GroEL/GroES chaperone and Lon protease regulate expression of the Vibrio fischeri lux operon in Escherichia coli

Chaperone GroEL/GroES and Lon protease were shown to play a role in regulating the expression of the Vibrio fischeri lux operon cloned in Escherichia coli cells. The E. coli groE mutant carrying a plasmid with the full-length V. fischeri lux regulon showed a decreased bioluminescence. The bioluminescence intensity was high in E. coli cells with mutant lonA and the same plasmid. Bioluminescence induction curves lacked the lag period characteristic of lon ⁺ strains. Regulatory luxR of V. fischeri was cloned in pGEX-KG to produce the hybrid gene GST-luxR. The product of its expression, GST-LuxR, was isolated together with GroEL and Lon upon affinity chromatography on a column with glutathione-agarose, suggesting complexation of LuxR with these proteins. It was assumed that GroEL/GroES is involved in LuxR folding, while Lon protease degrades LuxR before its folding into an active globule or after denaturation.     

Chaperone mediated solubilization of 69-kDa recombinant maltodextrin glucosidase in Escherichia coli

Aims:  To investigate the factors affecting expression and solubilization of Escherichia coli maltodextrin glucosidase in E. coli .
Methods and Results:  Expression level and solubilization of the recombinant E. coli maltodextrin glucosidase was studied in E. coli at different temperatures, in presence of overexpressed GroEL, GroES and externally supplemented glycerol. Aggregation of maltodextrin glucosidase in the cytoplasm was partially prevented by the co-expression of GroEL and GroES, and using externally supplemented glycerol or lowering the culture temperature. Co-expression of GroEL and GroES or simultaneous presence of overexpressed GroEL, GroES and externally supplemented glycerol together resulted significant increase of the activity of maltodextrin glucosidase. The growth rate of E. coli was inhibited by the formation of inclusion bodies whereas the presence of overexpressed GroEL, GroES alone or together with glycerol enhanced the growth rate of E. coli substantially.
Conclusions:  The results indicated that lowering the temperature, use of GroEL, GroES and glycerol could be few controlling factors for the solubilization of recombinant aggregation-prone maltodextrin glucosidase in E. coli.
Significance and Impact of the Study:  Our study could help in developing the strategy for enhancing the production of soluble industrial enzymes and finding the therapeutic agents against protein misfolding diseases.     

Structures of oversized genomes of phage P1 derivatives carrying lac genes of Escherichia coli K12

Physical analyses of two newly isolated oversized P1lac phage genomes showed that they are partly diploid in P1 genes and that they carry a 60–70-kb segment of host DNA. The transposable element γδ is present at one of the junctions between host and P1 DNA, and IS1 is at the other junction. These elements must thus have been actively involved in the formation of these P1lac prophages. The genome of a third oversized P1lac has a segment with dispensable P1 genes deleted. The absence of any known recombinogenic element at one of its junctions between P1 and host DNA suggests non-homologous recombination to have been involved in its formation. Non-homologous recombination might have also taken place in one of the final steps of the formation of the former P1lac genomes.     

Reconstitution of the Mycobacterium tuberculosis pupylation pathway in Escherichia coli

Prokaryotic ubiquitin-like protein (Pup) is a post-translational modifier that attaches to more than 50 proteins in Mycobacteria. Proteasome accessory factor A (PafA) is responsible for Pup conjugation to substrates, but the manner in which proteins are selected for pupylation is unknown. To address this issue, we reconstituted the pupylation of model Mycobacterium proteasome substrates in Escherichia coli, which does not encode Pup or PafA. Surprisingly, Pup and PafA were sufficient to pupylate at least 51 E. coli proteins in addition to the mycobacterial proteins. These data suggest that pupylation signals are intrinsic to targeted proteins and might not require Mycobacterium-specific cofactors for substrate recognition by PafA in vivo.     

Expression of leucine genes from an extremely thermophilic bacterium in Escherichia coli

Summary The organisation of the leucine genes in Thermus thermophilus HB8 was analysed by examining the ability of recombinant DNAs to complement Escherichia coli mutations. The arrangement of the genes is different from that in the mesophilic bacteria E. coli and Salmonella typhimurium. The promoter responsible for the expression of the leuB, leuC and leuD genes of Thermus HB8 in E. coli was identified. The sequence of Thermus DNA containing this promoter revealed structural similarities to the promoter and attenuator regions of the E. coli leucine operon.     

The mechanism of sugar-mediated catabolite repression of the propionate catabolic genes in Escherichia coli

Carbon catabolite repression (CCR) is a well-known phenomenon that involves the preferential utilization of glucose as a carbon source. Cyclic adenosine monophosphate (cAMP) and the cAMP receptor protein (CRP) mediate CCR. Recently, a second CCR hierarchy that leads to the preferential consumption of arabinose over xylose, mediated by arabinose-bound AraC, has been identified. In this study, we report yet another CCR hierarchy that causes the preferential utilization of sugars (arabinose, galactose, glucose, mannose, and xylose) over a short-chain fatty acid (propionate). Expression of the propionate catabolic (prpBCDE) genes is down-regulated in the presence of these sugars. Sugar-mediated repression of the propionate catabolic genes is independent of sugar-specific regulators such as AraC and dependent on global regulators of sugar transport such as the cAMP-CRP complex and the Phosphotransferase System (PTS). Inhibition of the prpBCDE promoter is encountered during rapid sugar uptake and metabolism. This unique regulatory crosstalk between sugar metabolism and fatty acid metabolism may help provide new insights into CRP-dependent catabolite repression acting in conjunction with non-carbohydrate metabolism.     

Docking analysis insights quercetin can be a non-antibiotic adjuvant by inhibiting Mmr drug efflux pump in Mycobacterium sp. and its homologue EmrE in Escherichia coli

Drug efflux pumps (EP) like Mmr in Mycobacterium transported drugs out of cell, a main reason for drug resistance developing in Mycobacterium tuberculosis. In this in silico study, mainly analysed EP inhibitory potential of a plant-derived flavonoid, quercetin, through docking analysis. Mmr present in Mycobacterium smegmatis and M. tuberculosis, and its homologue EmrE of Escherichia coli was used. Initially, homology modelling of EP monomers and dimers constructed from M. smegmatis, M. tuberculosis and E. coli; the stabilities of models were analysed from Ramachandran plots prepared in PROCHECK. Docking analysis of quercetin with EP protein showed that in all three organisms, the residues for function and stability are important and quercetin had best interactions comparing to compounds such as, verapamil, reserpine, chlorpromazine, Carbonyl Cyanide m- Chloro Phenylhydrazone. Molecular dynamics and simulation studies showed that during the entire course of simulation quercetin-Mmr complex were stable. It insights quercetin can act as a non-antibiotic adjuvant for treatment of tuberculosis by bring down the efflux of drug from bacteria.     

Organisation of the Escherichia coli chromosome between genes glnS and glnU, V

Summary Analysis of bacteriophage DNA and of subcloned plasmid DNA has allowed the localisation of the following genes, located at 16 min on the Escherichia coli chromosome, within a restriction map of the region: glnS, nagE, nagB, nagA, asnB, metT, leuW, glnU, glnU, metT, glnV, glnV.     

Characterization of three genes in the dam-containing operon of Escherichia coli

The dam-containing operon in Escherichia coli is located at 74 min on the chromosomal map and contains the genes aroK, aroB, a gene called urf74.3, dam and trpS. We have determined the nucleotide sequence between the dam and trpS genes and show that it encodes two proteins with molecular weights of 24 and 27 kDa. Furthermore, we characterize the three genes urf74.3, 24kDa, 27kDa and the proteins they encode. The predicted amino acid sequences of the 24 and 27 kDa proteins are similar to those of the CbbE and CbbZ proteins, respectively, of the Alcaligenes eutrophus cbb operon, which encodes enzymes involved in the Calvin cycle. In separate experiments, we have shown that the 24 kDa protein has d-ribulose-5-phosphate epimerase activity (similar to CbbE), and we call the gene rpe. Similarly, the 27 kDa protein has 2-phosphoglycolate phosphatase activity (similar to CbbZ), and we name the gene gph. The Urf74.3 protein, with a predicted molecular weight of 46 kDa, migrated as a 70 kDa product under denaturing conditions. Overexpression of Urf74.3 induced cell filamentation, indicating that Urf74.3 directly or indirectly interferes with cell division. We present evidence for translational coupling between aroB and urf74.3 and also between rpe and gph. Proteins encoded in the dam superoperon appear to be largely unrelated: Dam, and perhaps Urf74.3, are involved in cell cycle regulation, AroK, AroB, and TrpS function in aromatic amino acid biosynthesis, whereas Rpe and Gph are involved in carbohydrate metabolism.