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Heterologous Hosts (heterologous + hosts)
Selected AbstractsA novel DNA modification by sulphurMOLECULAR MICROBIOLOGY, Issue 5 2005Xiufen Zhou Summary Streptomyces lividans has a novel DNA modification, which sensitises its DNA to degradation during electrophoresis (the Dnd phenotype). The entire gene cluster (dnd) involved in this modification was localized on an 8 kb DNA fragment and was expressed in a S. lividans deletion mutant (dnd) and in several heterologous hosts. Disruption of the dnd locus abolishes the Dnd phenotype, and gain of the dnd locus conferred the Dnd phenotype respectively. Extensive analysis of the dnd gene cluster revealed five open reading frames, whose hypothetic functions suggested an incorporation of sulphur or a sulphur-containing substance into S. lividans genome, yet in an unknown manner. The Dnd phenotype was also discovered to exist in DNA of widespread bacterial species of variable origin and diverse habitat. Similarly organized gene clusters were found in several bacterial genomes representing different genera and in eDNA of marine organisms, suggesting such modification as a widespread phenomenon. A coincidence between the Dnd phenotype and DNA modification by sulphur was demonstrated to occur in several representative bacterial genomes by the in vivo35S-labelling experiments. [source] The dynamic microbe: green fluorescent protein brings bacteria to lightMOLECULAR MICROBIOLOGY, Issue 5 2002Carolyn M. Southward Summary The demonstration that the green fluorescent protein (GFP) from the jellyfish Aequorea victoria required no jellyfish-specific cofactors and could be expressed as a fluorescent protein in heterologous hosts including both prokaryotes and eukaryotes sparked the development of GFP as one of the most common reporters in use today. Over the past several years, the utility of GFP as a reporter has been optimized through the isolation and engineering of variants with increased folding rates, different in vivo stabilities and colour variants with altered excitation and emission spectral properties. One of the great utilities of GFP is as a probe for characterizing spatial and temporal dynamics of gene expression, protein localization and protein,protein interactions in living cells. The innovative application of GFP as a reporter in bacteria has made a significant contribution to microbial cell biology. This review will highlight recent studies that demonstrate the potential of GFP for real-time analysis of gene expression, protein localization and the dynamics of signalling transduction pathways through protein,protein interactions. [source] Nitric oxide scavenging and detoxification by the Mycobacterium tuberculosis haemoglobin, HbN in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 5 2002Ranjana Pathania Summary Nitric oxide (NO), generated in large amounts within the macrophages, controls and restricts the growth of internalized human pathogen, Mycobacterium tuberculosis H37Rv. The molecular mechanism by which tubercle bacilli survive within macrophages is currently of intense interest. In this work, we have demonstrated that dimeric haemoglobin, HbN, from M. tuberculosis exhibits distinct nitric oxide dioxygenase (NOD) activity and protects growth and cellular respiration of heterologous hosts, Escherichia coli and Mycobacterium smegmatis, from the toxic effect of exogenous NO and the NO-releasing compounds. A flavohaemoglobin (HMP)-deficient mutant of E. coli, unable to metabolize NO, acquired an oxygen-dependent NO consumption activity in the presence of HbN. On the basis of cellular haem content, the specific NOD activity of HbN was nearly 35-fold higher than the single-domain Vitreoscilla haemoglobin (VHb) but was sevenfold lower than the two-domain flavohaemoglobin. HbN-dependent NO consumption was sustained with repeated addition of NO, demonstrating that HbN is catalytically reduced within E. coli. Aerobic growth and respiration of a flavohaemoglobin (HMP) mutant of E. coli was inhibited in the presence of exogenous NO but remained insensitive to NO inhibition when these cells produced HbN, VHb or flavohaemoglobin. M. smegmatis, carrying a native HbN very similar to M. tuberculosis HbN, exhibited a 7.5-fold increase in NO uptake when exposed to gaseous NO, suggesting NO-induced NOD activity in these cells. In addition, expression of plasmid-encoded HbN of M. tuberculosis in M. smegmatis resulted in 100-fold higher NO consumption activity than the isogenic control cells. These results provide strong experimental evidence in support of NO scavenging and detoxification function for the M. tuberculosis HbN. The catalytic NO scavenging by HbN may be highly advantageous for the survival of tubercle bacilli during infection and pathogenesis. [source] Efficient experimental design and micro-scale medium enhancement of 6-deoxyerythronolide B production through Escherichia coliBIOTECHNOLOGY PROGRESS, Issue 5 2009Michael Pistorino Abstract The recent use of heterologous hosts to produce natural products has shown significant potential, although limitations still exist regarding optimal production titers. In this study, we utilize micro-scale cultures and well-defined screening methods to identify key medium components that influence the heterologous production of the complex polyketide 6-deoxyerythronolide B (6dEB) through E. coli. It was determined that tryptone had a significant effect on 6dEB production and could supplement substrate requirements and improve recombinant protein levels of the essential deoxyerythronolide B synthase (DEBS) which catalyze 6dEB conversion. As a result, the study (1) demonstrates the feasibility of micro-scale cultures to study E. coli 6dEB production and effectively model larger-scale cultures; (2) identifies an enhanced medium which generates over 160 mg L,1 6dEB (a 22-fold improvement over current culture media); and (3) provides new insight and understanding related to the heterologous production of 6dEB from E. coli. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | ||||||||||