191 research outputs found
Elucidation of the prodiginine biosynthetic pathway in Streptomyces coelicolor A3(2)
The prodiginine antibiotics are produced by eubacteria, in particular members of
the actinomycete family. Interest in this group of compounds has been stimulated by
their antitumour, immunosuppressant and antimalarial activities at non-toxic levels.
Streptomyces coelicolor A3(2) produces two prodiginines: undecylprodiginine and its
carbocyclic derivative streptorubin B, which are both derived from the two intermediates
4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC) and 2-undecylpyrrole (2-UP).
The red gene cluster of S. coelicolor contains 23 genes responsible for prodiginine
biosynthesis.
PCR-targeting was used to generate rapid in-frame deletions or replacements of
several genes in the S. coelicolor red cluster. Using this method redI, redJ, redK, the A
domain encoding region of redL, redT and redV were disrupted. Prodiginine production
by these mutants was analysed by LC-MS allowing roles for the genes investigated to be
hypothesised. A major focus was investigating the function of RedH (proposed to
catalyse the condensation of 2-UP and MBC) and RedG (proposed to be responsible for
the oxidative carbocyclisation of undecylprodiginine to form streptorubin B) by genetic
complementation of existing mutants and heterologous expression of the genes in S.
venezuelae coupled with feeding of synthetic MBC and 2-UP. The results of these
experiments clearly defined the roles of RedH in the condensation of MBC and 2-UP
and RedG in the oxidative carbocyclisation of undecylprodiginine.
Streptomyces longispororuber is known to produce undecylprodiginine (like S.
coelicolor) and a carbocyclic undecylprodiginine derivative called metacycloprodigiosin (streptorubin A), which contains a 12-membered carbocycle instead of the 10-membered
carbocycle of streptorubin B. A S. longispororuber fosmid library was constructed, from
which a clone containing a previously identified redG orthologue was isolated and
partially sequenced. Expression of the S. longispororuber redG orthologue in the S.
coelicolor redG mutant resulted in production of metacycloprodigiosin instead of
streptorubin B showing that RedG and its S. longispororuber orthologue catalyse
carbocyclisation reactions during prodiginine biosynthesis.
Another aim of the work was to investigate redU, a gene from the red cluster
that encodes a phosphopantetheinyl transferase (PPTase). PPTases are responsible for
post-translational modification of acyl carrier proteins (ACPs) and peptidyl carrier
proteins (PCPs). A pre-existing redU mutant and two newly constructed mutants lacking
PPTases encoded elsewhere in the S. coelicolor genome were analysed to investigate the
role of PPTases in S. coelicolor metabolite biosynthesis. Production of prodiginines,
actinorhodins, methylenomycins, calcium dependent antibiotics, coelichelin and grey
spore pigment was investigated as ACPs and PCPs are involved in biosynthesis of these
compounds. Different specific PPtases were found to be required to modify the
ACP/PCP domains/proteins in the biosynthesis of these metabolites
Elucidation of the prodiginine biosynthetic pathway in Streptomyces coelicolor A3(2)
The prodiginine antibiotics are produced by eubacteria, in particular members of the actinomycete family. Interest in this group of compounds has been stimulated by their antitumour, immunosuppressant and antimalarial activities at non-toxic levels. Streptomyces coelicolor A3(2) produces two prodiginines: undecylprodiginine and its carbocyclic derivative streptorubin B, which are both derived from the two intermediates 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC) and 2-undecylpyrrole (2-UP). The red gene cluster of S. coelicolor contains 23 genes responsible for prodiginine biosynthesis. PCR-targeting was used to generate rapid in-frame deletions or replacements of several genes in the S. coelicolor red cluster. Using this method redI, redJ, redK, the A domain encoding region of redL, redT and redV were disrupted. Prodiginine production by these mutants was analysed by LC-MS allowing roles for the genes investigated to be hypothesised. A major focus was investigating the function of RedH (proposed to catalyse the condensation of 2-UP and MBC) and RedG (proposed to be responsible for the oxidative carbocyclisation of undecylprodiginine to form streptorubin B) by genetic complementation of existing mutants and heterologous expression of the genes in S. venezuelae coupled with feeding of synthetic MBC and 2-UP. The results of these experiments clearly defined the roles of RedH in the condensation of MBC and 2-UP and RedG in the oxidative carbocyclisation of undecylprodiginine. Streptomyces longispororuber is known to produce undecylprodiginine (like S. coelicolor) and a carbocyclic undecylprodiginine derivative called metacycloprodigiosin (streptorubin A), which contains a 12-membered carbocycle instead of the 10-membered carbocycle of streptorubin B. A S. longispororuber fosmid library was constructed, from which a clone containing a previously identified redG orthologue was isolated and partially sequenced. Expression of the S. longispororuber redG orthologue in the S. coelicolor redG mutant resulted in production of metacycloprodigiosin instead of streptorubin B showing that RedG and its S. longispororuber orthologue catalyse carbocyclisation reactions during prodiginine biosynthesis. Another aim of the work was to investigate redU, a gene from the red cluster that encodes a phosphopantetheinyl transferase (PPTase). PPTases are responsible for post-translational modification of acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). A pre-existing redU mutant and two newly constructed mutants lacking PPTases encoded elsewhere in the S. coelicolor genome were analysed to investigate the role of PPTases in S. coelicolor metabolite biosynthesis. Production of prodiginines, actinorhodins, methylenomycins, calcium dependent antibiotics, coelichelin and grey spore pigment was investigated as ACPs and PCPs are involved in biosynthesis of these compounds. Different specific PPtases were found to be required to modify the ACP/PCP domains/proteins in the biosynthesis of these metabolites.EThOS - Electronic Theses Online ServiceUniversity of WarwickGBUnited Kingdo
A baseline evaluation of oceanographic and sea ice conditions in the Hudson Bay Complex during 2016-2018
In this paper, we examine sea surface temperatures (SSTs) and sea ice conditions in the Hudson Bay Complex as a baseline evaluation for the BaySys 2016â2018 field program time frame. Investigated in particular are spatiotemporal patterns in SST and sea ice state and dynamics, with rankings of the latter to highlight extreme conditions relative to the examined 1981â2010 climatology. Results from this study show that SSTs in northwestern Hudson Bay from May to July, 2016â2018, are high relative to the climatology for SST (1982â2010). SSTs are also warmer in 2016 and 2017 than in 2018 relative to their climatology. Similarly, unusually low sea ice cover existed from August to December of 2016 and July to September of 2017, while unusually high sea ice cover existed in January, February, and October of 2018. The ice-free season was approximately 20 days longer in 2016 than in 2018. Unusually high ice-drift speeds occurred in April of 2016 and 2017 and in May of 2018, coinciding with strong winds in 2016 and 2018 and following strong winds in March 2017. Strong meridional circulation was observed in spring of 2016 and winter of 2017, while weak meridional circulation existed in 2018. In a case study of an extreme event, a blizzard from 7 to 9 March 2017, evaluated using Lagrangian dispersion statistics, is shown to have suppressed sea ice deformation off the coast of Churchill. These results are relevant to describing and planning for possible future pathways and scenarios under continued climate change and river regulation
Atmospheric Forcing Drives the Winter Sea Ice Thickness Asymmetry of Hudson Bay
Recently, we highlighted the presence of a strong westâeast asymmetry in sea ice thickness across Hudson Bay that is driven by cyclonic circulation. Building on this work, we use satellite altimetry and a unique set of in situ observations of ice thickness from three moored upward looking sonars to examine the role of atmospherically driven ice dynamics in producing contrasting regional ice thickness patterns. Ultimately, northânorthwesterly winds coupled with numerous reversals during winter 2016/2017 led to thicker ice in southern Hudson Bay, while enhanced westânorthwesterly winds during winter 2017/2018 led to thicker ice in eastern Hudson Bay that delayed breakup and onset of the summer shipping season to coastal communities. Extending the analysis over the 40âyear satellite observation period, we find that these two different patterns of atmospheric forcing alter the timing of breakup by 30 days in eastern Hudson Bay and offer some skill in seasonal predictions of breakup
A baseline evaluation of atmospheric and river discharge conditions in the Hudson Bay Complex during 2016-2018
In this article, we examine atmospheric and river discharge conditions within the Hudson Bay Complex for the BaySys 2016â2018 field program time frame. Investigated in particular is a subset of European Centre for Medium-Range Weather Forecasts (ECMWF) Re-Analysis - Interim (ERA-Interim) atmospheric forcing variables, namely 2-m surface temperature, 10-m surface winds, precipitation, and sea-level pressure, in addition to river discharge. Results from this assessment show that 2016 was characterized by unusually warm conditions (terrestrial and marine) throughout the annual cycle; 2017 by strong cyclone activity in March and high precipitation in January, October, and November; and 2018 by cold and windy conditions throughout the annual cycle. Evaluation of terrestrial conditions showed higher than normal land surface temperatures (the Hudson Bay physical watershed) for all of the 2016â2018 period (excluding a colder than normal spell AugustâNovember 2018), particularly in January (2016 and 2017), higher than normal precipitation in October (2016 and 2017), and higher than normal terrestrial discharge to the Hudson Bay Complex in March (2016 and 2017), with drier than average June through October (2016â2018)
Simulated impacts of relative climate change and river discharge regulation on sea ice and oceanographic conditions in the Hudson Bay Complex
In this analysis, we examine relative contributions from climate change and river discharge regulation to changes in marine conditions in the Hudson Bay Complex using a subset of five atmospheric forcing scenarios from the Coupled Model Intercomparison Project Phase 5 (CMIP5), river discharge data from the Hydrological Predictions for the Environment (HYPE) model, both naturalized (without anthropogenic intervention) and regulated (anthropogenically controlled through diversions, dams, reservoirs), and output from the Nucleus for European Modeling of the Ocean Ice-Ocean model for the 1981â2070 time frame. Investigated in particular are spatiotemporal changes in sea surface temperature, sea ice concentration and thickness, and zonal and meridional sea ice drift in response to (i) climate change through comparison of historical (1981â2010) and future (2021â2050 and 2041â2070) simulations, (ii) regulation through comparison of historical (1981â2010) naturalized and regulated simulations, and (iii) climate change and regulation combined through comparison of future (2021â2050 and 2041â2070) naturalized and regulated simulations. Also investigated is use of the diagnostic known as e-folding time spatial distribution to monitor changes in persistence in these variables in response to changing climate and regulation impacts in the Hudson Bay Complex. Results from this analysis highlight bay-wide and regional reductions in sea ice concentration and thickness in southwest and northeast Hudson Bay in response to a changing climate, and east-west asymmetry in sea ice drift response in support of past studies. Regulation is also shown to amplify or suppress the climate change signal. Specifically, regulation amplifies sea surface temperatures from April to August, suppresses sea ice loss by approximately 30% in March, contributes to enhanced sea ice drift speed by approximately 30%, and reduces meridional circulation by approximately 20% in January due to enhanced zonal drift. Results further suggest that the offshore impacts of regulation are amplified in a changing climate
Sediment-laden sea ice in southern Hudson Bay: Entrainment, transport, and biogeochemical implications
During a research expedition in Hudson Bay in June 2018, vast areas of thick (>10 m), deformed sediment-laden sea ice were encountered unexpectedly in southern Hudson Bay and presented difficult navigation conditions for the Canadian Coast Guard Ship Amundsen. An aerial survey of one of these floes revealed a maximum ridge height of 4.6 m and an average freeboard of 2.2 m, which corresponds to an estimated total thickness of 18 m, far greater than expected within a seasonal ice cover. Samples of the upper portion of the ice floe revealed that it was isothermal and fresh in areas with sediment present on the surface. Fine-grained sediment and larger rocks were visible on the ice surface, while a pronounced sediment band was observed in an ice core. Initial speculation was that this ice had formed in the highly dynamic Nelson River estuary from freshwater, but ÎŽ^{18}O isotopic analysis revealed a marine origin. In southern Hudson Bay, significant tidal forcing promotes both sediment resuspension and new ice formation within a flaw lead, which we speculate promotes the formation of this sediment-laden sea ice. Historic satellite imagery shows that sediment-laden sea ice is typical of southern Hudson Bay, varying in areal extent from 47 to 118 km2 during June. Based on an average sediment particle concentration of 0.1 mg mL^{â1} in sea ice, an areal extent of 51,924 km2 in June 2018, and an estimated regional end-of-winter ice thickness of 1.5 m, we conservatively estimated that a total sediment load of 7.8 Ă 106 t, or 150 t km^{â2}, was entrained within sea ice in southern Hudson Bay during winter 2018. As sediments can alter carbon concentrations and light transmission within sea ice, these first observations of this ice type in Hudson Bay imply biogeochemical impacts for the marine system
Structural basis for chain release from the enacyloxin polyketide synthase
Modular polyketide synthases and nonribosomal peptide synthetases are molecular assembly lines consisting of several multienzyme subunits that undergo dynamic self-assembly to form a functional mega-complex. N- and C-terminal docking domains are usually responsible for mediating interactions between subunits. Here we show that communication between two nonribosomal peptide synthetase subunits responsible for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii, is mediated by an intrinsically disordered short linear motif and a Ă-hairpin docking domain. The structures, interactions and dynamics of these subunits are characterised using several complementary biophysical techniques, providing extensive insights into binding and catalysis. Bioinformatics analyses reveal that short linear motif/Ă-hairpin docking domain pairs mediate subunit interactions in numerous nonribosomal peptide and hybrid polyketide-nonribosomal peptide synthetases, including those responsible for assembling several important drugs. Short linear motifs and Ă-hairpin docking domains from heterologous systems are shown to interact productively, highlighting the potential of such interfaces as tools for biosynthetic engineering
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