Secretion and folding of human growth hormone in Escherichia coli

Abstract

Phytoremediation is the use of plants for the treatment of environmental pollution, including chlorinated organics. although conceptually very attractive, removal and biodegradation of chlorinated pollutants by plants is a rather slow and inefficient process resulting in incomplete treatment and potential release of toxic metabolites into the environment. In order to overcome inherent limitations of plant metabolic capabilities, plants have been genetically modified, following a strategy similar to the development of transgenic crops: genes from bacteria, fungi, and mammals involved in the metabolism of organic contaminants, such as cytochrome p-450 and glutathione substrate catabolic genes, natural or engineered, for the simultaneous remediation of a range of pollutants, such as usually found in contaminated sites, e.g., chlorinated solvent, metals, and nitroaromatics. In addition, biodegradation of many xenobiotics are catalyzed by similar, broad-substrate enzymes, such as cytochrome P-450 monoxygenases, glutathione S-transferases, and fungal peroxidases, that can potentially be used for the treatment of multiple pollutants. Moreover, the introduction of multiple transgenes involved in different phases of the metabolism of xenobiotics in plants, i.e., uptake by roots and the different phases of the green liver model, would allow enhancing both the removal and metabolism of several toxic compounds and could therefore help overcome a major limitation inherent to phytoremediation, i.e., the threat that accumulated toxic compounds would volatilize or otherwise contaminate the food chain. An important barrier to the application of transgenic plants for bioremediation in the field is associated with the true or perceived risk of horizontal gene transfer to related wild or cultivated plants. Therefore, it is likely that the next generation of transgenic plants will involve systems preventing such a transfer, for instance by the introduction of transgenes into chloroplastic DNA or the use of conditional lethality genes (Davison, 2005). Since bacteria naturally exchange plasmids via conjugation, endophytes that gain genes involved in pollutant degradation might not be considered ‘genetically modified’ and may be subject to fewer restrictions in usage.     

Interconnecting solvent quality,transcription, and chromosome folding in Escherichia coli

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Catching prompt metabolite dynamics in Escherichia coli with the BioScope at oxygen rich conditions

The design and application of a BioScope, a mini plug-flow reactor for carrying out pulse response experiments, specifically designed for Escherichia coli is presented. Main differences with the previous design are an increased volume-specific membrane surface for oxygen transfer and significantly decreased sampling intervals. The characteristics of the new device (pressure drop, residence time distribution, plug-flow behavior and O₂ mass transfer) were determined and evaluated. Subsequently, 2.8 mM glucose perturbation experiments on glucose-limited aerobic E. coli chemostat cultures were carried out directly in the chemostat as well as in the BioScope (for two time frames: 8 and 40 s). It was ensured that fully aerobic conditions were maintained during the perturbation experiments. To avoid metabolite leakage during quenching, metabolite quantification (glycolytic and TCA-cycle intermediates and nucleotides) was carried out with a differential method, whereby the amounts measured in the filtrate were subtracted from the amounts measured in total broth. The dynamic metabolite profiles obtained from the BioScope perturbations were very comparable with the profiles obtained from the chemostat perturbation. This agreement demonstrates that the BioScope is a promising device for studying in vivo kinetics in E. coli that shows much faster response (<10 s) in comparison with eukaryotes.     

Variation in periodic replication of the chromosome in Escherichia coli B/rTT.

A method using 5-bromouracil photolysis induction with 313 nm radiation was employed to estimate the variation in the period between successive rounds of DNA replication in rapidly growing cultures of Escherichia coli$\text{B}\text{rTT}$ The coefficient of variation of this period was 9.3%, which is significantly less than the corresponding value of about 20% reported for variation in the cell interdivision period. Thus chromosome replication is much more tightly controlled than is cell division. The reduced variability of the DNA replication cycle indicates that the period (D) between termination of a round of DNA replication and cell division and the following period ending in initiation of the next round of DNA replication (B) are riot independent of each other but tend to have compensatory variations. The results suggest that other events in the cell cycle are related more closely to DNA replication rather than to the much less regular event of cell division.     

Protein folding in the periplasm of Escherichia coli

With the discovery of molecular chaperones and the development of heterologous gene expression techniques, protein folding in bacteria has come into focus as a potentially limiting factor in expression and as a topic of interest in its own right. Many proteins of importance in biotechnology contain disulphide bonds, which form in the Escherichia coli periplasm, but most work on protein folding in the periplasm of E. coli is very recent and is often speculative. This MicroReview gives a short overview of the possible fates of a periplasmic protein from the moment it is translocated, as well as of the E. coli proteins involved in this process. After an introduction to the specific physiological situation in the periplasm of E. coli, we discuss the proteins that might help other proteins to obtain their correctly folded conformation — disulphide isomerase, rotamase, parts of the translocation apparatus and putative periplasmic chaperones — and briefly cover the guided assembly of multi-subunit structures. Finally, our MicroReview turns to the fate of misfolded proteins: degradation by periplasmic proteases and aggregation phenomena.     

Disulfide-dependent folding and export of Escherichia coli DsbC

DsbC, a member of the Dsb family in the periplasm of Gram-negative bacteria, is not only a disulfide isomerase but also a chaperone. Five DsbC mutants with Cys in the active site sequence of Cys(98)-Gly-Tyr-Cys(101) and the nonactive site disulfide Cys(141)-Cys(163) replaced by Ser have been studied. The results show that the active site Cys residues are necessary for enzyme activities but not required for chaperone activity, while the lack of the nonactive site disulfide results in a decreased chaperone activity in assisting the reactivation of denatured d-glyceraldehyde-3-phosphate dehydrogenase but has no effect on enzyme activities. Wild-type DsbC was overexpressed and correctly processed as a soluble periplasmic protein. Mutation in one of these Cys residues results in aggregation or extracellular/membrane locations, but does not affect the proper processing. DsbC mutated in either Cys residue of nonactive site disulfide shows higher sensitivity to unfolding by guanidine hydrochloride and slower refolding compared with wild-type DsbC and the active site Cys mutants. The above results provide experimental evidence for structural role of the nonactive site disulfide in folding and biological activities of DsbC.     

Beta-lactamase production by Escherichia coli under different growth conditions

Facultative anaerobic bacteria, such as Escherichia coli, are more resistant to cephalosporin antibiotics during anaerobic growth. Strict anaerobic ambience reduces beta-lactamase production or the enzyme affinities for their substrates. A different balance between DNA gyrase and topoisomerase I activity, during aerobic and anaerobic growth condition, could be related to the bacteria behavior.     

Progressive segregation of the Escherichia coli chromosome

We have followed the fate of 14 different loci around the Escherichia coli chromosome in living cells at slow growth rate using a highly efficient labelling system and automated measurements. Loci are segregated as they are replicated, but with a marked delay. Most markers segregate in a smooth temporal progression from origin to terminus. Thus, the overall pattern is one of continuous segregation during replication and is not consistent with recently published models invoking extensive sister chromosome cohesion followed by simultaneous segregation of the bulk of the chromosome. The terminus, and a region immediately clockwise from the origin, are exceptions to the overall pattern and are subjected to a more extensive delay prior to segregation. The origin region and nearby loci are replicated and segregated from the cell centre, later markers from the various positions where they lie in the nucleoid, and the terminus region from the cell centre. Segregation appears to leave one copy of each locus in place, and rapidly transport the other to the other side of the cell centre.     

Recombinant protein folding and misfolding in Escherichia coli

The past 20 years have seen enormous progress in the understanding of the mechanisms used by the enteric bacterium Escherichia coli to promote protein folding, support protein translocation and handle protein misfolding. Insights from these studies have been exploited to tackle the problems of inclusion body formation, proteolytic degradation and disulfide bond generation that have long impeded the production of complex heterologous proteins in a properly folded and biologically active form. The application of this information to industrial processes, together with emerging strategies for creating designer folding modulators and performing glycosylation all but guarantee that E. coli will remain an important host for the production of both commodity and high value added proteins.     

Macrodomain organization of the Escherichia coli chromosome

We have explored the Escherichia coli chromosome architecture by genetic dissection, using a site-specific recombination system that reveals the spatial proximity of distant DNA sites and records interactions. By analysing the percentages of recombination between pairs of sites scattered over the chromosome, we observed that DNA interactions were restricted to within subregions of the chromosome. The results indicated an organization into a ring composed of four macrodomains and two less-structured regions. Two of the macrodomains defined by recombination efficiency are similar to the Ter and Ori macrodomains observed by FISH. Two newly characterized macrodomains flank the Ter macrodomain and two less-structured regions flank the Ori macrodomain. Also the interactions between sister chromatids are rare, suggesting that chromosome segregation quickly follows replication. These results reveal structural features that may be important for chromosome dynamics during the cell cycle.     

Segregation of the Escherichia coli chromosome terminus

We studied the segregation of the replication terminus of the Escherichia coli chromosome by time-lapse and still photomicroscopy. The replicated termini lie together at the cell centre. They rapidly segregate away from each other immediately before cell division. At fast growth rate, the copies move progressively and quickly toward the centres of the new-born cells. At slow growth rate, the termini usually remain near the inner cell pole and migrate to the cell centre in the middle of the cell cycle. A terminus domain of about 160kb, roughly centred on the dif recombination site, segregated as a unit at cell division. Sequences outside this domain segregated before division, giving two separate foci in predivision cells. Resolution of chromosome dimers via the terminus dif site requires the XerC recombinase and an activity of the FtsK protein that is thought to align the dif sequences at the cell centre. We found that anchoring of the termini at the cell centre and proper segregation at cell division occurred normally in the absence of recombination via the XerC recombinase. Anchoring and proper segregation were, however, frequently disrupted when the C-terminal domain of FtsK was truncated.     

Kinetic models for the growth of Escherichia coli with mixtures of sugars under carbon-limited conditions

In natural environments, heterotrophic microorganisms encounter complex mixtures of carbon sources, each of which is present only at very low concentrations. Under such conditions no significant growth could be expected if cells utilized only one of the available carbon compounds as suggested by the principle of diauxic growth. Indeed, there is much evidence that microbial cells utilize many carbon sources simultaneously. In order to predict bacterial growth under such conditions we developed a model describing the specific growth rate as a function of the individual concentrations of several simultaneously utilized carbon substrates. Together with multisubstrate models previously published, this model was evaluated for its ability to describe growth of Escherichia coli during the simultaneous utilization of mixtures of sugars in carbon-limited continuous culture. Using the micromax and Ks constants determined for single substrate growth with six different sugars, the model was able for most experiments to adequately describe the specific growth rate of the culture, i.e., the experimentally set dilution rate, from the measured concentrations of the individual sugars. The model provides an explanation why bacteria can still grow relatively fast under environmental conditions where the concentrations of carbon substrates are usually extremely low.     

The effect of growth rate and other growth conditions on the lipid composition of Escherichia coli

Escherichia coli was grown as a continuous culture at various defined conditions of temperature, pH, aeration rate and dilution rate. The lipids were extracted from disrupted cells and the relative fatty acid content of the individual and total phospholipids was determined. The lipid composition of E. coli was shown to change with the fermentation conditions. Interestingly, E. coli adapted to high growth rates and to low oxygen tension by changing the lipid composition of the membrane in exactly the same way, thus indicating a common effect.     

Sister chromosome cohesion of Escherichia coli

We analysed Escherichia coli cells synchronized for initiation of chromosomal DNA replication by fluorescence in situ hybridization (FISH) using fluorescent DNA probes corresponding to various chromosomal regions. Sister copies of regions in an approximately oriC-proximal half of the chromosome are cohesive with each other after replication until the late period of chromosome replication. Sister copies of regions relatively close to the terminus are also separated from each other in the same late period of replication. It is important that sister copies in all the tested regions are thus separated from each other nearly all at once in the late period of chromosome replication. These results are consistent with results obtained by FISH in randomly growing cultures. Cohesion of sister copies in an oriC-close region is observed in a dam null mutant lacking DNA adenine methyltransferase the same as in the parental isogenic dam+ strain, indicating that the cohesion is independent of DNA adenine methyltransferase. This further implies that hemimethylated DNA-binding proteins, such as SeqA, are not involved in the cohesion. On the other hand, the cohesion of sister copies of the oriC-close region was not observed in mukB null mutant cells, suggesting that MukB might be involved in the chromosome cohesion.