Use of Time Lapse Microscopy in the Genetic Characterisation of SCO1389 and SCO5750 in Streptomyces Coelicolor Growth and Development

Vinod Jyothikumar

Research output: ThesisDoctoral Thesis

Abstract

Streptomycetes have a complex morphogenetic programme end up in the formation of aerial hyphae that develop into chains of spores. After spore dispersal, environmental signals trigger dormant spores to germinate to establish a new colony. This complexity of streptomycete microcolonies makes studying the dynamic processes that contribute to growth and development a challenging procedure. In order to study the mechanisms that underpin streptomycete growth, we have developed a system for studying hyphal extension, protein trafficking, and sporulation by time-lapse microscopy.Heterogeneous distribution of phospholipids along bacterial membrane results in the formation of domains enriched in anionic phospholipids at the cell poles and at the mid cell. Cardiolipin plays a key role in organization of bacterial membranes and forms membrane domains that participate in interaction with multi-protein complexes involved in cell division, energy metabolism, and membrane transport. The goals of this study were to genetic characterization SCO5750 with localization of cardiolipin, and SCO1389, that synthesizes this anionic phospholipid and so change membrane lipid composition. SCO1389 gene from cosmid St1A8A derivative was disrupted by replacement with an apramycin-resistant gene followed by exchange with the homologous chromosomal region. Most of recombination resulted in single crossover and none had undergone the second crossover event to delete the SCO1389 gene. SCO1389 could not be deleted without causing lethality, except when a second SCO1389 copy is expressed in the same cells, which implies that SCO1389 is involved in an essential primary metabolic process, likely membrane phospholipid biosynthesis. The proper genomic disruptions were confirmed by Southern blot hybridization. Depletion of SCO1389 caused failure in erection of aerial hyphae and severely retards growth whilst there was enough genetic evidence that SCO1389 is essential for growth and development, it was not clear why cells cannot grow in the absence of this gene. Over expression of SCO1389 weakens hyphal tips, mis-shaped aerial hyphae and large anucleated spores. A SCO1389-egfp translational fusion was constructed, which showed distinctive regions of strong fluorescence mostly in the substrate hyphae and in few young aerial hyphae. Use of the cardiolipin specific fluorescent dye 10-N-nonyl-acridine orange (NAO) revealed cardiolipin rich domains in the Escherichia coli membrane (Mileykovskaya et al., 2000). Staining of Streptomyces cells with NAO showed that there were green fluorescence domains in the branch points, substrate hyphae, hyphal tips, aerial hyphae and in spores. These fluorescence domains were scarcely detectable in cells of the SCO1389-disrupted mutant. The red shift fluorescence due to stacking of two dye molecules showed same results like the green shift in mutants and M145 strains. Statistical analysis shows altering the expression level in SCO1389 affects the growth and branching in the strain RJ118b (induced by anhydrotetracycline-atc) and RJ110 (induced by thiostrepton).
LanguageEnglish
Awarding Institution
  • University Of Strathclyde
Supervisors/Advisors
  • Herron, Paul, Supervisor
Award date19 Nov 2009
Publication statusPublished - 19 Nov 2009

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Streptomyces coelicolor
Hyphae
Growth and Development
Microscopy
Microscopic examination
Cardiolipins
Spores
Membranes
Antennas
Phospholipids
Genes
Fluorescence
Acridine Orange
Thiostrepton
Growth
Cosmids
Biosynthesis
Substrates
Membrane Lipids
Fluorescent Dyes

Cite this

@phdthesis{3ab8fce86b04455c8cdf4d7a5d171f5a,
title = "Use of Time Lapse Microscopy in the Genetic Characterisation of SCO1389 and SCO5750 in Streptomyces Coelicolor Growth and Development",
abstract = "Streptomycetes have a complex morphogenetic programme end up in the formation of aerial hyphae that develop into chains of spores. After spore dispersal, environmental signals trigger dormant spores to germinate to establish a new colony. This complexity of streptomycete microcolonies makes studying the dynamic processes that contribute to growth and development a challenging procedure. In order to study the mechanisms that underpin streptomycete growth, we have developed a system for studying hyphal extension, protein trafficking, and sporulation by time-lapse microscopy.Heterogeneous distribution of phospholipids along bacterial membrane results in the formation of domains enriched in anionic phospholipids at the cell poles and at the mid cell. Cardiolipin plays a key role in organization of bacterial membranes and forms membrane domains that participate in interaction with multi-protein complexes involved in cell division, energy metabolism, and membrane transport. The goals of this study were to genetic characterization SCO5750 with localization of cardiolipin, and SCO1389, that synthesizes this anionic phospholipid and so change membrane lipid composition. SCO1389 gene from cosmid St1A8A derivative was disrupted by replacement with an apramycin-resistant gene followed by exchange with the homologous chromosomal region. Most of recombination resulted in single crossover and none had undergone the second crossover event to delete the SCO1389 gene. SCO1389 could not be deleted without causing lethality, except when a second SCO1389 copy is expressed in the same cells, which implies that SCO1389 is involved in an essential primary metabolic process, likely membrane phospholipid biosynthesis. The proper genomic disruptions were confirmed by Southern blot hybridization. Depletion of SCO1389 caused failure in erection of aerial hyphae and severely retards growth whilst there was enough genetic evidence that SCO1389 is essential for growth and development, it was not clear why cells cannot grow in the absence of this gene. Over expression of SCO1389 weakens hyphal tips, mis-shaped aerial hyphae and large anucleated spores. A SCO1389-egfp translational fusion was constructed, which showed distinctive regions of strong fluorescence mostly in the substrate hyphae and in few young aerial hyphae. Use of the cardiolipin specific fluorescent dye 10-N-nonyl-acridine orange (NAO) revealed cardiolipin rich domains in the Escherichia coli membrane (Mileykovskaya et al., 2000). Staining of Streptomyces cells with NAO showed that there were green fluorescence domains in the branch points, substrate hyphae, hyphal tips, aerial hyphae and in spores. These fluorescence domains were scarcely detectable in cells of the SCO1389-disrupted mutant. The red shift fluorescence due to stacking of two dye molecules showed same results like the green shift in mutants and M145 strains. Statistical analysis shows altering the expression level in SCO1389 affects the growth and branching in the strain RJ118b (induced by anhydrotetracycline-atc) and RJ110 (induced by thiostrepton).",
author = "Vinod Jyothikumar",
year = "2009",
month = "11",
day = "19",
language = "English",
school = "University Of Strathclyde",

}

TY - THES

T1 - Use of Time Lapse Microscopy in the Genetic Characterisation of SCO1389 and SCO5750 in Streptomyces Coelicolor Growth and Development

AU - Jyothikumar, Vinod

PY - 2009/11/19

Y1 - 2009/11/19

N2 - Streptomycetes have a complex morphogenetic programme end up in the formation of aerial hyphae that develop into chains of spores. After spore dispersal, environmental signals trigger dormant spores to germinate to establish a new colony. This complexity of streptomycete microcolonies makes studying the dynamic processes that contribute to growth and development a challenging procedure. In order to study the mechanisms that underpin streptomycete growth, we have developed a system for studying hyphal extension, protein trafficking, and sporulation by time-lapse microscopy.Heterogeneous distribution of phospholipids along bacterial membrane results in the formation of domains enriched in anionic phospholipids at the cell poles and at the mid cell. Cardiolipin plays a key role in organization of bacterial membranes and forms membrane domains that participate in interaction with multi-protein complexes involved in cell division, energy metabolism, and membrane transport. The goals of this study were to genetic characterization SCO5750 with localization of cardiolipin, and SCO1389, that synthesizes this anionic phospholipid and so change membrane lipid composition. SCO1389 gene from cosmid St1A8A derivative was disrupted by replacement with an apramycin-resistant gene followed by exchange with the homologous chromosomal region. Most of recombination resulted in single crossover and none had undergone the second crossover event to delete the SCO1389 gene. SCO1389 could not be deleted without causing lethality, except when a second SCO1389 copy is expressed in the same cells, which implies that SCO1389 is involved in an essential primary metabolic process, likely membrane phospholipid biosynthesis. The proper genomic disruptions were confirmed by Southern blot hybridization. Depletion of SCO1389 caused failure in erection of aerial hyphae and severely retards growth whilst there was enough genetic evidence that SCO1389 is essential for growth and development, it was not clear why cells cannot grow in the absence of this gene. Over expression of SCO1389 weakens hyphal tips, mis-shaped aerial hyphae and large anucleated spores. A SCO1389-egfp translational fusion was constructed, which showed distinctive regions of strong fluorescence mostly in the substrate hyphae and in few young aerial hyphae. Use of the cardiolipin specific fluorescent dye 10-N-nonyl-acridine orange (NAO) revealed cardiolipin rich domains in the Escherichia coli membrane (Mileykovskaya et al., 2000). Staining of Streptomyces cells with NAO showed that there were green fluorescence domains in the branch points, substrate hyphae, hyphal tips, aerial hyphae and in spores. These fluorescence domains were scarcely detectable in cells of the SCO1389-disrupted mutant. The red shift fluorescence due to stacking of two dye molecules showed same results like the green shift in mutants and M145 strains. Statistical analysis shows altering the expression level in SCO1389 affects the growth and branching in the strain RJ118b (induced by anhydrotetracycline-atc) and RJ110 (induced by thiostrepton).

AB - Streptomycetes have a complex morphogenetic programme end up in the formation of aerial hyphae that develop into chains of spores. After spore dispersal, environmental signals trigger dormant spores to germinate to establish a new colony. This complexity of streptomycete microcolonies makes studying the dynamic processes that contribute to growth and development a challenging procedure. In order to study the mechanisms that underpin streptomycete growth, we have developed a system for studying hyphal extension, protein trafficking, and sporulation by time-lapse microscopy.Heterogeneous distribution of phospholipids along bacterial membrane results in the formation of domains enriched in anionic phospholipids at the cell poles and at the mid cell. Cardiolipin plays a key role in organization of bacterial membranes and forms membrane domains that participate in interaction with multi-protein complexes involved in cell division, energy metabolism, and membrane transport. The goals of this study were to genetic characterization SCO5750 with localization of cardiolipin, and SCO1389, that synthesizes this anionic phospholipid and so change membrane lipid composition. SCO1389 gene from cosmid St1A8A derivative was disrupted by replacement with an apramycin-resistant gene followed by exchange with the homologous chromosomal region. Most of recombination resulted in single crossover and none had undergone the second crossover event to delete the SCO1389 gene. SCO1389 could not be deleted without causing lethality, except when a second SCO1389 copy is expressed in the same cells, which implies that SCO1389 is involved in an essential primary metabolic process, likely membrane phospholipid biosynthesis. The proper genomic disruptions were confirmed by Southern blot hybridization. Depletion of SCO1389 caused failure in erection of aerial hyphae and severely retards growth whilst there was enough genetic evidence that SCO1389 is essential for growth and development, it was not clear why cells cannot grow in the absence of this gene. Over expression of SCO1389 weakens hyphal tips, mis-shaped aerial hyphae and large anucleated spores. A SCO1389-egfp translational fusion was constructed, which showed distinctive regions of strong fluorescence mostly in the substrate hyphae and in few young aerial hyphae. Use of the cardiolipin specific fluorescent dye 10-N-nonyl-acridine orange (NAO) revealed cardiolipin rich domains in the Escherichia coli membrane (Mileykovskaya et al., 2000). Staining of Streptomyces cells with NAO showed that there were green fluorescence domains in the branch points, substrate hyphae, hyphal tips, aerial hyphae and in spores. These fluorescence domains were scarcely detectable in cells of the SCO1389-disrupted mutant. The red shift fluorescence due to stacking of two dye molecules showed same results like the green shift in mutants and M145 strains. Statistical analysis shows altering the expression level in SCO1389 affects the growth and branching in the strain RJ118b (induced by anhydrotetracycline-atc) and RJ110 (induced by thiostrepton).

M3 - Doctoral Thesis

ER -