Elongation factor P is required for EIIGlc translation in Corynebacterium glutamicum due to an essential polyproline motif.

Translating ribosomes require elongation factor P (EF-P) to incorporate consecutive prolines (XPPX) into nascent peptide chains. The proteome of Corynebacterium glutamicum ATCC 13032 contains a total of 1,468 XPPX motifs, many of which are found in proteins involved in primary and secondary metabolism. We show here that synthesis of EIIGlc , the glucose-specific permease of the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) encoded by ptsG, is strongly dependent on EF-P, as an efp deletion mutant grows poorly on glucose as sole carbon source. The amount of EIIGlc is strongly reduced in this mutant, which consequently results in a lower rate of glucose uptake. Strikingly, the XPPX motif is essential for the activity of EIIGlc , and substitution of the prolines leads to inactivation of the protein. Finally, translation of GntR2, a transcriptional activator of ptsG, is also dependent on EF-P. However, its reduced amount in the efp mutant can be compensated for by other regulators. These results reveal for the first time a translational bottleneck involving production of the major glucose transporter EIIGlc , which has implications for future strain engineering strategies.

Comprehensive analysis of transcriptome and metabolome analysis reveal new targets of Glaesserella parasuis glucose-specific enzyme IIBC (PtsG).

The ptsG (hpIIBCGlc) gene, belonging to the glucose-specific phosphotransferase system, encodes the bacterial glucose-specific enzyme IIBC. In this study, the effects of a deletion of the ptsG gene were investigated by metabolome and transcriptome analyses. At the transcriptional level, we identified 970 differentially expressed genes between ΔptsG and sc1401 (Padj<0.05) and 2072 co-expressed genes. Among these genes, those involved in methane metabolism, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, pyruvate metabolism, phosphotransferase system (PTS), biotin metabolism, Two-component system and Terpenoid backbone biosynthesis showed significant changes in the ΔptsG mutant strain. Metabolome analysis revealed that a total of 310 metabolites were identified, including 20 different metabolites (p < 0.05). Among them, 15 metabolites were upregulated and 5 were downregulated in ΔptsG mutant strain. Statistical analysis revealed there were 115 individual metabolites having correlation, of which 89 were positive and 26 negative. These metabolites include amino acids, phosphates, amines, esters, nucleotides, benzoic acid and adenosine, among which amino acids and phosphate metabolites dominate. However, not all of these changes were attributable to changes in mRNA levels and must also be caused by post-transcriptional regulatory processes. The knowledge gained from this lays the foundation for further study on the role of ptsG in the pathogenic process of Glaesserella parasuis (G.parasuis).

The ptsG Gene Encoding the Major Glucose Transporter of Bacillus cereus C1L Participates in Root Colonization and Beneficial Metabolite Production to Induce Plant Systemic Disease Resistance.

Rhizosphere interactions between microorganisms and plants have great influence on plant health. Bacillus cereus C1L, an induced systemic resistance (ISR)-eliciting rhizobacterium from Lilium formosanum, can protect monocot and dicot plants from disease challenges. To identify the ISR-involved bacterial genes, the systemic protection effect of transposon-tagged mutants of B. cereus C1L against southern corn leaf blight (SCLB) was surveyed, and a mutant of the ptsG gene encoding glucose-specific permease of the phosphotransferase system was severely impaired in the abilities of disease suppression and root colonization. The ptsG mutant lost the preferential utilization of glucose and showed reduction of glucose-assisted growth in minimal medium. A promoter-based reporter assay revealed that ptsG expression could be activated by certain sugar constituents of maize root exudates, among which B. cereus C1L exhibited the highest chemotactic response toward glucose, whereas neither of them could attract the ptsG mutant. Additionally, ptsG deficiency almost completely abolished glucose uptake of B. cereus C1L. Metabolite analysis indicated that the lack of ptsG undermined glucose-induced accumulation of acetoin and 2,3-butanediol in B. cereus C1L, both eliciting maize ISR against SCLB. Pretreatments with B. cereus C1L, ptsG mutant, acetoin, and 2,3-butanediol enhanced defense-related reactive oxygen species accumulation and callose deposition at different levels that were positively correlated to their ISR-eliciting activities. Thus, glucose uptake-mediating ptsG participates in ISR elicitation by endowing B. cereus C1L with the full capacities for root colonization and beneficial glucose metabolite production, providing a clue regarding how ISR-mediating rhizobacteria create a mutually beneficial relationship with various plant species.

Depletion of Glucose Activates Catabolite Repression during Pneumonic Plague.

Bacterial pathogenesis depends on changes in metabolic and virulence gene expression in response to changes within a pathogen's environment. The plague-causing pathogen, Yersinia pestis, requires expression of the gene encoding the Pla protease for progression of pneumonic plague. The catabolite repressor protein Crp, a global transcriptional regulator, may serve as the activator of pla in response to changes within the lungs as disease progresses. By using gene reporter fusions, the spatial and temporal activation of the crp and pla promoters was measured in a mouse model of pneumonic plague. In the lungs, crp was highly expressed in bacteria found within large aggregates resembling biofilms, while pla expression increased over time independent of the aggregated state. Increased expression of crp and pla correlated with a reduction in lung glucose levels. Deletion of the glucose-specific phosphotransferase system EIIBC (PtsG) of Y. pestis rescued glucose levels in the lungs, resulting in reduced expression of both crp and pla We propose that activation of pla expression during pneumonic plague is driven by an increase of both Crp and cAMP levels following consumption of available glucose in the lungs by Y. pestis Thus, Crp operates as a sensor linking the nutritional environment of the host to regulation of virulence gene expression.IMPORTANCE Using Yersinia pestis as a model for pneumonia, we discovered that glucose is rapidly consumed, leading to a catabolite-repressive environment in the lungs. As a result, expression of the gene encoding the plasminogen activator protease, a target of the catabolite repressor protein required for Y. pestis pathogenesis, is activated. Interestingly, expression of the catabolite repressor protein itself was also increased in the absence of glucose but only in biofilms. The data presented here demonstrate how a bacterial pathogen senses changes within its environment to coordinate metabolism and virulence gene expression.

Metabolic engineering of Escherichia coli to produce succinate from soybean hydrolysate under anaerobic conditions.

It is of great economic interest to produce succinate from low-grade carbon sources, which can enhance the competitiveness of the biological route. In this study, succinate producer Escherichia coli CT550/pHL413KF1 was further engineered to efficiently use the mixed sugars from non-food based soybean hydrolysate to produce succinate under anaerobic conditions. Since many common E. coli strains fail to use galactose anaerobically even if they can use it aerobically, the glucose, and galactose related sugar transporters were deactivated individually and evaluated. The PTS system was found to be important for utilization of mixed sugars, and galactose uptake was activated by deactivating ptsG. In the ptsG- strain, glucose, and galactose were used simultaneously. Glucose was assimilated mainly through the mannose PTS system while galactose was transferred mainly through GalP in a ptsG- strain. A new succinate producing strain, FZ591C which can efficiently produce succinate from the mixed sugars present in soybean hydrolysate was constructed by integration of the high succinate yield producing module and the galactose utilization module into the chromosome of the CT550 ptsG- strain. The succinate yield reached 1.64 mol/mol hexose consumed (95% of maximum theoretical yield) when a mixed sugars feedstock was used as a carbon source. Based on the three monitored sugars, a nominal succinate yield of 1.95 mol/mol was observed as the strain can apparently also use some other minor sugars in the hydrolysate. In this study, we demonstrate that FZ591C can use soybean hydrolysate as an inexpensive carbon source for high yield succinate production under anaerobic conditions, giving it the potential for industrial application.

Simultaneous uptake of lignocellulose-based monosaccharides by Escherichia coli.

Lignocellulosic waste is a naturally abundant biomass and is therefore an attractive material to use in second generation biorefineries. Microbial growth on the monosaccharides present in hydrolyzed lignocellulose is however associated with several obstacles whereof one is the lack of simultaneous uptake of the sugars. We have studied the aerobic growth of Escherichia coli on D-glucose, D-xylose, and L-arabinose and for simultaneous uptake to occur, both the carbon catabolite repression mechanism (CCR) and the AraC repression of xylose uptake and metabolism had to be removed. The strain AF1000 is a MC4100 derivative that is only able to assimilate arabinose after a considerable lag phase, which is unsuitable for commercial production. This strain was successfully adapted to growth on L-arabinose and this led to simultaneous uptake of arabinose and xylose in a diauxic growth mode following glucose consumption. In this strain, a deletion in the phosphoenolpyruvate:phosphotransferase system (PTS) for glucose uptake, the ptsG mutation, was introduced. The resulting strain, PPA652ara simultaneously consumed all three monosaccharides at a maximum specific growth rate of 0.59 h(-1) , 55% higher than for the ptsG mutant alone. Also, no residual sugar was present in the cultivation medium. The potential of PPA652ara is further acknowledged by the performance of AF1000 during fed-batch processing on a mixture of D-glucose, D-xylose, and L-arabinose. The conclusion is that without the removal of both layers of carbon uptake control, this process results in accumulation of pentoses and leads to a reduction of the specific growth rate by 30%.

Enhancing human-like collagen accumulation by deleting the major glucose transporter ptsG in recombinant Escherichia coli BL21.

Collagen has been proven to be a valuable biomedical material for many medical applications. Human-like collagen (HLC) is a novel important biomedical material with diverse medical applications. In this work, recombinant Escherichia coli BL21 3.7 ∆ptsG was constructed, the characters of ptsG mutant strain were analyzed, and real-time quantitative polymerase chain reaction (PCR) was applied to investigate the effect of ptsG gene deletion on the transcriptional level of the phosphotransferase system (PTS) genes responsible for glucose transport. The HLC production and cell growth ability were 1.33- and 1.24-fold higher than those of its parent strain in the fermentation medium, respectively, and 1.16- and 1.17-fold in the modified minimal medium individually. The acetate accumulation decreased by 42%-56% compared to its parent strain in the fermentation medium, and 70%-87% in the modified minimal medium. The results of RT-qPCR showed that the transcriptional level of crr, ptsH, ptsI, and blgF in ptsG mutant all decreased dramatically, which inferred a decrease in the glucose uptake rate, but the transcriptional level of FruB and manX increased slightly, which demonstrated the activation of fructose- and mannose-specific transport pathways in the ptsG mutant. This study demonstrates that ptsG deletion is an effective strategy to reduce acetate accumulation and increase biomass and HLC production.

Dynamic and balanced regulation of the thrABC operon gene for efficient synthesis of L-threonine.

L-threonine is an essential amino acid used widely in food, cosmetics, animal feed and medicine. The thrABC operon plays an important role in regulating the biosynthesis of L-theronine. In this work, we systematically analyzed the effects of separating thrAB and thrC in different proportions on strain growth and L-threonine production in Escherichia coli firstly. The results showed that higher expression of thrC than thrAB enhanced cell growth and L-threonine production; however, L-threonine production decreased when the thrC proportion was too high. The highest L-threonine production was achieved when the expression intensity ratio of thrAB to thrC was 3:5. Secondly, a stationary phase promoter was also used to dynamically regulate the expression of engineered thrABC. This strategy improved cell growth and shortened the fermentation period from 36 h to 24 h. Finally, the acetate metabolic overflow was reduced by deleting the ptsG gene, leading to a further increase in L-threonine production. With these efforts, the final strain P 2.1 -2901ΔptsG reached 40.06 g/L at 60 h fermentation, which was 96.85% higher than the initial control strain TH and the highest reported titer in shake flasks. The maximum L-threonine yield and productivity was obtained in reported fed-batch fermentation, and L-threonine production is close to the highest titer (127.30 g/L). In this work, the expression ratio of genes in the thrABC operon in E. coli was studied systematically, which provided a new approach to improve L-threonine production and its downstream products.

Dynamics of the canonical RNA degradosome components during glucose stress.

The RNA Degradosome (RNAD) is a multi-enzyme complex, which performs important functions in post-transcriptional regulation in Escherichia coli with the assistance of regulatory sRNAs and the RNA chaperone Hfq. Although the interaction of the canonical RNAD components with RNase E has been extensively studied, the dynamic nature of the interactions in vivo remains largely unknown. In this work, we explored the rearrangements upon glucose stress using fluorescence energy transfer (hetero-FRET). Results revealed differences in the proximity of the canonical components with 1% (55.5 mM) glucose concentration, with the helicase RhlB and the glycolytic enzyme Enolase exhibiting the largest changes to the C-terminus of RNase E, followed by PNPase. We quantified ptsG mRNA decay and SgrS sRNA synthesis as they mediate bacterial adaptation to glucose stress conditions. We propose that once the mRNA degradation is completed, the RhlB, Enolase and PNPase decrease their proximity to the C-terminus of RNase E. Based on the results, we present a model where the canonical components of the RNAD coalesce when the bacteria is under glucose-6-phosphate stress and associate it with RNA decay. Our results demonstrate that FRET is a helpful tool to study conformational rearrangements in enzymatic complexes in bacteria in vivo.

Simultaneous conversion of glucose and xylose to 3-hydroxypropionic acid in engineered Escherichia coli by modulation of sugar transport and glycerol synthesis.

Escherichia coli expressing the Lactobacillus brevis dhaB1B2B3 and dhaR1R2 clusters and Pseudomonas aeruginosa aldhH was engineered to produce 3-HP from glucose and xylose via the glycerol biosynthetic pathway. Glycerol, a key precursor for 3-HP biosynthesis was produced by overexpression of the GPD1 and GPP2 genes from Saccharomyces cerevisiae. For relief of carbon catabolite repression, deletion of the chromosomal ptsG gene and overexpression of the endogenous xylR gene rendered engineered E. coli JHS01300/pCPaGGRm to utilize glucose and xylose simultaneously and to produce glycerol at 0.48 g/g yield and 0.35 g/L-h productivity. Finally, engineered E. coli JHS01300/pELDRR+pCPaGGRm produced 29.4 g/L of 3-HP with 0.54 g/L-h productivity and 0.36 g/g yield in a sugar-limited fed-batch fermentation. It was concluded that dual modulation of sugar transport and glycerol biosynthesis is a promising strategy for efficient conversion of glucose and xylose to 3-HP.

Simultaneous utilization of glucose and mannose from woody hydrolysate for free fatty acid production by metabolically engineered Escherichia coli.

In this study, the Escherichia coli strain MG1655 with fadD mutant (named as ML103), and MG1655 with fadD and ptsG double mutant (named as ML190) carrying the plasmid with the acyl-ACP thioesterase (TE) from Ricinus communis (pXZ18) or the plasmid with the combination of the TE and the native (3R)-hydroxyacyl-ACP dehydrase (fabZ) (pXZ18Z), produced free fatty acids (FFAs) efficiently using mannose as the sole carbon source. Due to the carbon catabolite repression (CCR) regulation, ML103(pXZ18) utilized glucose and mannose sequentially in the mixed sugar culture, while ML190(pXZ18) and ML190(pXZ18Z), with ptsG mutation, used glucose and mannose simultaneously. The highest total FFA concentration from the mixed sugar culture reached 2.96g/L by ML190(pXZ18Z). Furthermore, the strain ML190(pXZ18Z) can produce 2.86g/L FFAs with a high yield of 0.23g/g using hydrolysate mainly contained glucose and mannose from a commercial plant.

In silico deletion of PtsG gene in Escherichia coli genome-scale model predicts increased succinate production from glycerol.

Systems metabolic engineering and in silico analyses are necessary to study gene knockout candidate for enhanced succinic acid production by Escherichia coli. Metabolically engineered E. coli has been reported to produce succinate from glucose and glycerol. However, investigation on in silico deletion of ptsG/b1101 gene in E. coli from glycerol using minimization of metabolic adjustment algorithm with the OptFlux software platform has not yet been elucidated. Herein we report what is to our knowledge the first direct predicted increase in succinate production following in silico deletion of the ptsG gene in E. coli GEM from glycerol with the OptFlux software platform. The result indicates that the deletion of this gene in E. coli GEM predicts increased succinate production that is 20% higher than the wild-type control model. Hence, the mutant model maintained a growth rate that is 77% of the wild-type parent model. It was established that knocking out of the ptsG/b1101 gene in E. coli using glucose as substrate enhanced succinate production, but the exact mechanism of this effect is still obscure. This study informs other studies that the deletion of ptsG/b1101 gene in E. coli GEM predicted increased succinate production, enabling a model-driven experimental inquiry and/or novel biological discovery on the underground metabolic role of this gene in E. coli central metabolism in relation to increasing succinate production when glycerol is the substrate.

Efficient free fatty acid production from woody biomass hydrolysate using metabolically engineered Escherichia coli.

Four engineered Escherichia coli strains, ML103(pXZ18), ML103(pXZ18Z), ML190(pXZ18) and ML190(pXZ18Z), were constructed to investigate free fatty acid production using hydrolysate as carbon source. These strains exhibited efficient fatty acid production when xylose was used as the sole carbon source. For mixed sugars, ML103 based strains utilized glucose and xylose sequentially under the carbon catabolite repression (CCR) regulation, while ML190 based strains, with ptsG mutation, used glucose and xylose simultaneously. The total free fatty acid concentration and yield of the strain ML190(pXZ18Z) based on the mixed sugar reached 3.64 g/L and 24.88%, respectively. Furthermore, when hydrolysate from a commercial plant was used as the carbon source, the strain ML190(pXZ18Z) can produce 3.79 g/L FFAs with a high yield of 21.42%.

The small RNA SgrS: roles in metabolism and pathogenesis of enteric bacteria.

Bacteria adapt to ever-changing habitats through specific responses to internal and external stimuli that result in changes in gene regulation and metabolism. One internal metabolic cue affecting such changes in Escherichia coli and related enteric species is cytoplasmic accumulation of phosphorylated sugars such as glucose-6-phosphate or the non-metabolizable analog α-methylglucoside-6-phosphate. This "glucose-phosphate stress" triggers a dedicated stress response in γ-proteobacteria including several enteric pathogens. The major effector of this stress response is a small RNA (sRNA), SgrS. In E. coli and Salmonella, SgrS regulates numerous mRNA targets via base pairing interactions that result in alterations in mRNA translation and stability. Regulation of target mRNAs allows cells to reduce import of additional sugars and increase sugar efflux. SgrS is an unusual sRNA in that it also encodes a small protein, SgrT, which inhibits activity of the major glucose transporter. The two functions of SgrS, base pairing and production of SgrT, reduce accumulation of phosphorylated sugars and thereby relieve stress and promote growth. Examination of SgrS homologs in many enteric species suggests that SgrS has evolved to regulate distinct targets in different organisms. For example, in Salmonella, SgrS base pairs with sopD mRNA and represses production of the encoded effector protein, suggesting that SgrS may have a specific role in the pathogenesis of some γ-proteobacteria. In this review, we outline molecular mechanisms involved in SgrS regulation of its target mRNAs. We also discuss the response to glucose-phosphate stress in terms of its impact on metabolism, growth physiology, and pathogenesis.