Acetate metabolism in Escherichia coli strains lacking phosphoenolpyruvate: carbohydrate phosphotransferase system; evidence of carbon recycling strategies and futile cycles

The ptsHIcrr operon was deleted from Escherichia coli wild-type JM101 to generate strain PB11 (PTS(-)). In a mutant derived from PB11 that partially recovered its growth capacity on glucose by an adaptive evolution process (PB12, PTS(-)Glc(+)), part of the phosphoenolpyruvate not used in glucose transport has been utilized for the synthesis of aromatic compounds. In this report, it is shown that on acetate as a carbon source, PB11 displayed a specific growth rate (mu) higher than PB12 (0.21 and 0.13 h(-1), respectively) while JM101 had a mu of 0.28 h(-1). To understand these growth differences on acetate, we compared the expression profiles of central metabolic genes by RT-PCR analysis. Obtained data revealed that some gluconeogenic genes were downregulated in both PTS(-) strains as compared to JM101, while most glycolytic genes were upregulated in PB12 in contrast to PB11 and JM101. Furthermore, inactivation of gluconeogenic genes, like ppsA, sfcA, and maeB,and poxB gene that codes for pyruvate oxidase, has differential impacts in the acetate metabolism of these strains. Results indicate that growth differences on acetate in the PTS(-) derivatives are due to potential carbon recycling strategies, mainly in PB11, and futile carbon cycles, especially in PB12.     

Metabolic engineering and protein directed evolution increase the yield of L-phenylalanine synthesized from glucose in Escherichia coli

L-phenylalanine (L-Phe) is an aromatic amino acid with diverse commercial applications. Technologies for industrial microbial synthesis of L-Phe using glucose as a starting raw material currently achieve a relatively low conversion yield (Y(Phe/Glc)). The purpose of this work was to study the effect of PTS (phosphotransferase transport system) inactivation and overexpression of different versions of feedback inhibition resistant chorismate mutase-prephenate dehydratase (CM-PDT) on the yield (Y(Phe/Glc)) and productivity of L-Phe synthesized from glucose. The E. coli JM101 strain and its mutant derivative PB12 (PTS(-)Glc(+) phenotype) were used as hosts. PB12 has an inactive PTS, but is capable of transporting and phosphorylating glucose by using an alternative system constituted by galactose permease (GalP) and glucokinase activities (Glk). JM101 and PB12 were transformed with three plasmids, harboring genes that encode for a feedback inhibition resistant DAHP synthase (aroG(fbr)), transketolase (tktA) and either a truncated CM-PDT (pheA(fbr)) or its derived evolved genes (pheA(ev1) or pheA(ev2)). Resting-cells experiments with these engineered strains showed that JM101 and PB12 strains expressing either pheA(ev1) or pheA(ev2) genes produced l-Phe from glucose with Y(Phe/Glc) of 0.21 and 0.33 g/g, corresponding to 38 and 60% of the maximum theoretical yield (0.55 g/g), respectively. In addition, in both engineered strains the reached q(Phe) high levels of 40 mg/g-dcw.h. The metabolic engineering strategy followed in this work, including a strain with an inactive PTS, resulted in a positive impact over the Y(Phe/Glc), enhancing it nearly 57% compared with its PTS(+) counterpart. This is the first report wherein PTS inactivation was a successful strategy to improve the Y(Phe/Glc).     

ppGpp-dependent stationary phase induction of genes on Salmonella pathogenicity island 1

We have examined expression of the genes on Salmonella pathogenicity island 1 (SPI1) during growth under the physiologically well defined standard growth condition of Luria-Bertani medium with aeration. We found that the central regulator hilA and the genes under its control are expressed at the onset of stationary phase. Interestingly, the two-component regulatory genes hilC/hilD, sirA/barA, and ompR, which are known to modulate expression from the hilA promoter (hilAp) under so-called "inducing conditions" (Luria-Bertani medium containing 0.3 m NaCl without aeration), acted under standard conditions at the stationary phase induction level. The induction of hilAp depended not on RpoS, the stationary phase sigma factor, but on the stringent signal molecule ppGpp. In the ppGpp null mutant background, hilAp showed absolutely no activity. The stationary phase induction of hilAp required spoT but not relA. Consistent with this requirement, hilAp was also induced by carbon source deprivation, which is known to transiently elevate ppGpp mediated by spoT function. The observation that amino acid starvation elicited by the addition of serine hydroxamate did not induce hilAp in a RelA(+) SpoT(+) strain suggested that, in addition to ppGpp, some other alteration accompanying entry into the stationary phase might be necessary for induction. It is speculated that during the course of infection Salmonella encounters various stressful environments that are sensed and translated to the intracellular signal, ppGpp, which allows expression of Salmonella virulence genes, including SPI1 genes.     

Participation of the Entner-Doudoroff pathway in Escherichia coli strains with an inactive phosphotransferase system (PTS- Glc+) in gluconate and glucose batch cultures

The activity of the enzymes of the central metabolic pathways has been the subject of intensive analysis; however, the Entner-Doudoroff (ED) pathway has only recently begun to attract attention. The metabolic response to edd gene knockout in Escherichia coli JM101 and PTS- Glc+ was investigated in gluconate and glucose batch cultures and compared with other pyruvate kinase and PTS mutants previously constructed. Even though the specific growth rates between the strain carrying the edd gene knockout and its parent JM101 and PTS- Glc+ edd and its parent PTS- Glc+ were very similar, reproducible changes in the specific consumption rates and biomass yields were obtained when grown on glucose. These results support the participation of the ED pathway not only on gluconate metabolism but on other metabolic and biochemical processes in E. coli. Despite that gluconate is a non-PTS carbohydrate, the PTS- Glc+ and derived strains showed important reductions in the specific growth and gluconate consumption rates. Moreover, the overall activity of the ED pathway on gluconate resulted in important increments in PTS- Glc+ and PTS- Glc+ pykF mutants. Additional results obtained with the pykA pykF mutant indicate the important contribution of the pyruvate kinase enzymes to pyruvate synthesis and energy production in both carbon sources.     

Analysis of carbon metabolism in Escherichia coli strains with an inactive phosphotransferase system by (13)C labeling and NMR spectroscopy

We have developed Escherichia coli strains that internalize glucose utilizing the GalP permease instead of the phosphoenolpyruvate:carbohydrate phosphotransferase system. It has been demonstrated that a strain with these modifications (PTS(-)Glc(+)) can direct more carbon flux into the aromatic pathway than the wild-type parental strain (N. Flores et al., 1996, Nat. Biotechnol. 14, 620-623; G. Gosset et al., 1996, J. Ind. Microbiol. 17, 47-52; J. L. Baéz et al., 2001, Biotechnol. Bioeng. 73, 530-535). In this study, we have determined and compared the carbon fluxes of a wild-type strain (JM101), a PTS(-)Glc(-) strain, and two isogenic PTS(-)Glc(+) derivatives named PB12 and PB13 by combining genetic, biochemical, and NMR approaches. It was determined that in these strains a functional glk gene in the chromosome is required for rapid glucose consumption; furthermore, glucokinase-specific activities were higher than in the wild-type strain. (13)C labeling and NMR analysis allowed the determination of differences in vivo which include higher glycolytic fluxes of 93.1 and 89.2% compared with the 76.6% obtained for the wild-type E. coli. In PB12 and PB13 we found a flux through the malic enzymes of 4 and 10%, respectively, compared to zero in the wild-type strain. While flux through the Pck enzyme was absent in PB12 and PB13, in the wild type it was 7.7%. Finally, it was found that in the JM101 and PB12 strains both the oxidative and the nonoxidative branches of the pentose phosphate pathway contributed to ribose 5-phosphate synthesis, whereas in PB13 this pentose was synthesized almost exclusively through the oxidative branch. The determined carbon fluxes correlate with biochemical and genetic characterizations.     

The constancy of global regulation across a species: the concentrations of ppGpp and RpoS are strain-specific in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Sigma factors and the alarmone ppGpp control the allocation of RNA polymerase to promoters under stressful conditions. Both ppGpp and the sigma factor σ^S (RpoS) are potentially subject to variability across the species Escherichia coli. To find out the extent of strain variation we measured the level of RpoS and ppGpp using 31 E. coli strains from the ECOR collection and one reference K-12 strain.     

RNA聚合酶σ~S亚基的研究进展

σ~S(RpoS)是大肠杆菌RNA聚合酶的一个亚基.在压力情况下,如高温、酸、渗透冲击、营养缺陷和生长进入稳定期等能够被诱导,并在一定程度上能够取代σ~(70)与核心酶结合形成全酶,从而激活多数σ~S-依赖的基因的转录.对RpoS调控的基因和它们的启动子序列、调控方式以及它自身的调控进行了简单的综述.     

Influence of the RpoS (KatF) sigma factor on maintenance of viability and culturability of Escherichia coli and Salmonella typhimurium in seawater.

The sigma factor RpoS is essential for stationary-phase-specific, multiple-stress resistance. We compared the viabilities (direct viable counts) and culturabilities (colony counts) in seawater of Escherichia coli and Salmonella typhimurium strains and those in which rpoS was deleted or which were deficient in guanosine 3',5'-bispyrophosphate (ppGpp) synthesis (relA spoT). RpoS, possibly via ppGpp regulation, positively influenced the culturability of these bacteria in oligotrophic seawater. This influence closely depended, however, upon the growth state of the cells and the conditions under which they were grown prior to their transfer to seawater. The protective effect of RpoS was observed only in stationary-phase cells grown at low osmolarity. A previous exposure of cells to high osmolarity (0.5 M NaCl) also had a strong influence on the effect of RpoS on cell culturability in seawater. Both E. coli and S. typhimurium RpoS mutants lost the ability to acquire a high resistance to seawater, as observed in both logarithmic-phase and stationary-phase RpoS+ cells grown at high osmolarity. A previous growth of S. typhimurium cells under anoxic conditions also modulated the incidence of RpoS on their culturability. When grown anaerobically at high osmolarity, logarithmic-phase S. typhimurium RpoS+ cells partly lost their resistance to seawater through preadaptation to high osmolarity. When grown anaerobically at high osmolarity until stationary phase, both RpoS+ and RpoS- cells retained very high levels of both viability and culturability and then did not enter the viable but nonculturable state for over 8 days in seawater because of an RpoS-independent, unknown mechanism.     

The relationship between the spoT gene, the synthesis of stable RNA, ribosomal proteins, and the beta beta' subunits of RNA polymerase following a nutritional shiftup of Escherichia coli.

The level of ppGpp and rates of synthesis of stable RNA, ribosomal protein, and the beta and beta' subunits of RNA polymerase were measured following a nutritional shiftup in Escherichia coli strains, NF 929 (spoT+) and NF 930 (spoT-). In the spoT+ strain, ppGpp levels decreased 50% within 2 min following shiftup, and the rates of synthesis of stable RNA, ribosomal proteins, and the beta and beta' subunits of RNA polymerase increased with little or no lag. In contrast, in the spoT- strain, ppGpp levels transiently increased 40% during the first 6 min following shiftup. An inhibition in the rate of stable RNA synthesis and a delay in the increased synthesis of ribosomal proteins and beta and beta' subunits occurred concurrently with the transient increase in ppGpp. In addition, the DNA-dependent synthesis in vitro of the beta and beta' subunits of RNA polymerase was inhibited by physiological levels of ppGpp. Because of the timing and magnitude of the changes in ppGpp levels in the spoT- strain versus the timing when the new rates of stable RNA, ribosomal protein, and beta and beta' subunits synthesis are reached, it is concluded that ppGpp is not the sole element regulating the expression of these genes.     

Residual guanosine 3',5'-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations

It was known previously that 1) the relA gene of Escherichia coli encodes an enzyme capable of guanosine 3',5'-bispyrophosphate (ppGpp) synthesis, 2) an uncharacterized source of ppGpp synthesis exists in relA null strains, and 3) cellular degradation of ppGpp is mainly due to a manganese-dependent ppGpp 3'-pyrophosphohydrolase encoded by the spoT gene. Here, the effects of spoT gene insertions and deletions are compared with analogous alterations in neighboring genes in the spo operon and found to be lethal in relA+ strains as well as slower growing in relAl backgrounds than delta relA hosts. Cells with null alleles in both the relA and spoT genes are found no longer to accumulate ppGpp after glucose exhaustion or after chelation of manganese ions by picolinic acid addition; the inability to form ppGpp is reversed by a minimal spoT gene on a multicopy plasmid. Strains apparently lacking ppGpp show a complex phenotype including auxotrophy for several amino acids and morphological alterations. We propose that the SpoT protein can either catalyze or control the alternative pathway of ppGpp synthesis in addition to its known role as a (p)ppGpp 3'-pyrophosphohydrolase. We favor the possibility that the SpoT protein is a bifunctional enzyme capable of catalyzing either ppGpp synthesis or degradation.     

Analysis of differentially upregulated proteins in ptsHIcrr− and rppH− mutants in Escherichia coli during an adaptive laboratory evolution experiment

The previous deletion of the cytoplasmic components of the phosphotransferase system (PTS) in Escherichia coli JM101 resulted in the PTS⁻ derivative strain PB11 with severely impaired growth capability in glucose as the sole carbon source. Previous adaptive laboratory evolution (ALE) experiment led to select a fast-growing strain named PB12 from PB11. Comparative genome analysis of PB12 showed a chromosomal deletion, which result in the loss of several genes including rppH which codes for the RNA pyrophosphohydrolase RppH, involved in the preparation of hundreds of mRNAs for further degradation by RNase E. Previous inactivation of rppH in PB11 (PB11rppH⁻) improved significantly its growing capabilities and increased several mRNAs respect its parental strain PB11. These previous results led to propose to the PB11rppH⁻ mutant as an intermediate between PB11 and PB12 strains merged during the early ALE experiment. In this contribution, we report the metabolic response to the PTS⁻ and rppH⁻ mutations in the deep of a proteomic approach to understanding the relevance of rppH⁻ phenotype during an ALE experiment. Differentially upregulated proteins between the wild-type JM101/PB11, PB11/PB11rppH⁻, and PB11/PB12 comparisons led to identifying 45 proteins between strain comparisons. Downregulated or upregulated proteins in PB11rppH⁻ were found expressed at an intermediate level with respect to PB11 and PB12. Many of these proteins were found involved in non-previously metabolic traits reported in the study of the PTS⁻ strains, including glucose, amino acids, ribose transport; amino acid biosynthesis; NAD biosynthesis/salvage pathway, biosynthesis of Ac-CoA precursors; detoxification and degradation pathways; stress response; protein synthesis; and possible mutator activities between comparisons. No changes were found in the expression of galactose permease GalP, previously proposed as the primary glucose transporter in the absence of PTS selected by the PTS⁻ derivatives during the ALE experiment. This result suggests that the evolving PTS⁻ population selected other transporters such as LamB, MglB, and ManX instead of GalP for glucose uptake during the early ALE experiment. Analysis of the biological relevance of the metabolic traits developed by the studied strains provided valuable information to understand the relevance of the rppH⁻ mutation in the PTS⁻ background during an ALE experiment as a strategy for the selection of valuable phenotypes for metabolic engineering purposes.