Difference in the distribution pattern of substrate enzymes in the metabolic network of Escherichia coli, according to chaperonin requirement

<p>Abstract</p> <p>Background</p> <p>Chaperonins are important in living systems because they play a role in the folding of proteins. Earlier comprehensive analyses identified substrate proteins for which folding requires the chaperonin GroEL/GroES (GroE) in <it>Escherichia coli</it>, and they revealed that many chaperonin substrates are metabolic enzymes. This result implies the importance of chaperonins in metabolism. However, the relationship between chaperonins and metabolism is still unclear.</p> <p>Results</p> <p>We investigated the distribution of chaperonin substrate enzymes in the metabolic network using network analysis techniques as a first step towards revealing this relationship, and found that as chaperonin requirement increases, substrate enzymes are more laterally distributed in the metabolic. In addition, comparative genome analysis showed that the chaperonin-dependent substrates were less conserved, suggesting that these substrates were acquired later on in evolutionary history.</p> <p>Conclusions</p> <p>This result implies the expansion of metabolic networks due to this chaperonin, and it supports the existing hypothesis of acceleration of evolution by chaperonins. The distribution of chaperonin substrate enzymes in the metabolic network is inexplicable because it does not seem to be associated with individual protein features such as protein abundance, which has been observed characteristically in chaperonin substrates in previous works. However, it becomes clear by considering this expansion process due to chaperonin. This finding provides new insights into metabolic evolution and the roles of chaperonins in living systems.</p

Revealing the Functions of the Transketolase Enzyme Isoforms in Rhodopseudomonas palustris Using a Systems Biology Approach

BACKGROUND: Rhodopseudomonas palustris (R. palustris) is a purple non-sulfur anoxygenic phototrophic bacterium that belongs to the class of proteobacteria. It is capable of absorbing atmospheric carbon dioxide and converting it to biomass via the process of photosynthesis and the Calvin-Benson-Bassham (CBB) cycle. Transketolase is a key enzyme involved in the CBB cycle. Here, we reveal the functions of transketolase isoforms I and II in R. palustris using a systems biology approach. METHODOLOGY/PRINCIPAL FINDINGS: By measuring growth ability, we found that transketolase could enhance the autotrophic growth and biomass production of R. palustris. Microarray and real-time quantitative PCR revealed that transketolase isoforms I and II were involved in different carbon metabolic pathways. In addition, immunogold staining demonstrated that the two transketolase isoforms had different spatial localizations: transketolase I was primarily associated with the intracytoplasmic membrane (ICM) but transketolase II was mostly distributed in the cytoplasm. Comparative proteomic analysis and network construction of transketolase over-expression and negative control (NC) strains revealed that protein folding, transcriptional regulation, amino acid transport and CBB cycle-associated carbon metabolism were enriched in the transketolase I over-expressed strain. In contrast, ATP synthesis, carbohydrate transport, glycolysis-associated carbon metabolism and CBB cycle-associated carbon metabolism were enriched in the transketolase II over-expressed strain. Furthermore, ATP synthesis assays showed a significant increase in ATP synthesis in the transketolase II over-expressed strain. A PEPCK activity assay showed that PEPCK activity was higher in transketolase over-expressed strains than in the negative control strain. CONCLUSIONS/SIGNIFICANCE: Taken together, our results indicate that the two isoforms of transketolase in R. palustris could affect photoautotrophic growth through both common and divergent metabolic mechanisms

The cumulative impact of chaperone mediated protein- folding during evolution

Molecular chaperones support protein folding and unfolding along with assembly and translocation of protein complexes. Chaperones have been recognized as important mediators between organismal genotype and phenotype as well as important maintainers of cellular fitness under environmental conditions that induce high mutational loads. This thesis presents recent studies revealing that the folding assistance supplied by chaperones is evident in genomic sequences, thus implicating chaperone-mediated folding as an influential factor during protein evolution. Furthermore the evolution and the symbiogenic origin of the eukaryotic chaperone repertoire are elucidated. Protein interaction with chaperones ensures a proper folding and function, yet an adaptation to obligatory dependence on such assistance may be irreversible, representing an evolutionary trap. Correlation between chaperone requirement and protein expression level indicate that the evolution of substrate-chaperone interaction is bounded by the required substrate abundance within the cell. Accumulating evidence suggests that the utility of chaperones is governed by a delicate balance between their help in mitigating the risks of protein misfolding and aggregate formation on the one hand, and the slower rate of protein maturation and the energetic cost of chaperone synthesis on the other

Studies on Methylmalonyl-CoA Mutase from Escherichia coli

Methylmalonyl-CoA mutase (MCM, E.C. 5.4.99.2), a coenzyme B12-dependent enzyme, catalyses the inter conversion of succinyl-CoA and methylmalonyl- CoA. The gene (sbm) encoding this enzyme is found in Escherichia coli (E. coli) at 62.3min on the E. coli chromosome. However, the metabolic role of this enzyme in the organism is not known. This project involves an investigation into this metabolic obscurity. The sbm gene is part of a four gene operon which also includes argK (or ygfD) that codes for a protein kinase catalysing the phosphorylation of two periplasmic binding proteins involved in cationic amino acid transport, ygfG that codes for methylmalonyl-CoA decarboxylase and ygfH that codes for propionyl-CoA: succinyl-CoA transferase. From existing literature we suspect that this operon, including the sbm gene, could be involved in the utilisation of unusual carbon sources such as succinate and propionate. An insertion mutant of the sbm gene created by transposon mediated mutagenesis was used for investigating the role of this gene. The wild type E. coli K12 strain, E. coli TR6524 and the mutant E. coli K12 (sbm::MudJ) were used in this study. Growth of the two strains (E. coli TR6524 and FA1P1) in minimal media with three different concentrations (0.05, 0.5, 5.0μg/mL) of vitamin B12 and in the presence succinate, propionate or glucose as the sole source of carbon, was studied. Growth was typical in media with glucose with no major differences in the growth pattern of the wild type and mutant strain. However, the two strains exhibited a differential growth pattern in media containing succinate, with the wild type growing faster than the mutant, indicating the role of the sbm gene in the utilisation of this carbon source. Growth in media containing propionate as the sole carbon source indicated only marginal differences in the growth pattern of the wild type and mutant strain. This result possibly suggests that the other pathways for propionate utilisation in E. coli compensate for the lack of a functional Sbm protein in the mutant strain. Promoter analysis indicated the presence of a promoter induced by σS, a transcription factor involved in the expression of proteins under stress or stationary phase growth conditions. Reverse transcription polymerase chain reaction (RT-PCR) studies of the genes of the sbm operon (sbm-argK-ygfGygfH) under the same growth conditions were carried out. Densitometric analysis of the PCR products suggested that the transcription level of sbm was higher in E. coli grown in succinate as compared to when grown in glucose and not as much when grown in propionate indicating a transcriptional level control of the sbm gene expression during the utilisation of succinate. RT-PCR studies also indicated a higher level of transcription of the gene in the stationary phase of the culture during the utilisation of succinate. Real time reverse transcription PCR (QPCR) analysis was used for the absolute quantification of the transcription of the genes of the sbm operon. An increase in the mRNA levels corresponding to the sbm, argK and ygfG genes was observed as E. coli TR6524 growth reached stationary phase, in the presence of succinate or propionate as the sole source of carbon as compared to glucose, In contrast, the highest mRNA levels corresponding to the ygfH gene were observed in the early log-phase of growth. This indicated a differential transcriptional level control of the genes within the operon. This study further established the possible role of this operon in the utilisation of succinate and propionate. The MCM enzyme activity measurement in the whole cell extracts of the wild type E. coli K12, grown under the above mentioned conditions, led to the first ever measurement of MCM activity in wild type E. coli. These measurements also revealed a four fold increase of the MCM specific activity in the case of growth in succinate (4.76x10-3U/mg) and a two fold increase for growth in propionate (2.79x10-3U/mg) compared to that observed with growth in glucose (1.37x10-3U/mg), indicating a significant level of involvement of the enzyme in succinate utilisation, and to a lesser extent in propionate utilisation. The proteomic analysis to understand the gene expression pattern of E. coli TR6524 was carried out using cells harvested at the stationary phase. The results showed that growth conditions induced the expression of transport related (HisJ, DppA) and energy generating proteins (PckA, AceF) required by E. coli to cope with the stressful growth conditions. However, Sbm was not identified among the limited protein spots that were analysed. Finally, E. coli K12 sbm gene was successfully cloned into B. cereus SPV leading to the development of a metabolically engineered polyhydroxyalkanoate producing strain of B. cereus. The intention was to provide the bacteria with a natural intracellular source of propionyl-CoA, leading to the production of the P(3HB-co-3HV) copolymer from structurally non related carbon sources like glucose. Hence, this work has initiated investigation into the metabolic role of the sbm gene product in E. coli. In addition, it has also led to the use of this gene product in metabolic engineering applications

Monitoring bacterial physiology during recombinant protein production using reporter gene technology

This work presents an evaluation of the applicability of gene reporter technology to monitor Escherichia coli stress in industrial conditions with special interest in recombinant protein production. Different reporter plasmids containing promoter sequences of genes of the heatshock response were utilized to monitor chaperone expression upon different sources of stress such as exposure to chemicals, temperature and anaerobic growth. Activation of the heat shock response was monitored by $\beta$-galactosidase activity from the reporter plasmid pQF50groE. Cultures responded to heat-shock, anaerobiosis and $\beta$-mercaptoethanol by increasing the expression of $\sigma$$^{32}$-related genes. The performance of fluorescence reporters containing varieties of GFP was measured by fluorimetry and flow cytometry. Low copy number plasmids were demonstrated to be better suited than medium-high copy plasmids to report stress in industrial conditions. Reporter plasmids containing the promoters of the chaperones DnaK and GroES were utilized to measure E. coli stress in reducing environments and during recombinant protein production. It was demonstrated that the production strategy caused an impact in the host physiology which determined the outcome of the process. Flow cytometry showed excellent potential to obtain reliable measurements providing data of reporter activity cell death and cell morphology