scholarly journals Cosmid based mutagenesis causes genetic instability in Streptomyces coelicolor, as shown by targeting of the lipoprotein signal peptidase gene

2016 ◽  
Author(s):  
John T Munnoch ◽  
David A. Widdick ◽  
Govind Chandra ◽  
Iain C. Sutcliffe ◽  
Tracy Palmer ◽  
...  
Keyword(s):  
Cell Division ◽  
Gene Duplication ◽  
Small Rna ◽  
Genetic Instability ◽  
Genetic Manipulation ◽  
Streptomyces Coelicolor ◽  
Extracellular Proteins ◽  
Signal Peptidase ◽  
Wild Type ◽  
Developmental Defects

AbstractBacterial lipoproteins are a class of extracellular proteins tethered to cell membranes by covalently attached lipids. Deleting the lipoprotein signal peptidase (lsp) gene in Streptomyces coelicolor results in growth and developmental defects that cannot be restored by reintroducing the lsp. We report resequencing of the genomes of the wild-type M145 and the cis-complemented Δlsp mutant (BJT1004), mapping and identifying secondary mutations, including an insertion into a novel putative small RNA, scr6809. Disruption of scr6809 led to a range of developmental phenotypes. However, these secondary mutations do not increase the efficiency of disrupting lsp suggesting they are not lsp specific suppressors. Instead we suggest that these were induced by introducing the cosmid St4A10Δlsp as part of the Redirect mutagenesis protocol, which transiently duplicates a number of important cell division genes. Disruption of lsp using no gene duplication resulted in the previously observed phenotype. We conclude that lsp is not essential in S. coelicolor but loss of lsp does lead to developmental defects due to the loss of lipoproteins from the cell. Significantly, our results indicate the use of cosmid libraries for the genetic manipulation of bacteria can lead to unexpected phenotypes not necessarily linked to the gene or pathway of interest.

scholarly journals Branching of sporogenic aerial hyphae in sflA and sflB mutants of Streptomyces coelicolor correlates to ectopic localization of DivIVA and FtsZ in time and space

2020 ◽  
Author(s):  
Le Zhang ◽  
Joost Willemse ◽  
Paula Yagüe ◽  
Ellen de Waal ◽  
Dennis Claessen ◽  
...  
Keyword(s):  
Cell Division ◽  
Streptomyces Coelicolor ◽  
Constitutive Expression ◽  
Hyphal Growth ◽  
Lateral Wall ◽  
Specific Cell ◽  
Wild Type ◽  
Aerial Hyphae ◽  
In The Wild ◽  
Z Ring

ABSTRACTBacterial cytokinesis starts with the polymerization of the tubulin-like FtsZ, which forms the cell division scaffold. SepF aligns FtsZ polymers and also acts as a membrane anchor for the Z-ring. While in most bacteria cell division takes place at midcell, during sporulation of Streptomyces many septa are laid down almost simultaneously in multinucleoid aerial hyphae. The genomes of streptomycetes encode two additional SepF paralogs, SflA and SflB, which can interact with SepF. Here we show that the sporogenic aerial hyphae of sflA and sflB mutants of Streptomyces coelicolor frequently branch, a phenomenon never seen in the wild-type strain. The branching coincided with ectopic localization of DivIVA along the lateral wall of sporulating aerial hyphae. Constitutive expression of SflA and SflB largely inhibited hyphal growth, further correlating SflAB activity to that of DivIVA. SflAB localized in foci prior to and after the time of sporulation-specific cell division, while SepF co-localized with active septum synthesis. Foci of FtsZ and DivIVA frequently persisted between adjacent spores in spore chains of sflA and sflB mutants, at sites occupied by SflAB in wild-type cells. This may be caused by the persistance of SepF multimers in the absence of SflAB. Taken together, our data show that SflA and SflB play an important role in the control of growth and cell division during Streptomyces development.

scholarly journals Cosmid based mutagenesis causes genetic instability in Streptomyces coelicolor, as shown by targeting of the lipoprotein signal peptidase gene

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
John T. Munnoch ◽  
David A. Widdick ◽  
Govind Chandra ◽  
Iain C. Sutcliffe ◽  
Tracy Palmer ◽  
...  
Keyword(s):  
Genetic Instability ◽  
Streptomyces Coelicolor ◽  
Signal Peptidase

scholarly journals Sporulation-specific cell division defects in ylmE mutants of Streptomyces coelicolor are rescued by additional deletion of ylmD

2017 ◽  
Author(s):  
Le Zhang ◽  
Joost Willemse ◽  
Paul A. Hoskisson ◽  
Gilles P. van Wezel
Keyword(s):  
Cell Division ◽  
Streptomyces Coelicolor ◽  
High Heat ◽  
Heat Sensitivity ◽  
Specific Cell ◽  
Developmental Defects ◽  
Ftsz Ring ◽  
Severe Effect ◽  
Aberrant Phenotype ◽  
Electron And Fluorescence Microscopy

ABSTRACTCell division during the reproductive phase of the Streptomyces life-cycle requires tight coordination between synchronous formation of multiple septa and DNA segregation. One remarkable difference with most other bacterial systems is that cell division in Streptomyces is positively controlled by the recruitment of FtsZ by SsgB. Here we show that deletion of ylmD (SCO2081) or ylmE (SCO2080), which lie in operon with ftsZ in the dcw cluster of actinomycetes, has major consequences for sporulation-specific cell division in Streptomyces coelicolor. Electron and fluorescence microscopy demonstrated that ylmE mutants have a highly aberrant phenotype with defective septum synthesis, and produce very few spores with low viability and high heat sensitivity. FtsZ-ring formation was also highly disturbed in ylmE mutants. Deletion of ylmD had a far less severe effect on sporulation. Interestingly, the additional deletion of ylmD restored sporulation to the ylmE null mutant. YlmD and YlmE are not part of the divisome, but instead localize diffusely in aerial hyphae, with differential intensity throughout the sporogenic part of the hyphae. Taken together, our work reveals a function for YlmD and YlmE in the control of sporulation-specific cell division in S. coelicolor, whereby the presence of YlmD alone results in major developmental defects.

scholarly journals ssgA Is Essential for Sporulation ofStreptomyces coelicolor A3(2) and Affects Hyphal Development by Stimulating Septum Formation

2000 ◽  
Vol 182 (20) ◽  
pp. 5653-5662 ◽  
Author(s):  
Gilles P. van Wezel ◽  
Jannes van der Meulen ◽  
Shinichi Kawamoto ◽  
Ruud G. M. Luiten ◽  
Henk K. Koerten ◽  
...  
Keyword(s):  
Cell Division ◽  
Morphological Changes ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Wild Type ◽  
Septum Formation ◽  
Transmission Electron ◽  
In The Wild ◽  
Regulator Genes

ABSTRACT The role of ssgA in cell division and development of streptomycetes was analyzed. An ssgA null mutant ofStreptomyces coelicolor produced aerial hyphae but failed to sporulate, and ssgA can therefore be regarded as a novelwhi gene. In addition to the morphological changes, antibiotic production was also disturbed, with strongly reduced actinorhodin production. These defects could be complemented by plasmid-borne ssgA. In the wild-type strain, transcription of ssgA was induced by nutritional shift-down and was shown to be linked to that of the upstream-located gene ssgR, which belongs to the family of iclR-type transcriptional regulator genes. Analysis of mycelium harvested from liquid-grown cultures by transmission electron microscopy showed that septum formation had strongly increased in ssgA-overexpressing strains in comparison to wild-type S. coelicolor and that spore-like compartments were produced at high frequency. Furthermore, the hyphae were significantly wider and contained irregular and often extremely thick septa. These data underline the important role forssgA in Streptomyces cell division.

scholarly journals Introduction of the Foreign Transposon Tn4560 in Streptomyces coelicolor Leads to Genetic Instability near the Native Insertion Sequence IS1649

2007 ◽  
Vol 189 (24) ◽  
pp. 9108-9116 ◽  
Author(s):  
Elizabeth M. Widenbrant ◽  
Camilla M. Kao
Keyword(s):  
Genetic Instability ◽  
Type Strain ◽  
Insertion Sequence ◽  
Structural Changes ◽  
Wild Type Strain ◽  
Streptomyces Coelicolor ◽  
Wild Type ◽  
Insertion Elements ◽  
Foreign Dna

ABSTRACT We report an altered pattern of genetic instability for Streptomyces coelicolor when the bacterium harbored a foreign transposon, Tn4560. Deletions, amplifications, and circularizations of the linear 8.7-Mb chromosome occurred more frequently at sites adjacent to native insertion elements, notably IS1649. In contrast, deletions, amplifications, and circularizations of a wild-type strain happened at heterogeneous sites within the chromosome. In 50 strains examined, structural changes removed or duplicated hundreds of contiguous S. coelicolor genes, altering up to 33% of the chromosome. S. coelicolor shows a bias toward one type of genetic instability during this particular assault from the environment, the invasion of foreign DNA.

scholarly journals CytochromebdOxidase Has an Important Role in Sustaining Growth and Development ofStreptomyces coelicolorA3(2) under Oxygen-Limiting Conditions

2018 ◽  
Vol 200 (16) ◽  
Author(s):  
Marco Fischer ◽  
Dörte Falke ◽  
Carolin Naujoks ◽  
R. Gary Sawers
Keyword(s):  
Streptomyces Coelicolor ◽  
Reduction Rate ◽  
Growth Conditions ◽  
Developmental Cycle ◽  
Wild Type ◽  
Mycelium Growth ◽  
Developmental Defects ◽  
Content Type ◽  
Mycelia Growth ◽  
Genes Encoding

ABSTRACTStreptomyces coelicolorA3(2) is a filamentously growing, spore-forming, obligately aerobic actinobacterium that uses both a copperaa3-type cytochromecoxidase and a cytochromebdoxidase to respire oxygen. Using defined knockout mutants, we demonstrated that either of these terminal oxidases was capable of allowing the bacterium to grow and complete its developmental cycle. The genes encoding thebcccomplex and theaa3oxidase are clustered at a single locus. Using Western blot analyses, we showed that thebcc-aa3oxidase branch is more prevalent in spores than thebdoxidase. The level of the catalytic subunit, CydA, of thebdoxidase was low in spore extracts derived from the wild type, but it was upregulated in a mutant lacking thebcc-aa3supercomplex. This indicates that cytochromebdoxidase can compensate for the lack of the other respiratory branch. Components of both oxidases were abundant in growing mycelium. Growth studies in liquid medium revealed that a mutant lacking thebcc-aa3oxidase branch grew approximately half as fast as the wild type, while the oxygen reduction rate of the mutant remained close to that of the wild type, indicating that thebdoxidase was mainly functioning in controlling electron flux. Developmental defects were observed for a mutant lacking the cytochromebdoxidase during growth on buffered rich medium plates with glucose as the energy substrate. Evidence based on using the redox-cycling dye methylene blue suggested that cytochromebdoxidase is essential for the bacterium to grow and complete its developmental cycle under oxygen limitation.IMPORTANCERespiring with oxygen is an efficient means of conserving energy in biological systems. The spore-forming, filamentous actinobacteriumStreptomyces coelicolorgrows only aerobically, synthesizing two enzyme complexes for O2reduction, the cytochromebcc-aa3cytochrome oxidase supercomplex and the cytochromebdoxidase. We show in this study that the bacterium can survive with either of these respiratory pathways to oxygen. Immunological studies indicate that thebcc-aa3oxidase is the main oxidase present in spores, but thebdoxidase compensates if thebcc-aa3oxidase is inactivated. Both oxidases are active in mycelia. Growth conditions were identified, revealing that cytochromebdoxidase is essential for aerial hypha formation and sporulation, and this was linked to an important role of the enzyme under oxygen-limiting conditions.

scholarly journals Streptomyces coelicolor Undergoes Spontaneous Chromosomal End Replacement

2007 ◽  
Vol 189 (24) ◽  
pp. 9117-9121 ◽  
Author(s):  
Elizabeth M. Widenbrant ◽  
Hsiu-Hui Tsai ◽  
Carton W. Chen ◽  
Camilla M. Kao
Keyword(s):  
Genetic Instability ◽  
Type Strain ◽  
Wild Type Strain ◽  
Streptomyces Coelicolor ◽  
The Other ◽  
Wild Type ◽  
Genetic Changes

ABSTRACT We report a previously unobserved form of genetic instability for Streptomyces coelicolor, the replacement of one chromosome end by the other end. These genetic changes occurred spontaneously in both a wild-type strain and strains harboring a foreign transposon. Deleted and duplicated DNA comprises up to 33% of the genome.

scholarly journals Environmental RNAi pathways in the two-spotted spider mite

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Mosharrof Mondal ◽  
Jacob Peter ◽  
Obrie Scarbrough ◽  
Alex Flynt
Keyword(s):  
Gene Expression ◽  
Small Rna ◽  
Spider Mite ◽  
Genetic Manipulation ◽  
Model Organisms ◽  
Rnai Pathway ◽  
Plant Host ◽  
Rnai Technology ◽  
Two Spotted Spider Mite ◽  
Multicellular Organisms

Abstract Background RNA interference (RNAi) regulates gene expression in most multicellular organisms through binding of small RNA effectors to target transcripts. Exploiting this process is a popular strategy for genetic manipulation and has applications that includes arthropod pest control. RNAi technologies are dependent on delivery method with the most convenient likely being feeding, which is effective in some animals while others are insensitive. The two-spotted spider mite, Tetranychus urticae, is prime candidate for developing RNAi approaches due to frequent occurrence of conventional pesticide resistance. Using a sequencing-based approach, the fate of ingested RNAs was explored to identify features and conditions that affect small RNA biogenesis from external sources to better inform RNAi design. Results Biochemical and sequencing approaches in conjunction with extensive computational assessment were used to evaluate metabolism of ingested RNAs in T. urticae. This chelicerae arthropod shows only modest response to oral RNAi and has biogenesis pathways distinct from model organisms. Processing of synthetic and plant host RNAs ingested during feeding were evaluated to identify active substrates for spider mite RNAi pathways. Through cataloging characteristics of biochemically purified RNA from these sources, trans-acting small RNAs could be distinguished from degradation fragments and their origins documented. Conclusions Using a strategy that delineates small RNA processing, we found many transcripts have the potential to enter spider mite RNAi pathways, however, trans-acting RNAs appear very unstable and rare. This suggests potential RNAi pathway substrates from ingested materials are mostly degraded and infrequently converted into regulators of gene expression. Spider mites infest a variety of plants, and it would be maladaptive to generate diverse gene regulators from dietary RNAs. This study provides a framework for assessing RNAi technology in organisms where genetic and biochemical tools are absent and benefit rationale design of RNAi triggers for T.urticae.

scholarly journals Wild-Type Endoderm Abrogates the Ventral Developmental Defects Associated with GATA-4 Deficiency in the Mouse

1997 ◽  
Vol 189 (2) ◽  
pp. 270-274 ◽  
Author(s):  
Naoko Narita ◽  
Malgorzata Bielinska ◽  
David B. Wilson
Keyword(s):  
Wild Type ◽  
Developmental Defects

scholarly journals Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

2014 ◽  
Vol 80 (8) ◽  
pp. 2410-2416 ◽  
Author(s):  
Areen Banerjee ◽  
Ching Leang ◽  
Toshiyuki Ueki ◽  
Kelly P. Nevin ◽  
Derek R. Lovley
Keyword(s):  
Ethanol Production ◽  
Genetic Manipulation ◽  
Electron Flow ◽  
Model Organism ◽  
Inducible Expression ◽  
Carbon Flow ◽  
Wild Type ◽  
Content Type ◽  
Microbial Electrosynthesis ◽  
Clostridium Ljungdahlii

ABSTRACTThe development of tools for genetic manipulation ofClostridium ljungdahliihas increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox forC. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported forClostridium perfringens, was investigated. The plasmid pAH2, originally developed forC. perfringenswith agusAreporter gene, functioned as an effective lactose-inducible system inC. ljungdahlii. Lactose induction ofC. ljungdahliicontaining pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression ofadhE130-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression ofadhE1in a strain in whichadhE1and theadhE1homologadhE2had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression ofadhE2similarly failed to restore ethanol production, suggesting thatadhE1is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

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