scholarly journals Importance of Pyruvate Sensing and Transport for the Resuscitation of Viable but NonculturableEscherichia coliK-12

2018 ◽  
Vol 201 (3) ◽  
Author(s):  
Cláudia Vilhena ◽  
Eugen Kaganovitch ◽  
Alexander Grünberger ◽  
Magdalena Motz ◽  
Ignasi Forné ◽  
...  
Keyword(s):  
Response Regulator ◽  
Bacterial Species ◽  
Microfluidic System ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Content Type ◽  
E Coli ◽  
High Affinity ◽  
Vbnc Bacteria ◽  
K 12

ABSTRACTEscherichia coliand many other bacterial species can enter into a viable but nonculturable (VBNC) state, which is a survival strategy adopted by cells exposed to adverse environmental conditions. Pyruvate is known to be one factor that promotes resuscitation of VBNC cells. Here we studied the role of a pyruvate-sensing network, composed of the histidine kinase-response regulator systems BtsS/BtsR and YpdA/YpdB and the target genebtsT, encoding the high-affinity pyruvate/H+symporter BtsT, in the resuscitation of VBNCE. coliK-12 cells after exposure to cold for 120 days. Analysis of the proteome of VBNC cells revealed upregulation, relative to exponentially growing cells, of BtsT and other proteins involved in pyruvate metabolism. Provision of pyruvate stimulated protein and DNA biosynthesis, and thus resuscitation, in wild-type but notbtsSR ypdABmutant VBNC cells. This result was corroborated by time-dependent tracking of the resuscitation of individual VBNCE. colicells observed in a microfluidic system. Finally, transport assays revealed that14C-labeled pyruvate was rapidly taken up into VBNC cells by BtsT. These results provide the first evidence that pyruvate is taken up as a carbon source for the resuscitation of VBNCE. colicells.IMPORTANCEViable but nonculturable (VBNC) bacteria do not form colonies in standard medium but otherwise retain their metabolic activity and can express toxic proteins. Many bacterial genera, includingEscherichia,Vibrio, andListeria, have been shown to enter the VBNC state upon exposure to adverse conditions, such as low temperature, radiation, and starvation. Ultimately, these organisms pose a public health risk with potential implications for the pharmaceutical and food industries, as dormant organisms are especially difficult to selectively eliminate and VBNC bacteria can be resuscitated if placed in an environment with appropriate nutrition and temperature. Here we used a microfluidic system to monitor the resuscitation of single VBNC cells over time. We provide new molecular insights into the initiation of resuscitation by demonstrating that VBNCE. colicells rapidly take up pyruvate with an inducible high-affinity transporter, whose expression is triggered by the BtsSR-YpdAB sensing network.

scholarly journals Induction of Viable but Nonculturable Escherichia coli O157:H7 in the Phyllosphere of Lettuce: a Food Safety Risk Factor

2011 ◽  
Vol 77 (23) ◽  
pp. 8295-8302 ◽  
Author(s):  
Laura-Dorina Dinu ◽  
Susan Bach
Keyword(s):  
Escherichia Coli ◽  
Food Safety ◽  
Low Temperature ◽  
Cell Density ◽  
Prolonged Storage ◽  
Potential Hazard ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Content Type ◽  
E Coli

ABSTRACTEscherichia coliO157:H7 continues to be an important human pathogen and has been increasingly linked to food-borne illness associated with fresh produce, particularly leafy greens. The aim of this work was to investigate the fate ofE. coliO157:H7 on the phyllosphere of lettuce under low temperature and to evaluate the potential hazard of viable but nonculturable (VBNC) cells induced under such stressful conditions. First, we studied the survival of six bacterial strains following prolonged storage in water at low temperature (4°C) and selected two strains with different nonculturable responses for the construction ofE. coliO157:H7 Tn7gfptransformants in order to quantitatively assess the occurrence of human pathogens on the plant surface. Under a suboptimal growth temperature (16°C), bothE. coliO157:H7 strains maintained culturability on lettuce leaves, but under more stressful conditions (8°C), the bacterial populations evolved toward the VBNC state. The strain-dependent nonculturable response was more evident in the experiments with different inoculum doses (109and 106E. coliO157:H7 bacteria per g of leaf) when strain BRMSID 188 lost culturability after 15 days and strain ATCC 43895 lost culturability within 7 days, regardless of the inoculum dose. However, the number of cells entering the VBNC state in high-cell-density inoculum (approximately 55%) was lower than in low-cell-density inoculum (approximately 70%). We recorded the presence of verotoxin for 3 days in samples that contained a VBNC population of 4 to 5 log10cells but did not detect culturable cells. These findings indicate thatE. coliO157:H7 VBNC cells are induced on lettuce plants, and this may have implications regarding food safety.

scholarly journals Conversion of Norepinephrine to 3,4-Dihdroxymandelic Acid in Escherichia coli Requires the QseBC Quorum-Sensing System and the FeaR Transcription Factor

2017 ◽  
Vol 200 (1) ◽  
Author(s):  
Sasikiran Pasupuleti ◽  
Nitesh Sule ◽  
Michael D. Manson ◽  
Arul Jayaraman
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Quorum Sensing ◽  
Aromatic Amines ◽  
Response Regulator ◽  
Content Type ◽  
E Coli ◽  
Expression Of Genes ◽  
K 12 ◽  
Cognate Response Regulator

ABSTRACTThe detection of norepinephrine (NE) as a chemoattractant byEscherichia colistrain K-12 requires the combined action of the TynA monoamine oxidase and the FeaB aromatic aldehyde dehydrogenase. The role of these enzymes is to convert NE into 3,4-dihydroxymandelic acid (DHMA), which is a potent chemoattractant sensed by the Tsr chemoreceptor. These two enzymes must be induced by prior exposure to NE, and cells that are exposed to NE for the first time initially show minimal chemotaxis toward it. The induction of TynA and FeaB requires the QseC quorum-sensing histidine kinase, and the signaling cascade requires new protein synthesis. Here, we demonstrate that the cognate response regulator for QseC, the transcription factor QseB, is also required for induction. The related quorum-sensing kinase QseE appears not to be part of the signaling pathway, but its cognate response regulator, QseF, which is also a substrate for phosphotransfer from QseC, plays a nonessential role. The promoter of thefeaRgene, which encodes a transcription factor that has been shown to be essential for the expression oftynAandfeaB, has two predicted QseB-binding sites. One of these sites appears to be in an appropriate position to stimulate transcription from the P1promoter of thefeaRgene. This study unites two well-known pathways: one for expression of genes regulated by catecholamines (QseBC) and one for expression of genes required for metabolism of aromatic amines (FeaR, TynA, and FeaB). This cross talk allowsE. colito convert the host-derived and chemotactically inert NE into the potent bacterial chemoattractant DHMA.IMPORTANCEThe chemotaxis ofE. coliK-12 to norepinephrine (NE) requires the conversion of NE to 3,4-dihydroxymandleic acid (DHMA), and DHMA is both an attractant and inducer of virulence gene expression for a pathogenic enterohemorrhagicE. coli(EHEC) strain. The induction of virulence by DHMA and NE requires QseC. The results described here show that the cognate response regulator for QseC, QseB, is also required for conversion of NE into DHMA. Production of DHMA requires induction of a pathway involved in the metabolism of aromatic amines. Thus, the QseBC sensory system provides a direct link between virulence and chemotaxis, suggesting that chemotaxis to host signaling molecules may require that those molecules are first metabolized by bacterial enzymes to generate the actual chemoattractant.

scholarly journals Systematic Nomenclature for GGDEF and EAL Domain-Containing Cyclic Di-GMP Turnover Proteins of Escherichia coli: TABLE 1

2015 ◽  
Vol 198 (1) ◽  
pp. 7-11 ◽  
Author(s):  
Regine Hengge ◽  
Michael Y. Galperin ◽  
Jean-Marc Ghigo ◽  
Mark Gomelsky ◽  
Jeffrey Green ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
Content Type ◽  
E Coli ◽  
Diguanylate Cyclases ◽  
Genes Encoding ◽  
Systematic Nomenclature ◽  
K 12 ◽  
Encoding Genes

In recent years,Escherichia colihas served as one of a few model bacterial species for studying cyclic di-GMP (c-di-GMP) signaling. The widely usedE. coliK-12 laboratory strains possess 29 genes encoding proteins with GGDEF and/or EAL domains, which include 12 diguanylate cyclases (DGC), 13 c-di-GMP-specific phosphodiesterases (PDE), and 4 “degenerate” enzymatically inactive proteins. In addition, six new GGDEF and EAL (GGDEF/EAL) domain-encoding genes, which encode two DGCs and four PDEs, have recently been found in genomic analyses of commensal and pathogenicE. colistrains. As a group of researchers who have been studying the molecular mechanisms and the genomic basis of c-di-GMP signaling inE. coli, we now propose a general and systematicdgcandpdenomenclature for the enzymatically active GGDEF/EAL domain-encoding genes of this model species. This nomenclature is intuitive and easy to memorize, and it can also be applied to additional genes and proteins that might be discovered in various strains ofE. coliin future studies.

scholarly journals Unexpected Diversity of Escherichia coli SialateO-Acetyl Esterase NanS

2016 ◽  
Vol 198 (20) ◽  
pp. 2803-2809 ◽  
Author(s):  
Ariel Rangel ◽  
Susan M. Steenbergen ◽  
Eric R. Vimr
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Bacterial Species ◽  
Sialic Acids ◽  
Host Bacterium ◽  
Acetyl Esterase ◽  
Content Type ◽  
E Coli ◽  
Host Microbe Interactions ◽  
K 12

ABSTRACTThe sialic acids (N-acylneuraminates) are a group of nine-carbon keto-sugars existing mainly as terminal residues on animal glycoprotein and glycolipid carbohydrate chains. Bacterial commensals and pathogens exploit host sialic acids for nutrition, adhesion, or antirecognition, whereN-acetyl- orN-glycolylneuraminic acids are the two predominant chemical forms of sialic acids. Each form may be modified by acetyl esters at carbon position 4, 7, 8, or 9 and by a variety of less-common modifications. Modified sialic acids produce challenges for colonizing bacteria, because the chemical alterations toN-acetylneuraminic acid (Neu5Ac) confer increased resistance to sialidase and aldolase activities essential for the catabolism of host sialic acids. Bacteria withO-acetyl sialate esterase(s) utilize acetylated sialic acids for growth, thereby gaining a presumed metabolic advantage over competitors lacking this activity. Here, we demonstrate the esterase activity ofEscherichia coliNanS after purifying it as a C-terminal HaloTag fusion. Using a similar approach, we show thatE. colistrain O157:H7 Stx prophage or prophage remnants invariably include paralogs ofnanSoften located downstream of the Shiga-like toxin genes. These paralogs may include sequences encoding N- or C-terminal domains of unknown function where the NanS domains can act as sialateO-acetyl esterases, as shown by complementation of anE. colistrain K-12nanSmutant and the unimpaired growth of anE. coliO157nanSmutant onO-acetylated sialic acid. We further demonstrate thatnanShomologs inStreptococcusspp. also encode active esterase, demonstrating an unexpected diversity of bacterial sialateO-acetyl esterase.IMPORTANCEThe sialic acids are a family of over 40 naturally occurring 9-carbon keto-sugars that function in a variety of host-bacterium interactions. These sugars occur primarily as terminal carbohydrate residues on host glycoproteins and glycolipids. Available evidence indicates that diverse bacterial species use host sialic acids for adhesion or as sources of carbon and nitrogen. Our results show that the catabolism of the diacetylated form of host sialic acid requires a specialized esterase, NanS. Our results further show thatnanShomologs exist in bacteria other thanEscherichia coli, as well as part of toxigenicE. coliprophage. The unexpected diversity of these enzymes suggests new avenues for investigating host-bacterium interactions. Therefore, these original results extend our previous studies ofnanSto include mucosal pathogens, prophage, and prophage remnants. This expansion of thenanSsuperfamily suggests important, although as-yet-unknown, functions in host-microbe interactions.

scholarly journals Viable but Nonculturable Escherichia coli O157:H7 and Salmonella enterica in Fresh Produce: Rapid Determination by Loop-Mediated Isothermal Amplification Coupled with a Propidium Monoazide Treatment

2020 ◽  
Vol 86 (7) ◽  
Author(s):  
Lu Han ◽  
Kaidi Wang ◽  
Lina Ma ◽  
Pascal Delaquis ◽  
Susan Bach ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pure Culture ◽  
Fresh Produce ◽  
Isothermal Amplification ◽  
Propidium Monoazide ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Content Type ◽  
E Coli ◽  
Detection And Quantification

ABSTRACT Escherichia coli O157:H7 and Salmonella enterica are leading causes of foodborne outbreaks linked to fresh produce. Both species can enter the “viable but nonculturable” (VBNC) state that precludes detection using conventional culture-based or molecular methods. In this study, we assessed propidium monoazide-quantitative PCR (PMA-qPCR) assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. The performance of PMA-LAMP assays targeting the wzy gene of E. coli O157:H7 and the agfA gene of S. enterica and the performance of PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce, and spinach. No cross-reaction was observed in the specificity tests. The values representing the limit of detection (LOD) seen with PMA-LAMP were 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture and were 5.13 × 103 or 5.13 × 104 CFU/g for VBNC E. coli O157:H7 and 1.05 × 104 or 1.05 × 105 CFU/g for VBNC S. enterica in fresh produce, representing results comparable to those obtained by PMA-qPCR. Standard curves showed correlation coefficients ranging from 0.925 to 0.996, indicating a good quantitative capacity of PMA-LAMP for determining populations of both bacterial species in the VBNC state. The PMA-LAMP assay was completed with considerable economy of time (30 min versus 1 h) and achieved sensitivity and quantitative capacity comparable to those seen with a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive, and robust method for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. IMPORTANCE VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade detection in conventional plating assays. Both E. coli O157:H7 and S. enterica have been reported to enter the VBNC state under a range of environmental stress conditions and to resuscitate under favorable conditions and are a potential cause of human infections. PMA-LAMP methods developed in this study provide a rapid, sensitive, and specific way to determine levels of VBNC E. coli O157:H7 and S. enterica in fresh produce, which potentially decreases the risks related to the consumption of fresh produce contaminated by enteric pathogens in this state. PMA-LAMP can be further applied in the field study to enhance our understanding of the fate of VBNC pathogens in the preharvest and postharvest stages of fresh produce.

scholarly journals Genome-Based Comparison of Cyclic Di-GMP Signaling in Pathogenic and Commensal Escherichia coli Strains

2015 ◽  
Vol 198 (1) ◽  
pp. 111-126 ◽  
Author(s):  
Tatyana L. Povolotsky ◽  
Regine Hengge
Keyword(s):  
Escherichia Coli ◽  
Bacterial Species ◽  
Content Type ◽  
E Coli ◽  
Large Deletions ◽  
Diguanylate Cyclases ◽  
Core Set ◽  
Genes Encoding ◽  
K 12 ◽  
Encoding Genes

ABSTRACTThe ubiquitous bacterial second messenger cyclic di-GMP (c-di-GMP) has recently become prominent as a trigger for biofilm formation in many bacteria. It is generated by diguanylate cyclases (DGCs; with GGDEF domains) and degraded by specific phosphodiesterases (PDEs; containing either EAL or HD-GYP domains). Most bacterial species contain multiples of these proteins with some having specific functions that are based on direct molecular interactions in addition to their enzymatic activities.Escherichia coliK-12 laboratory strains feature 29 genes encoding GGDEF and/or EAL domains, resulting in a set of 12 DGCs, 13 PDEs, and four enzymatically inactive “degenerate” proteins that act by direct macromolecular interactions. We present here a comparative analysis of GGDEF/EAL domain-encoding genes in 61 genomes of pathogenic, commensal, and probioticE. colistrains (including enteric pathogens such as enteroaggregative, enterohemorrhagic, enteropathogenic, enterotoxigenic, and adherent and invasiveEscherichia coliand the 2011 German outbreak O104:H4 strain, as well as extraintestinal pathogenicE. coli, such as uropathogenic and meningitis-associatedE. coli). We describe additional genes for two membrane-associated DGCs (DgcX and DgcY) and four PDEs (the membrane-associated PdeT, as well as the EAL domain-only proteins PdeW, PdeX, and PdeY), thus showing the pangenome ofE. colito contain at least 35 GGDEF/EAL domain proteins. A core set of only eight proteins is absolutely conserved in all 61 strains: DgcC (YaiC), DgcI (YliF), PdeB (YlaB), PdeH (YhjH), PdeK (YhjK), PdeN (Rtn), and the degenerate proteins CsrD and CdgI (YeaI). In all other GGDEF/EAL domain genes, diverse point and frameshift mutations, as well as small or large deletions, were discovered in various strains.IMPORTANCEOur analysis reveals interesting trends in pathogenicEscherichia colithat could reflect different host cell adherence mechanisms. These may either benefit from or be counteracted by the c-di-GMP-stimulated production of amyloid curli fibers and cellulose. Thus, EAEC, which adhere in a “stacked brick” biofilm mode, have a potential for high c-di-GMP accumulation due to DgcX, a strongly expressed additional DGC. In contrast, EHEC and UPEC, which use alternative adherence mechanisms, tend to have extra PDEs, suggesting that low cellular c-di-GMP levels are crucial for these strains under specific conditions. Overall, our study also indicates that GGDEF/EAL domain proteins evolve rapidly and thereby contribute to adaptation to host-specific and environmental niches of various types ofE. coli.

scholarly journals Escherichia coli K-12 Lacks a High-Affinity Assimilatory Cysteine Importer

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Yidan Zhou ◽  
James A. Imlay
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Chemical Reactivity ◽  
Enteric Bacteria ◽  
Natural Environments ◽  
Content Type ◽  
E Coli ◽  
High Affinity ◽  
Anoxic Environments ◽  
K 12

ABSTRACT The most direct route by which microbes might assimilate sulfur would be by importing cysteine. However, alone among the amino acids, cysteine does not have well-characterized importers. We determined that Escherichia coli can rapidly import cysteine, but in our experiments, it did so primarily through the LIV ATP-driven system that is dedicated to branched-chain amino acids. The affinity of this system for cysteine is far lower than for Leu, Ile, and Val, and so in their presence, cysteine is excluded. Thus, this transport is unlikely to be relevant in natural environments. Growth studies, transcriptomics, and transport assays failed to detect any high-affinity importer that is dedicated to cysteine assimilation. Enteric bacteria do not contain the putative cysteine importer that was identified in Campylobacter jejuni. This situation is surprising, because E. coli deploys ion- and/or ATP-driven transporters that import cystine, the oxidized form of cysteine, with high affinity and specificity. We conjecture that in oxic environments, molecular oxygen oxidizes environmental cysteine to cystine, which E. coli imports. In anoxic environments where cysteine is stable, the cell chooses to assimilate hydrogen sulfide instead. Calculations suggest that this alternative is almost as economical, and it avoids the toxic effects that can result when excess cysteine enters the cell. IMPORTANCE This investigation discovered that Escherichia coli lacks a transporter dedicated to the assimilation of cysteine, an outcome that is in striking contrast to the many transporters devoted to the other 19 amino acids. We ascribe the lack of a high-affinity cysteine importer to two considerations. First, the chemical reactivity of this amino acid is unique, and its poorly controlled import can have adverse consequences for the cell. Second, our analysis suggests that the economics of biosynthesis depend sharply upon whether the cell is respiring or fermenting. In the anoxic habitats in which cysteine might be found, the value of import versus biosynthesis is strongly reduced compared to that in oxic habitats. These studies may explain why bacteria choose to synthesize rather than to import other useful biomolecules as well.

scholarly journals Tailoring a Global Iron Regulon to a Uropathogen

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Rajdeep Banerjee ◽  
Erin Weisenhorn ◽  
Kevin J. Schwartz ◽  
Kevin S. Myers ◽  
Jeremy D. Glasner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Urinary Tract ◽  
Amino Acid ◽  
Regulatory Network ◽  
Iron Homeostasis ◽  
Iron Limitation ◽  
Content Type ◽  
E Coli ◽  
General Stress ◽  
K 12

ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

scholarly journals Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

2014 ◽  
Vol 81 (1) ◽  
pp. 130-138 ◽  
Author(s):  
James Kirby ◽  
Minobu Nishimoto ◽  
Ruthie W. N. Chow ◽  
Edward E. K. Baidoo ◽  
George Wang ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Xylose Reductase ◽  
Pentose Phosphate ◽  
Terpene Biosynthesis ◽  
Content Type ◽  
E Coli ◽  
Pathway Expression ◽  
Terpene Synthesis ◽  
Selection For

ABSTRACTTerpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of adxsdeletion inEscherichia coligrown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-typeE. coliyajOgene, annotated as a putative xylose reductase, or via various mutations in the nativeribBgene.In vitroanalysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway inE. colifor production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

scholarly journals Combined Action of Human Commensal Bacteria and Amorphous Silica Nanoparticles on the Viability and Immune Responses of Dendritic Cells

2017 ◽  
Vol 24 (10) ◽  
Author(s):  
Giulia Malachin ◽  
Elisa Lubian ◽  
Fabrizio Mancin ◽  
Emanuele Papini ◽  
Regina Tavano
Keyword(s):  
Dendritic Cells ◽  
Silica Nanoparticles ◽  
Amorphous Silica ◽  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Commensal Bacteria ◽  
Content Type ◽  
E Coli ◽  
Synergistic Induction ◽  
Ifn Γ

ABSTRACT Dendritic cells (DCs) regulate the host-microbe balance in the gut and skin, tissues likely exposed to nanoparticles (NPs) present in drugs, food, and cosmetics. We analyzed the viability and the activation of DCs incubated with extracellular media (EMs) obtained from cultures of commensal bacteria (Escherichia coli, Staphylococcus epidermidis) or pathogenic bacteria (Pseudomonas aeruginosa, Staphylococcus aureus) in the presence of amorphous silica nanoparticles (SiO2 NPs). EMs and NPs synergistically increased the levels of cytotoxicity and cytokine production, with different nanoparticle dose-response characteristics being found, depending on the bacterial species. E. coli and S. epidermidis EMs plus NPs at nontoxic doses stimulated the secretion of interleukin-1β (IL-1β), IL-12, IL-10, and IL-6, while E. coli and S. epidermidis EMs plus NPs at toxic doses stimulated the secretion of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), IL-4, and IL-5. On the contrary, S. aureus and P. aeruginosa EMs induced cytokines only when they were combined with NPs at toxic concentrations. The induction of maturation markers (CD86, CD80, CD83, intercellular adhesion molecule 1, and major histocompatibility complex class II) by commensal bacteria but not by pathogenic ones was improved in the presence of noncytotoxic SiO2 NP doses. DCs consistently supported the proliferation and differentiation of CD4+ and CD8+ T cells secreting IFN-γ and IL-17A. The synergistic induction of CD86 was due to nonprotein molecules present in the EMs from all bacteria tested. At variance with this finding, the synergistic induction of IL-1β was prevalently mediated by proteins in the case of E. coli EMs and by nonproteins in the case of S. epidermidis EMs. A bacterial costimulus did not act on DCs after adsorption on SiO2 NPs but rather acted as an independent agonist. The inflammatory and immune actions of DCs stimulated by commensal bacterial agonists might be altered by the simultaneous exposure to engineered or environmental NPs.

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