the wound microenvironment in E. faecalis wound pathogenesis and how these metabolic pathways can be targeted to better control wound infections.

Identification of a Novel Lactose-Specific PTS Operon in Bacillus licheniformis and Development of Derivative Artificial Operon Modules.

Bacillus licheniformis plays a crucial role as a microbial host in the food industry and shows promising potential as a probiotic for human intestinal regulation. It exhibits a remarkable ability to utilize lactose as its sole carbon source. Despite its significance, the lactose-related metabolic pathway in this strain remains unclear. In this study, we identified a novel lactose-specific operon (lacDCAB) in B. licheniformis, consisting of the lacD gene that encodes a unique 6-phospho-ß-galactosidase belonging to the GH4 family, and the lacCAB genes encoding a lactose-specific PTS1 system. Notably, we constructed and assessed an array library of transport and catabolic modules specifically for lactose utilization. Among these modules, PDS-lacD-P2-pts1 demonstrated the highest specific lactose consumption rate of 0.64 g/(L·h·OD), which was 8 times higher than that of the control strain. Furthermore, we developed a dual carbon source transport model based on the PDS-lacD-P2-pts1 assembly module, which highlighted efficient coutilization of glucose/sucrose, lactose/sucrose, lactose/galactose, and lactose/2,3-butanediol. This study provides insight into the lactose-specific metabolic pathway of B. licheniformis and presents a promising strategy for enhancing lactose utilization efficiency and mixed carbon source coutilization.

Utilization of glycoprotein-derived N-acetylglucosamine-L-asparagine during Enterococcus faecalis infection depends on catabolic and transport enzymes of the glycosylasparaginase locus.

Enterococcus faecalis is a Gram-positive clinical pathogen causing severe infections. Its survival during infection depends on its ability to utilize host-derived metabolites, such as protein-deglycosylation products. We have identified in E. faecalis OG1RF a locus (ega) involved in the catabolism of the glycoamino acid N-acetylglucosamine-L-asparagine. This locus is separated into two transcription units, genes egaRP and egaGBCD1D2, respectively. RT-qPCR experiments revealed that the expression of the ega locus is regulated by the transcriptional repressor EgaR. Electromobility shift assays evidenced that N-acetylglucosamine-L-asparagine interacts directly with the EgaR protein, which leads to the transcription of the ega genes. Growth studies with egaG, egaB and egaC mutants confirmed that the encoded proteins are necessary for N-acetylglucosamine-L-asparagine catabolism. This glycoamino acid is transported and phosphorylated by a specific phosphotransferase system EIIABC components (OG1RF_10751, EgaB, EgaC) and subsequently hydrolyzed by the glycosylasparaginase EgaG, which generates aspartate and 6-P-N-acetyl-ß-d-glucosaminylamine. The latter can be used as a fermentable carbon source by E. faecalis. Moreover, Galleria mellonella larvae had a significantly higher survival rate when infected with ega mutants compared to the wild-type strain, suggesting that the loss of N-acetylglucosamine-L-asparagine utilization affects enterococcal virulence.

Microbial genes outperform species and SNVs as diagnostic markers for Crohn's disease on multicohort fecal metagenomes empowered by artificial intelligence.

Dysbiosis of gut microbial community is associated with the pathogenesis of CD and may serve as a promising noninvasive diagnostic tool. We aimed to compare the performances of the microbial markers of different biological levels by conducting a multidimensional analysis on the microbial metagenomes of CD. We collected fecal metagenomic datasets generated from eight cohorts that altogether include 870 CD patients and 548 healthy controls. Microbial alterations in CD patients were assessed at multidimensional levels including species, gene, and SNV level, and then diagnostic models were constructed using artificial intelligence algorithm. A total of 227 species, 1047 microbial genes, and 21,877 microbial SNVs were identified that differed between CD and controls. The species, gene, and SNV models achieved an average AUC of 0.97, 0.95, and 0.77, respectively. Notably, the gene model exhibited superior diagnostic capability, achieving an average AUC of 0.89 and 0.91 for internal and external validations, respectively. Moreover, the gene model was specific for CD against other microbiome-related diseases. Furthermore, we found that phosphotransferase system (PTS) contributed substantially to the diagnostic capability of the gene model. The outstanding performance of PTS was mainly explained by genes celB and manY, which demonstrated high predictabilities for CD with metagenomic datasets and was validated in an independent cohort by qRT-PCR analysis. Our global metagenomic analysis unravels the multidimensional alterations of the microbial communities in CD and identifies microbial genes as robust diagnostic biomarkers across geographically and culturally distinct cohorts.

Structural Basis of the Mechanisms of Action and Immunity of Lactococcin A, a Class IId Bacteriocin.

Lactococcin A (LcnA), a class IId bacteriocin, induces membrane leakage and cell death by specifically binding to the membrane receptor-mannose phosphotransferase system (man-PTS), as is the case for pediocin-like (class IIa) bacteriocins. The cognate immunity protein of bacteriocins, which protects the producer cell from its own bacteriocin, recognizes and binds to the bacteriocin-man-PTS complex, consequently blocking membrane leakage. We previously deciphered the mode of action and immunity of class IIa bacteriocins. Here, we determined the structure of the ternary complex of LcnA, LciA (i.e., the immunity protein), and its receptor, i.e., the man-PTS of Lactococcus lactis (ll-man-PTS). An external loop on the membrane-located component IIC of ll-man-PTS was found to prevent specific binding of the N-terminal region of LcnA to the site recognized by pediocin-like bacteriocins. Thus, the N-terminal ß-sheet region of LcnA recognized an adjacent site on the extracellular side of ll-man-PTS, with the LcnA C-terminal hydrophobic helix penetrating into the membrane. The cytoplasmic cleft formed within the man-PTS Core and Vmotif domains induced by embedded LcnA from the periplasmic side is adopted by the appropriate angle between helices H3 and H4 of the N terminus of LciA. The flexible C terminus of LciA then blocks membrane leakage. To summarize, our findings reveal the molecular mechanisms of action and immunity of LcnA and LciA, laying a foundation for further design of class IId bacteriocins. IMPORTANCE Class IId (lactococcin-like) bacteriocins and class IIa (pediocin-like) bacteriocins share a few similarities: (i) both induce membrane leakage and cell death by specifically binding the mannose phosphotransferase system (man-PTS) on their target cells, and (ii) cognate immunity proteins recognize and bind to the bacteriocin-man-PTS complex to block membrane leakage. However, class IId bacteriocins lack the "pediocin box" motif, which is typical of class IIa bacteriocins, and basically target only lactococcal cells; in contrast, class IIa bacteriocins target diverse bacterial cells, but not lactococcal cells. We previously solved the structure of class IIa bacteriocin-receptor-immunity ternary complex from Lactobacillus sakei. Here, we determined the structure of the ternary complex of class IId bacteriocin LcnA, its cognate immunity protein LciA, and its receptor, the man-PTS of Lactococcus lactis. By comparing the interactions between man-PTS and class IIa and class IId bacteriocins, this study affords some clues to better understand the specificity of bacteriocins targeting the mannose phosphotransferase system.

A Retro-Aldol Reaction Prompted the Evolvability of a Phosphotransferase System for the Utilization of a Rare Sugar.

The evolution of the bacterial phosphotransferase system (PTS) linked to glycolysis is dependent on the availability of naturally occurring sugars. Although bacteria exhibit sugar specificities based on carbon catabolite repression, the acquisition and evolvability of the cellular sugar preference under conditions that are suboptimal for growth (e.g., environments rich in a rare sugar) are poorly understood. Here, we generated Escherichia coli mutants via a retro-aldol reaction to obtain progeny that can utilize the rare sugar d-tagatose. We detected a minimal set of adaptive mutations in the d-fructose-specific PTS to render E. coli capable of d-tagatose utilization. These E. coli mutant strains lost the tight regulation of both the d-fructose and N-acetyl-galactosamine PTS following deletions in the binding site of the catabolite repressor/activator protein (Cra) upstream from the fruBKA operon and in the agaR gene, encoding the N-acetylgalactosamine (GalNAc) repressor, respectively. Acquired d-tagatose catabolic pathways then underwent fine-tuned adaptation via an additional mutation in 1-phosphofructose kinase to adjust metabolic fluxes. We determined the evolutionary trajectory at the molecular level, providing insights into the mechanism by which enteric bacteria evolved a substrate preference for the rare sugar d-tagatose. Furthermore, the engineered E. coli mutant strain could serve as an in vivo high-throughput screening platform for engineering non-phosphosugar isomerases to produce rare sugars. IMPORTANCE Microorganisms generate energy through glycolysis, which might have preceded a rapid burst of evolution, including the evolution of cellular respiration in the primordial biosphere. However, little is known about the evolvability of cellular sugar preferences. Here, we generated Escherichia coli mutants via a retro-aldol reaction to obtain progeny that can utilize the rare sugar d-tagatose. Consequently, we identified mutational hot spots and determined the evolutionary trajectory at the molecular level. This provided insights into the mechanism by which enteric bacteria evolved substrate preferences for various sugars, accounting for the widespread occurrence of these taxa. Furthermore, the adaptive laboratory evolution-induced cellular chassis could serve as an in vivo high-throughput screening platform for engineering tailor-made non-phosphorylated sugar isomerases to produce low-calorigenic rare sugars showing antidiabetic, antihyperglycemic, and antitumor activities.

Combined engineering of l-sorbose dehydrogenase and fermentation optimization to increase 2-keto-l-gulonic acid production in Escherichia coli.

One-step fermentation to produce 2-keto-l-gulonic acid (2-KLG), the precursor of vitamin C, is a long-term goal. Improvement of the enzyme's activity through engineering could benefit 2-KLG production. This study aimed to conduct a semi-rational design of l-sorbose dehydrogenase (SDH) through structure-directed, to screen mutants that could enhance the 2-KLG titer. First, the predicted structure of SDH was obtained using AlphaFold2. The key mutation sites in the substrate pocket were identified by Ala scanning. Subsequently, the mutant V336I/V368A was obtained by iterative saturation mutagenesis, which increased the yield of 2-KLG 1.9-fold. Finally, 5.03 g/L of 2-KLG was obtained by a two-stage temperature control fermentation method, and the conversion rate was 50%. Furthermore, experiments showed that knockdown of the l-sorbose-associated phosphotransferase system delays 2-KLG production. The results show that the production of 2-KLG was effectively increased through a combination of SDH engineering and fermentation optimization.

Erythritol alters phosphotransferase gene expression and inhibits the in vitro growth of Staphylococcus coagulans isolated from canines with pyoderma.

Staphylococcus coagulans (SC) belongs to a group of coagulase-positive staphylococci occasionally isolated from the skin lesions of dogs with pyoderma. We recently revealed that erythritol, a sugar alcohol, inhibited the growth of SC strain JCM7470. This study investigated the molecular mechanisms involved in this growth inhibition of JCM7470 by erythritol, and determine whether erythritol inhibits the growth of SC isolated from the skin of dogs with pyoderma. Comprehensive analysis of the gene expression of JCM7470 in the presence of erythritol revealed that erythritol upregulated the expression of glcB and ptsG genes, both of which encode phosphotransferase system (PTS) glucoside- and glucose-specific permease C, B, and A domains (EIICBA), respectively, associated with sugar uptake. Moreover, erythritol suppressed in vitro growth of all 27 SC strains isolated from the skin lesions of canine pyoderma, including 13 mecA gene-positive and 14 mecA gene-negative strains. Finally, the growth inhibition of the SC clinical isolates by erythritol was restored by the addition of glucose. In summary, we revealed that erythritol promotes PTS gene expression and suppresses the in vitro growth of SC clinical isolates from dogs with pyoderma. Restoration of the erythritol-induced growth inhibition by glucose suggested that glucose starvation may contribute to the growth inhibition of SC.

Effects of galactosyltransferase on EPS biosynthesis and freeze-drying resistance of Lactobacillus acidophilus NCFM.

Galactosyltransferase (GalT) is an important enzyme in synthesizing exopolysaccharide (EPS), the major polymer of biofilms protecting cells from severe conditions. However, the contribution to, and regulatory mechanism of GalT, in stressor resistance are still unclear. Herein, we successfully overexpressed GalT in Lactobacillus acidophilus NCFM by genetic engineering. The GalT activity and freeze-drying survival rate of the recombinant strain were significantly enhanced. The EPS yield also increased by 17.8%, indicating a positive relationship between freeze-drying resistance and EPS. RNA-Seq revealed that GalT could regulate the flux of the membrane transport system, pivotal sugar-related metabolic pathways, and promote quorum sensing to facilitate EPS biosynthesis, which enhanced freeze-drying resistance. The findings concretely prove that the mechanism of GalT regulating EPS biosynthesis plays an important role in protecting lactic acid bacteria from freeze-drying stress.

Engineering D-glucose utilization in Azospirillum brasilense Sp7 promotes rice root colonization.

Bacteria of the genus Azospirillum include several plant associated bacteria which often promote the growth of their host plants. Although the host range of Azospirillum brasilense Sp7 is much wider than its close relative Azospirillum lipoferum 4B, it lacks the ability to efficiently utilize D-glucose for its growth. By comparing the genomes of both the species, the genes of A. lipoferum 4B responsible for conferring D-glucose utilization ability in A. brasilese Sp7 were identified by cloning individual or a combination of genes in a broad host range expression vector, mobilizing them in A. brasilense Sp7 and examining the ability of exconjugants to use D-glucose as sole carbon source for growth. These genes also included the homologs of genes involved in N-acetyl glucosamine utilization in Pseudomonas aeruginosa PAO1. A transcriptional fusion of the 5 genes encoding glucose-6-phosphate dehydrogenase and 4 components of glucose phosphotransferase system were able to improve D-glucose utilization ability in A. brasilense Sp7. The A. brasilense Sp7 strain engineered with D-glucose utilization ability showed significantly improved root colonization of rice seedling. The improvement in the ability of A. brasilense Sp7 to colonize rice roots is expected to bring benefits to rice by promoting its growth. KEY POINTS: • Genes required for glucose utilization in Azospirillum lipoferum were identified. • A gene cassette encoding glucose utilization was constructed. • Transfer of gene cassette in A. brasilense improves glucose utilization and rice root colonization..

Enzyme 1 of the phosphoenolpyruvate:sugar phosphotransferase system is involved in resistance to MreB disruption in wild-type and ∆envC cells.

Cell wall synthesis in bacteria is determined by two protein complexes: the elongasome and divisome. The elongasome is coordinated by the actin homolog MreB while the divisome is organized by the tubulin homolog FtsZ. While these two systems must coordinate with each other to ensure that elongation and division are coregulated, this cross talk has been understudied. Using the MreB depolymerizing agent, A22, we found that multiple gene deletions result in cells exhibiting increased sensitivity to MreB depolymerization. One of those genes encodes for EnvC, a part of the divisome that is responsible for splitting daughter cells after the completion of cytokinesis through the activation of specific amidases. Here we show this increased sensitivity to A22 works through two known amidase targets of EnvC: AmiA and AmiB. In addition, suppressor analysis revealed that mutations in enzyme 1 of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can suppress the effects of A22 in both wild-type and envC deletion cells. Together this work helps to link elongation, division, and metabolism.

Harnessing Escherichia coli's Native Machinery for Detection of Vitamin C (Ascorbate) Deficiency.

Vitamin C (l-ascorbate) deficiency is a global public health issue most prevalent in resource-limited regions, creating a need for an inexpensive detection platform. Here, we describe efforts to engineer whole-cell and cell-free ascorbate biosensors. Both sensors used the protein UlaR, which binds to a metabolite of ascorbate and regulates transcription. The whole-cell sensor could detect lower, physiologically relevant concentrations of ascorbate, which we attributed to intact functionality of a phosphotransferase system (PTS) that transports ascorbate across the cell membrane and phosphorylates it to form UlaR's ligand. We used multiple strategies to enhance cell-free PTS functionality (which has received little previous attention), improving the cell-free sensor's performance, but the whole-cell sensor remained more sensitive. These efforts demonstrated an advantage of whole-cell sensors for detection of molecules─like ascorbate─transformed by a PTS, but also proof of principle for cell-free sensors requiring membrane-bound components like the PTS. In addition, the cell-free sensor was functional in plasma, setting the stage for future implementation of ascorbate sensors for clinically relevant biofluids in field-deployable formats.

The bacteriocin Angicin interferes with bacterial membrane integrity through interaction with the mannose phosphotransferase system.

In a natural environment, bacteria are members of multispecies communities. To compete with rival species, bacteria produce antimicrobial peptides (AMPs), called bacteriocins. Bacteriocins are small, cationic, ribosomally synthesized peptides, which normally inhibit closely related species of the producing organism. Bacteriocin production is best studied in lactic bacteria (LAB). Streptococcus anginosus, belonging to LAB, produces the potent bacteriocin Angicin, which shows inhibitory activity against other streptococci, Listeria monocytogenes and vancomycin resistant Enterococcus faecium (VRE). Furthermore, Angicin shows a high resistance toward pH changes and heat, rendering it an interesting candidate for food preservation or clinical applications. The inhibitory activity of Angicin depends on the presence of a mannose phosphotransferase system (Man-PTS) in target cells, since L. monocytogenes harboring a deletion in an extracellular loop of this system is no longer sensitive to Angicin. Furthermore, we demonstrated by liposome leakage and pHluorin assays that Angicin destroys membrane integrity but shows only low cytotoxicity against human cell lines. In conclusion, we show that Angicin has a detrimental effect on the membrane of target organisms by using the Man-PTS as a receptor.