| 1. |
- Behle, Anna, et al.
(författare)
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Manipulation of topoisomerase expression inhibits cell division but not growth and reveals a distinctive promoter structure in Synechocystis
- 2022
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 50:22, s. 12790-12808
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Tidskriftsartikel (refereegranskat)abstract
- In cyanobacteria DNA supercoiling varies over the diurnal cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knockdown of gyrase subunits and overexpression of topoisomerase I (TopoI). Cell division was blocked but cell growth continued in all strains. The small endogenous plasmids were only transiently relaxed, then became strongly supercoiled in the TopoI overexpression strain. Transcript abundances showed a pronounced 5'/3' gradient along transcription units, incl. the rRNA genes, in the gyrase knockdown strains. These observations are consistent with the basic tenets of the homeostasis and twin-domain models of supercoiling in bacteria. TopoI induction initially led to downregulation of G+C-rich and upregulation of A+T-rich genes. The transcriptional response quickly bifurcated into six groups which overlap with diurnally co-expressed gene groups. Each group shows distinct deviations from a common core promoter structure, where helically phased A-tracts are in phase with the transcription start site. Together, our data show that major co-expression groups (regulons) in Synechocystis all respond differentially to DNA supercoiling, and suggest to re-evaluate the long-standing question of the role of A-tracts in bacterial promoters.
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| 2. |
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| 3. |
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| 4. |
- Fedriani, Rubén, 1991, et al.
(författare)
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The SOFIA Massive (SOMA) Star Formation Survey. IV. Isolated Protostars
- 2023
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Ingår i: Astrophysical Journal. - : American Astronomical Society. - 1538-4357 .- 0004-637X. ; 942:1
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Tidskriftsartikel (refereegranskat)abstract
- We present similar to 10-40 mu m SOFIA-FORCAST images of 11 isolated protostars as part of the SOFIA Massive (SOMA) Star Formation Survey, with this morphological classification based on 37 mu m imaging. We develop an automated method to define source aperture size using the gradient of its background-subtracted enclosed flux and apply this to build spectral energy distributions (SEDs). We fit the SEDs with radiative transfer models, developed within the framework of turbulent core accretion (TCA) theory, to estimate key protostellar properties. Here, we release the sedcreator python package that carries out these methods. The SEDs are generally well fitted by the TCA models, from which we infer initial core masses M ( c ) ranging from 20-430 M (circle dot), clump mass surface densities sigma(cl) similar to 0.3-1.7 g cm(-2), and current protostellar masses m (*) similar to 3-50 M (circle dot). From a uniform analysis of the 40 sources in the full SOMA survey to date, we find that massive protostars form across a wide range of clump mass surface density environments, placing constraints on theories that predict a minimum threshold sigma(cl) for massive star formation. However, the upper end of the m (*)-sigma(cl) distribution follows trends predicted by models of internal protostellar feedback that find greater star formation efficiency in higher sigma(cl) conditions. We also investigate protostellar far-IR variability by comparison with IRAS data, finding no significant variation over an similar to 40 yr baseline.
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| 5. |
- Kaczmarzyk, Danuta, et al.
(författare)
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Diversion of the long-chain acyl-ACP pool in Synechocystis to fatty alcohols through CRISPRi repression of the essential phosphate acyltransferase PlsX
- 2018
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Ingår i: Metabolic engineering. - : Academic Press. - 1096-7176 .- 1096-7184. ; 45, s. 59-66
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Tidskriftsartikel (refereegranskat)abstract
- Fatty alcohol production in Synechocystis sp. PCC 6803 was achieved through heterologous expression of the fatty acyl-CoA/ACP reductase Maqu2220 from the bacteria Marinobacter aquaeolei VT8 and the fatty acyl-ACP reductase DPW from the rice Oryza sativa. These platform strains became models for testing multiplex CRISPR-interference (CRISPRi) metabolic engineering strategies to both improve fatty alcohol production and to study membrane homeostasis. CRISPRi allowed partial repression of up to six genes simultaneously, each encoding enzymes of acyl-ACP-consuming pathways. We identified the essential phosphate acyltransferase enzyme PlsX (slr1510) as a key node in C18 fatty acyl-ACP consumption, repression of slr1510 increased octadecanol productivity threefold over the base strain and gave the highest specific titers reported for this host, 10.3 mg g−1 DCW. PlsX catalyzes the first committed step of phosphatidic acid synthesis, and has not been characterized in Synechocystis previously. We found that accumulation of fatty alcohols impaired growth, altered the membrane composition, and caused a build-up of reactive oxygen species.
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| 6. |
- Law, Chi Yan, 1990, et al.
(författare)
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Isolated Massive Star Formation in G28.20-0.05
- 2022
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Ingår i: Astrophysical Journal. - : American Astronomical Society. - 1538-4357 .- 0004-637X. ; 939:2
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Tidskriftsartikel (refereegranskat)abstract
- We report high-resolution 1.3 mm continuum and molecular line observations of the massive protostar G28.20-0.05 with Atacama Large Millimeter/submillimeter Array. The continuum image reveals a ring-like structure with 2000 au radius, similar to morphology seen in archival 1.3 cm Very Large Array observations. Based on its spectral index and associated H30α emission, this structure mainly traces ionized gas. However, there is evidence for ∼30 M ⊙ of dusty gas near the main millimeter continuum peak on one side of the ring, as well as in adjacent regions within 3000 au. A virial analysis on scales of ∼2000 au from hot core line emission yields a dynamical mass of ∼80 M ⊙. A strong velocity gradient in the H30α emission is evidence for a rotating, ionized disk wind, which drives a larger-scale molecular outflow. An infrared spectral energy distribution (SED) analysis indicates a current protostellar mass of m * ∼ 40 M ⊙ forming from a core with initial mass M c ∼ 300 M ⊙ in a clump with mass surface density of Σcl ∼ 0.8 g cm−2. Thus the SED and other properties of the system can be understood in the context of core accretion models. A structure-finding analysis on the larger-scale continuum image indicates G28.20-0.05 is forming in a relatively isolated environment, with no other concentrated sources, i.e., protostellar cores, above ∼1 M ⊙ found from ∼0.1 to 0.4 pc around the source. This implies that a massive star can form in relative isolation, and the dearth of other protostellar companions within the ∼1 pc environs is a strong constraint on massive star formation theories that predict the presence of a surrounding protocluster.
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| 7. |
- Liu, Mengyao, et al.
(författare)
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The SOFIA Massive (SOMA) Star Formation Survey. III. From Intermediate- to High-mass Protostars
- 2020
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Ingår i: Astrophysical Journal. - : American Astronomical Society. - 1538-4357 .- 0004-637X. ; 904:1
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Tidskriftsartikel (refereegranskat)abstract
- We present similar to 10-40 mm SOFIA-FORCAST images of 14 intermediate-mass protostar candidates as part of the SOFIA Massive (SOMA) Star Formation Survey. We build spectral energy distributions, also using archival Spitzer, Herschel, and IRAS data. We then fit the spectral energy distributions with radiative transfer models of Zhang & Tan, based on turbulent core accretion theory, to estimate key protostellar properties. With the addition of these intermediate-mass sources, based on average properties derived from SED fitting, SOMA protostars span luminosities from similar to 10(2) to 10(6) L-circle dot, current protostellar masses from similar to 0.5 to 35 M-circle dot, and ambient clump mass surface densities, Scl, from 0.1 to g cm(-2). A wide range of evolutionary states of the individual protostars and of the protocluster environments is also probed. We have also considered about 50 protostars identified in infrared dark clouds that are expected to be at the earliest stages of their evolution. With this global sample, most of the evolutionary stages of high- and intermediate-mass protostars are probed. The best-fitting models show no evidence that a threshold value of the protocluster clump mass surface density is required to form protostars up to similar to 25 M.. However, to form more massive protostars, there is tentative evidence that Sigma(cl) needs to be greater than or similar to 1 g cm(-2). We discuss how this is consistent with expectations from core accretion models that include internal feedback from the forming massive star.
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| 8. |
- Santos, Marina, et al.
(författare)
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CRISPRi as a Tool to Repress Multiple Copies of Extracellular Polymeric Substances (EPS)-Related Genes in the Cyanobacterium Synechocystis sp. PCC 6803
- 2021
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Ingår i: Life. - : MDPI AG. - 2075-1729. ; 11:11
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Tidskriftsartikel (refereegranskat)abstract
- The use of the versatile cyanobacterial extracellular polymeric substances (EPS) for biotechnological/biomedical applications implies an extensive knowledge of their biosynthetic pathways to improve/control polymer production yields and characteristics. The multiple copies of EPS-related genes, scattered throughout cyanobacterial genomes, adds another layer of complexity, making these studies challenging and time-consuming. Usually, this issue would be tackled by generating deletion mutants, a process that in cyanobacteria is also hindered by the polyploidy. Thus, the use of the CRISPRi multiplex system constitutes an efficient approach to addressing this redundancy. Here, three putative Synechocystis sp. PCC 6803 kpsM homologues (slr0977, slr2107, and sll0574) were repressed using this methodology. The characterization of the 3-sgRNA mutant in terms of fitness/growth and total carbohydrates, released and capsular polysaccharides, and its comparison with previously generated single knockout mutants pointed towards Slr0977 being the key KpsM player in Synechocystis EPS production. This work validates CRISPRi as a powerful tool to unravel cyanobacterial complex EPS biosynthetic pathways expediting this type of studies.
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| 9. |
- Shabestary, Kiyan, et al.
(författare)
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Targeted Repression of Essential Genes To Arrest Growth and Increase Carbon Partitioning and Biofuel Titers in Cyanobacteria
- 2018
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 7:7
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Tidskriftsartikel (refereegranskat)abstract
- Photoautotrophic production of fuels and chemicals by cyanobacteria typically gives lower volumetric productivities and titers than heterotrophic production. Cyanobacteria cultures become light limited above an optimal cell density, so that this substrate is not supplied to all cells sufficiently. Here, we investigate genetic strategies for a two-phase cultivation, where biofuel-producing Synechocystis cultures are limited to an optimal cell density through inducible CRISPR interference (CRISPRi) repression of cell growth. Fixed CO2 is diverted to ethanol or n-butanol. Among the most successful strategies was partial repression of citrate synthase gltA. Strong repression (>90%) of gitA at low culture densities increased carbon partitioning to n-butanol 5-fold relative to a nonrepression strain, but sacrificed volumetric productivity due to severe growth restriction. CO2 fixation continued for at least 3 days after growth was arrested. By targeting sgRNAs to different regions of the gitA gene, we could modulate GItA expression and carbon partitioning between growth and product to increase both specific and volumetric productivity. These growth arrest strategies can be useful for improving performance of other photoautotrophic processes.
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| 10. |
- Yang, Yao-Lun, et al.
(författare)
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Constraining the Infalling Envelope Models of Embedded Protostars: BHR 71 and Its Hot Corino
- 2020
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Ingår i: Astrophysical Journal. - : American Astronomical Society. - 1538-4357 .- 0004-637X. ; 891:1
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Tidskriftsartikel (refereegranskat)abstract
- The collapse of a protostellar envelope results in the growth of a protostar and the development of a protoplanetary disk, playing a critical role during the early stages of star formation. Characterizing the gas infall in the envelope constrains the dynamical models of star formation. We present unambiguous signatures of infall, probed by optically thick molecular lines, toward an isolated embedded protostar, BHR 71 IRS1. The three-dimensional radiative transfer calculations indicate that a slowly rotating infalling envelope model following the "inside-out" collapse reproduces the observations of both lines, as well as the low-velocity emission of the HCN line. The envelope has a model-derived ag lines, where outflows or a Keplerian disk may contribute. The ALMA observations serendipitously discover the emission of complex organic molecules (COMs) concentrated within a radius of 100 au, indicating that BHR 71 IRS1 harbors a hot corino. Eight species of COMs are identified, including CH3OH and CH3OCHO, along with H2CS, SO2 and HCN v(2) = 1. The emission of methyl formate and C-13-methanol shows a clear velocity gradient within a radius of 50 au, hinting at an unresolved Keplerian rotating disk.
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| 11. |
- Yao, Lun, et al.
(författare)
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Multiple Gene Repression in Cyanobacteria Using CRISPRi
- 2016
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Ingår i: ACS Photonics. - : American Chemical Society (ACS). - 2330-4022. ; 5:3, s. 207-212
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Tidskriftsartikel (refereegranskat)abstract
- We describe the application of clustered regularly interspaced short palindromic repeats interference (CRISPRi) for gene repression in the model cyanobacterium Synechcocystis sp. PCC 6803. The nuclease-deficient Cas9 from the type-II. CRISPR/Cas of Streptrococcus pyogenes was used to repress green fluorescent protein (GFP) to negligible levels. CRISPRi was also used to repress formation of carbon storage compounds polyhydroxybutryate (PHB) and glycogen during nitrogen starvation. As an example of the potential of CRISPRi for basic and applied cyanobacteria research, we simultaneously knocked down 4 putative aldehyde reductases and dehydrogenases at 50-95% repression. This work also demonstrates that tightly repressed promoters allow for inducible and reversible CRISPRi in cyanobacteria.
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| 12. |
- Yao, Lun, et al.
(författare)
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Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes
- 2020
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Ingår i: Nature Communications. - : Nature Research. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Cyanobacteria are model organisms for photosynthesis and are attractive for biotechnology applications. To aid investigation of genotype-phenotype relationships in cyanobacteria, we develop an inducible CRISPRi gene repression library in Synechocystis sp. PCC 6803, where we aim to target all genes for repression. We track the growth of all library members in multiple conditions and estimate gene fitness. The library reveals several clones with increased growth rates, and these have a common upregulation of genes related to cyclic electron flow. We challenge the library with 0.1 M L-lactate and find that repression of peroxiredoxin bcp2 increases growth rate by 49%. Transforming the library into an L-lactate-secreting Synechocystis strain and sorting top lactate producers enriches clones with sgRNAs targeting nutrient assimilation, central carbon metabolism, and cyclic electron flow. In many examples, productivity can be enhanced by repression of essential genes, which are difficult to access by transposon insertion.
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| 13. |
- Zhang, Bo lun, et al.
(författare)
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Experimental study on film cooling performance of a turbine blade tip with a trapezoidal slot cooling scheme in transonic flow using PSP technique
- 2022
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Ingår i: Experimental Thermal and Fluid Science. - : Elsevier BV. - 0894-1777. ; 130
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Forskningsöversikt (refereegranskat)abstract
- A turbine blade tip with a trapezoidal slot cooling scheme is first proposed in the current paper. The film cooling characteristics of a turbine blade tip with a trapezoidal slot scheme are experimentally studied by the pressure sensitive paint (PSP) technique in transonic flow. Varieties of density ratio, tip clearance gap and position of the trapezoidal slot scheme are selected as investigation parameters. The mainstream Reynolds number is 370,000 based on the blade axial chord length. The cascade exit Mach numbers are is 1.05. The results indicate that increasing the density ratio has a positive effect on the film cooling effectiveness of the whole blade tip with a pressure side trapezoidal slot scheme at a small tip clearance gap, whereas it decreases the film cooling effectiveness upstream of the blade tip at the large tip clearance gap. For the blade tip with a pressure side trapezoidal slot scheme, the film cooling effectiveness of the tip clearance gap of 1.5% near the trailing edge is higher than that of the other tip clearance gaps at the same density ratio condition. Changing the trapezoidal slot position from the tip pressure side to the tip midline increases the film cooling effectiveness near the trailing edge.
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| 14. |
- Zhang, Yichen, et al.
(författare)
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Massive Protostars in a Protocluster—A Multi-scale ALMA View of G35.20-0.74N
- 2022
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Ingår i: Astrophysical Journal. - : American Astronomical Society. - 1538-4357 .- 0004-637X. ; 936:1
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Tidskriftsartikel (refereegranskat)abstract
- We present a detailed study of the massive star-forming region G35.2-0.74N with Atacama Large Millimeter/submillimeter Array (ALMA) 1.3 mm multi-configuration observations. At 0.″2 (440 au) resolution, the continuum emission reveals several dense cores along a filamentary structure, consistent with previous ALMA 0.85 mm observations. At 0.″03 (66 au) resolution, we detect 22 compact sources, most of which are associated with the filament. Four of the sources are associated with compact centimeter continuum emission, and two of these are associated with H30α recombination line emission. The H30α line kinematics shows the ordered motion of the ionized gas, consistent with disk rotation and/or outflow expansion. We construct models of photoionized regions to simultaneously fit the multiwavelength free-free fluxes and the H30α total fluxes. The derived properties suggest the presence of at least three massive young stars with nascent hypercompact H ii regions. Two of these ionized regions are surrounded by a large rotating structure that feeds two individual disks, revealed by dense gas tracers, such as SO2, H2CO, and CH3OH. In particular, the SO2 emission highlights two spiral structures in one of the disks and probes the faster-rotating inner disks. The 12CO emission from the general region reveals a complex outflow structure, with at least four outflows identified. The remaining 18 compact sources are expected to be associated with lower-mass protostars forming in the vicinity of the massive stars. We find potential evidence for disk disruption due to dynamic interactions in the inner region of this protocluster. The spatial distribution of the sources suggests a smooth overall radial density gradient without subclustering, but with tentative evidence of primordial mass segregation.
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| 15. |
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