A sensitive method for confocal fluorescence microscopic visualization of starch granules in iodine stained samples

Ovečka, Miroslav; Bahaji, Abdellatif; Muñoz, Francisco José; Almagro, Goizeder; Ezquer, Ignacio; Baroja‐Fernández, Edurne; Li, Jun; Pozueta‐Romero, Javier

doi:10.4161/psb.21370

Cited by 22 publications

(19 citation statements)

References 21 publications

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“…4b), again appearing at internucleoid zones and potential division sites ( Fig. 3a) and not only at the cell periphery as reported previously for E. coli [41][42][43] . This indicates that small amounts of glycogen remain within these cells upon exiting lag phase 44,45 , possibly due to structural changes that are known to increase short chains within this polymer 19,21,39 and which would be expected to delay turnover 39 .…”

Section: Discussionsupporting

confidence: 85%

Disruption of glycogen metabolism alters cell size inEscherichia coli

Walt¹,

Bürgy²,

Engelbrecht³

et al. 2020

Preprint

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The availability of nutrients impacts cell size and growth rate in many organisms. Research in E. coli has traditionally focused on the influence of exogenous nutrient sources on cell size through their effect on growth and cell cycle progression. Utilising a set of mutants where three genes involved in glycogen degradation -glycogen phosphorylase (glgP), glycogen debranching enzyme (glgX) and maltodextrin phosphorylase (malP) -were disrupted, we examined if endogenous polyglucan degradation affects cell size. It was found that mutations to malP increased cell lengths and resulted in substantial heterogeneity of cell size. This was most apparent during exponential growth and the phenotype was unaccompanied by alterations in Z-ring occurrence, cellular FtsZ levels and generation times. ∆malP mutant cells did, however, accumulate increased DnaA amounts at late growth stages indicating a potential effect on DNA replication. Replication run-out experiments demonstrated that this was indeed the case, and that DNA replication was also affected in the other mutants. Bacteria with a disruption in glgX accumulated glycogen and protein inclusion bodies that coincided with each other at inter-nucleoid and polar regions.Robust adaptive survival strategies are employed by bacteria to promote fitness. Most notably, when nutrients are abundant, bacterial cells demonstrate increases in length, width and growth rate 1 which permit parental cells to promote vigour of future generations by giving rise to larger daughter cells 2 . Enhanced rates of macromolecular biosynthesis accompany nutrientdependent size increases to ensure that larger cells are born with more DNA, RNA and protein 3-6 . This positive scaling relationship between cell size and external nutrient-imposed growth rate has historically been termed Schaechter's nutrient growth law 1 . Escherichia coli is known to passively correct deviant changes to cell size by adding a constant volume of cellular material (cell unit) between division events, regardless of size at birth 7,8 which has led to the proposal of what is known as the adder model 9 .Under steady-state conditions, the cell unit postulated in the adder model is dictated by two mechanisms 10 . The first is an initiation adder that ensures DNA origin firing is triggered only when the cell has grown and accumulated protein factors, such as DnaA, to a threshold required to initiate DNA replication. Synchronous origin firing under conditions of slow growth results in the birth of two daughters, each containing one parental chromosome; however, in environments that promote rapid growth, cell division can occur at a rate faster than the time it takes to duplicate the genome. Bacteria overcome this problem by initiating overlapping replication cycles, so daughter cells are born with two, four or even eight replication origins that ensure genomic integrity is maintained when the cell is faced with rapid division rates 11 . Several studies have reported that the volume of the cell unit proposed in the adder model is prop...

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Section: Discussionsupporting

confidence: 85%

Disruption of glycogen metabolism alters cell size inEscherichia coli

Walt¹,

Bürgy²,

Engelbrecht³

et al. 2020

Preprint

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“…To test for the presence of starch, the cells were stained by a 2%KI, 1% I 2 (w/v) solution, incubated for 10 minutes in the dark and visualized (474/28 nm excitation, 525/50 nm emission) [58]. This method did not reveal the presence of starch or glycogen in G20.…”

Section: Methodsmentioning

confidence: 99%

System-Wide Adaptations of Desulfovibrio alaskensis G20 to Phosphate-Limited Conditions

et al. 2016

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The prevalence of lipids devoid of phosphorus suggests that the availability of phosphorus limits microbial growth and activity in many anoxic, stratified environments. To better understand the response of anaerobic bacteria to phosphate limitation and starvation, this study combines microscopic and lipid analyses with the measurements of fitness of pooled barcoded transposon mutants of the model sulfate reducing bacterium Desulfovibrio alaskensis G20. Phosphate-limited G20 has lower growth rates and replaces more than 90% of its membrane phospholipids by a mixture of monoglycosyl diacylglycerol (MGDG), glycuronic acid diacylglycerol (GADG) and ornithine lipids, lacks polyphosphate granules, and synthesizes other cellular inclusions. Analyses of pooled and individual mutants reveal the importance of the high-affinity phosphate transport system (the Pst system), PhoR, and glycolipid and ornithine lipid synthases during phosphate limitation. The phosphate-dependent synthesis of MGDG in G20 and the widespread occurrence of the MGDG/GADG synthase among sulfate reducing ∂-Proteobacteria implicate these microbes in the production of abundant MGDG in anaerobic environments where the concentrations of phosphate are lower than 10 μM. Numerous predicted changes in the composition of the cell envelope and systems involved in transport, maintenance of cytoplasmic redox potential, central metabolism and regulatory pathways also suggest an impact of phosphate limitation on the susceptibility of sulfate reducing bacteria to other anthropogenic or environmental stresses.

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“…This is achieved by the activity of the GRANULE-BOUND STARCH SYNTHASE (GBSS), the only starch synthase isoform required for amylose synthesis. Mutants carrying defects in the GBSS (or “waxy”) gene have been isolated in many species, including Arabidopsis [ 9 , 10 ], maize [ 11 ], rice [ 12 ], cassava [ 13 ], and potato [ 14 ], all of which produce amylose-free starch granules. Several properties distinguish GBSS from other starch synthase isoforms.…”

Section: Introductionmentioning

confidence: 99%

PROTEIN TARGETING TO STARCH Is Required for Localising GRANULE-BOUND STARCH SYNTHASE to Starch Granules and for Normal Amylose Synthesis in Arabidopsis

et al. 2015

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The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin) or linear (amylose). The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS) is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST) is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM). We discovered that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is exclusively involved in amylose synthesis.

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A sensitive method for confocal fluorescence microscopic visualization of starch granules in iodine stained samples

Cited by 22 publications

References 21 publications

Disruption of glycogen metabolism alters cell size inEscherichia coli

Disruption of glycogen metabolism alters cell size inEscherichia coli

System-Wide Adaptations of Desulfovibrio alaskensis G20 to Phosphate-Limited Conditions

PROTEIN TARGETING TO STARCH Is Required for Localising GRANULE-BOUND STARCH SYNTHASE to Starch Granules and for Normal Amylose Synthesis in Arabidopsis

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