Presented By 1 Department of Molecular Genetics, Weizmann Institute - - PowerPoint PPT Presentation

Presented By Swathy J.R 23.04.2016

1Department of Molecular Genetics, Weizmann Institute of Science,

Rehovot, Israel

2Department of Chemical Research Support, Chemical Research Support,

Weizmann Institute of Science, Rehovot, Israel.

2 March 2016

Fig. 1. Visualization

• f

cyanobacterial cells forming intracellular mineral inclusions. (A) Composite epifmuorescence and

• ptical

microscopy images showing rod-shaped auto fmuorescent cyanobacteria cells. (B) Optical image shows intracellular inclusions in cyanobacterial cells (higher magnifjcation in inset). (C to F) SEM images (secondary electron mode) showing cells

• f

the same morphotype, which systematically contain bright intracellular inclusions. (G to I) CSLM (G), phase contrast (H), and SEM (I) images of exactly the same area, showing that auto fmuorescent cells observed by CLSM contain the bright inclusions observed by SEM (inset). A difgerent, larger, and inclusion-deprived

SCIENCE VOL 336 27 APRIL 2012

In this paper

›Granules of Calcium carbonate were observed within and on the biofjlm surfaces. ›These calcium carbonate mineral scafgolds promote complex morphology and robustness to the biofjlms.

› Microbiologically induced calcium carbonate precipitation (MICP) is a bio-geochemical process that induces calcium carbonate precipitation within the soil matrix. › Calcium carbonate can be precipitated in three polymorphic forms, which in the

• rder
• f

their usual stabilities are calcite, aragonite and vaterite

Introduction

A byproduct of metab not clearly understoo A byproduct of metab not clearly understoo

Biofjlm Carbona te

Why this paper??

Calcite ν2 : 875 cm-1 ν3 : 1425 cm-1 ν4 : 713 cm-1

(a–c) T

• p view of a colony of an undomesticated strain of wild-type B. subtilis (NCIB 3610).

The colonies were grown on solid biomineralization-promoting medium without (a) or with (a and b) a calcium source, for 3 and 7 (a), 21 (b) days, at 30 °C, in a CO2-enriched

• environment. (b) Top view of a colony (left) and a magnifjcation of calcite crystals at the

periphery (right upper) or centre (right lower) of the colony. Images were taken by Stereo microscope with an objective of × 0.5 (a and b, left) or × 1 (b, right) Scale bar corresponds to 2 mm (a and b, left) or 100 µm(b, right). (c) The FTIR spectra of calcium carbonate minerals precipitated at the edges of the colony. ν2 and ν3 indicate characteristic vibrations. The results are of a representative experiment out of fjve independent repeats.

Complex colony morphology correlates with calcite precipitation in Bacillus subtilis.

XRD

(a, b) Upper panel: T

• p view of a biofjlm of wild-type B. subtilis. The biofjlms were grown

at 30 °C, in a CO2-enriched environment. (a) On solid biomineralization promoting medium with a calcium source for 1, 2, 5 days or without a calcium source for 5 days. (b) The bifjlms were grown on solid biomineralization-promoting medium with a calcium source for 10, 21 days or on MSgg and mLB medium for 5 days. Lower panel: MicroCT images of B. subtilis biofjlms. Scale bar corresponds to 2 mm. (c) Images representing the thickness of the calcium carbonate buildup underneath the wrinkles of wild-type and lcfA mutant. The images were obtained from the microCT by 2D slice cutting through the

(a) (b) (c)

Calcite and amorphous calcium carbonate have a distinct spatio- temporal organisation within the biofjlm.

3.5mm .5mm

Gene mutant : Partially defective in maturation and 3D structure. Planktonic growth was seen!

The biofjlm cells promote the formation of an alkaline microenvironment, required for calcium carbonate precipitation and morphogenesis.

(a, b) Wild-type and ureA-C mutant biofjlms were grown

• n

solid biomineralization- promoting medium with a calcium source, at 30 °C, in a CO2-enriched environment. (a) The intra-colony pH of wild- type (blue column) and ureA-C mutant strain (red column)

• n

solid biomineralization-promoting medium, with acidic environment (pH 5.5). The results represent the averages and S.D. of three independent experiments. (b) T

• p view of a biofjlm of wild-

type B. subtilis (upper panel) and its ureA-C mutant (lower panel) derivative that grows in either an acidic (pH 5.5) or neutral (pH 7) environment, for 3 or 7 days.

Images were taken by Stereo microscope with an objective × 0.5. Scale bar corresponds to 2 mm. The results are of a representative experiment out of three independent repeats. (c) Growth curves of wild-type (blue) and its ureA-C mutant derivatives (red) at 30 °C in liquid biomineralization-promoting

• medium. The results represent the averages and S.D. of six wells per strain, tested in three

independent experiments. Mutants unable to bufger the pH show fallings in colony morphology Featureless colonies

The extracellular matrix afgects amorphous calcium carbonate (ACC) and calcite distribution. Wrinkle morphology, CaCO3 in wrinkles were

• afgected. Edges shows precipitates. Planktonic

growth is not afgected. ∴ 3D structure is dependent on biomineralization

Exopolysaccharide mutant Amyloid mutant

CaCO3 afgects the matrix appearance.. vise versa is possible?

Exopolysaccharide double mutant

(a) Images represent biofjlm phenotypes of a wild-type strain and its derivative biofjlm formation mutants (mutants for extracellular matrix production): single mutants for epsH, ywqC-F and tasA and a double mutant for epsH and ywqC-F . Colonies were grown on solid biomineralization medium, in the absence or presence of a calcium source, for 19 days, at 30 °C. Left panel: images of biofjlm, taken with a Nikon D3. Scale bar corresponds to 1 mm; Right panel: MicroCT images of the wild-type biofjlm formation mutant strains. Scale bar corresponds to 2 mm. The results are of a representative

The extracellular matrix afgects amorphous calcium carbonate (ACC) and calcite distribution. Cont…

(b) The surface morphology of a calcite mineral. An environmental scanning electron micrograph (ESEM) image

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the calcite mineral extracted from the wild-type strain. The mineral was fractured, to expose its internal structure. Microscope magnifjcation × 12,600; Scale bars correspond to 5 µm. (c) The acidic polysaccharides interact with the mineral phase. Environmental scanning electron micrograph (ESEM) images of calcite crystals extracted from the edges of the colonies of the wild-type strain and from its extracellular matrix mutant derivatives (tasA, epsH and ywqC-F). The crystals were extracted and washed to remove organic matter. Microscope magnifjcation × 25,800. Scale bar corresponds to 2 µm. The results are

• f

a representative experiment out of four independent

• repeats. The red square highlights a

Elongated prismatic morphology

Amyloid mutant EPS mutant

Calcite reduced difgusion by 7 orders of magnitudes. Acts as a barrier towards antimicrobial agents - Ethanol 0.7% 30%

• Exact mechanism of

transport of calcium from medium to surface remains undetermined.

• Probably the initial

assembly of Ca2+

• ccurs inside the cells

– T

• be studied at a

single cell level. (as in the case of Magnetite )

Summary

› Robust bacterial biofjlm development needs:

– Organic extracellular matrix – Functional mineral deposits – Cellular microenvironment promoting controlled pH

› Calcium carbonate mineral granules on the biofjlm matrix contributes to the patterning and 3D structure morphogenesis. › A load bearing foundation, increased rigidity, resistance to shear stress and dispersal (intact protection) › An overall stability › Urease dependent metabolism in intra and extra cellular environment. › Excess Mg2+ or Ba2+ did not afgect the mineralization. › Cells in biofjlms are typically more resistant to antibiotics – Calcite formation is one of the factors as it reduces difgusion. › Endogenous accumulation of CO2 is naturally handled :

Cheng Wang et. al., An invisible soil acidifjcation: Critical role of soil carbonate and its impact on heavy metal bioavailability, Scientifjc Reports 5, Article number: 12735, doi:10.1038/srep12735, November 2015 Soil carbonates play a critical role in heavy metal transfer from soil to plants, implying that monitoring soil carbonate may be necessary in addition to soil pH for the evaluating soil quality and food safety. Yohey Suzuki et al., Formation and Geological Sequestration of Uranium Nanoparticles in Deep Granitic Aquifer, Scientifjc Reports | 6:22701 | DOI: 10.1038/srep22701 , March 2016 The microbiologically induced precipitation of calcium carbonate and U(IV) nanoparticles, can lead to long-term sequestration of uranium and

• ther radionuclides in contaminated aquifers

and deep geological repositories. Gordon E. Brown,jr. et.al., Mineral surfaces and bioavailability

• f

heavy metals: A molecular-scale perspective, Proc. Natl. Acad. Sci. USA Vol. 96, pp. 3388– 3395, March 1999 The importance of speciation of Lead, Arsenic and Selenium in the environment and their bioavailability

Carbonate s

Ronn S. Friedlander et. Al., Bacterial flagella explore microscale hummocks and hollows to increase adhesion, PNAS 2013 110 (14) 5624-5629; March 18, 2013,doi:10.1073/pnas.1219662110

Thank you…

Supplementary Information

Back up slides

• The carbonic

anhydrases, a metalloenzyme that catalyze the rapid interconversion of CO2 and water to bicarbonate and protons

• Mutants of the gene had mild impact whereas the
• verexpression increased calcite precipitation

Live vs dead cells to show the role of bacteria in mineralization and that this is not an chemically driven mechanism

In the absence of Ca2+, MgCO3 was formed

Figure 6. Biomineralization is required for formation

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complex morphology in Mycobacterium smegmatis.(a, b) Upper panel: top view of the biofjlm of the undomesticated M. smegmatis. The biofjlms were grown on solid biomineralization- promoting medium without (a) or with (a, b) a calcium source, for 3 (a), 5, 21 (b) days at 30 °C, in a CO-enriched environment. Images were taken with a Stereo microscope with an objective × 1. Scale bar corresponds to 500 µm. Lower panel: MicroCT images of the biofjlms formed by

• M. smegmatis. Scale bar corresponds to 2
• mm. The results are of a representative

experiment

• ut
• f

three independent

• repeats. (c) The FTIR spectra of calcium

carbonate minerals precipitated within the

• colony. The designation of the peaks as

v22, v3 and v is marked. The results are

• f

a representative experiment out of fjve independent repeats.