Using Mycobacterium smegmatis as a Bioindicator for Zinc-Limited Growth Conditions in Mycobacteria

요약

This protocol details preparation of Zn²⁺-limited media, growth vessels, and seed cultures to produce Zn²⁺-limited ribosomes in mycobacteria. Traces of Zn²⁺ lead to Zn²⁺-limiting growth and we describe morphogenic features used as bioindicators to verify Zn²⁺ limitation in vitro in the model bacterium, Mycobacterium smegmatis, and the human pathogen M. tuberculosis.

초록

Many bacteria build alternative ribosomes in Zn²⁺-limiting growth conditions by replacing Zn²⁺-binding ribosomal proteins with Zn²⁺-independent paralogs. Defining a system to study these alternative ribosomes has proven difficult because Zn²⁺ contamination in the laboratory is common. To address this issue, chelating agents are sometimes added to growth media, but this approach convolutes the biological response to gradual Zn²⁺ limitation and is associated with ribosome hibernation. Here, detailed instructions are outlined for preparing media and seeding cultures for Zn²⁺-limited growth without adding chelators. Following this method, the model bacterium, Mycobacterium smegmatis, undergoes morphogenesis, which depends on alternative ribosomes. Because morphogenesis is tractable and only occurs in Zn²⁺-limiting conditions, M. smegmatis can be used as a bioindicator to verify biologically relevant growth conditions. Three bioindicator phenotypes (cell density, cell length, and coenzyme F₄₂₀ fluorescence) that indicate Zn²⁺ limitation in the wild-type are described, and changes in these bioindicators for a deletion mutant that cannot build alternative ribosomes are outlined. Since trace Zn²⁺ contamination is difficult to control for each batch of media, and precise quantification of Zn²⁺ in each media preparation is overly burdensome, following this bioindicator phenotype is an accessible way to validate the preparation of Zn²⁺-limited growth media. To help identify proper conditions for Zn²⁺-limiting growth and alternative ribosome production, changes in the bioindicator phenotypes were profiled for Zn²⁺-contaminated or severely Zn²⁺-depleted preparations of Zn²⁺-limited media as well. Further details to achieve Zn²⁺-limiting growth and alternative ribosome production in M. tuberculosis are presented, along with the associated bioindicator phenotype. Overall, the detailed instructions and bioindicator phenotypes described here will help standardize the production of translationally active alternative ribosomes in mycobacteria.

서문

Zinc ion (Zn²⁺) is an essential micronutrient and a standard component of microbiological media. However, many bacteria endure Zn²⁺ limitation in the environment as Zn²⁺-limiting conditions are widespread; soil¹, marine², and niches within the host³ are often Zn²⁺-limited. As such, most bacteria have evolved mechanisms to overcome Zn²⁺ limitation and preserve cellular function⁴. Systems involved with maintaining Zn²⁺ homeostasis are under tight transcriptional control⁵ and are involved....

프로토콜

NOTE: The strains used in this study are wild-type M. smegmatis mc² 155 (gift from Dr. Robert Husson, Boston Children's Hospital) and the ΔaltRP deletion strain (originated from Prisic lab¹⁰). They can be cultured in a Biosafety Level 1 (BSL1) or a BSL2 laboratory, depending on local biosafety regulations and available resources. Here, the protocol is written for a BSL2 laboratory. If performed in a BSL1 laboratory, an aseptic technique with a Bunsen burner can replace a biosafety cabinet. In addition, researchers who wish to include other strains or conditions will need to calculate the volume of media nee....

결과

Bioindicator phenotypes of M. smegmatis
Wild-type M. smegmatis cultures grown in ZLM that achieve Zn²⁺-limiting growth have three main bioindicator phenotypes when compared to cells grown under Zn²⁺-replete conditions: [1] decreased OD₆₀₀, [2] elongated cells, and [3] decreased peak fluorescence at 420 nm. The mutant lacking AltRPs (ΔaltRP) does not have the same Zn²⁺-dependent morphogenesis as the wild-typ.......

토론

A major contribution to the knowledge gap on mechanistic underpinnings of Alt-ribosomes is the difficulty in achieving Zn²⁺-limiting conditions in vitro. Trace Zn²⁺ contamination in the laboratory setting is common; thus, achieving reproducible Zn²⁺-limited growth conditions is challenging. Zn²⁺ contamination can originate from several sources, although the most significant and common source of contamination and variability is from the ultrapure water used to rinse lab.......

자료

Name	Company	Catalog Number	Comments
1000 mL Vacuum Filtration Systems, Standard Line	VWR	10040-440
125 mL Erlenmeyer Shaker Flasks	Greiner Bio-One	679501
14 mL Culture Tubes, Plastic, with Dual-Position Caps	VWR	60818-725
250 mL Erlenmeyer Shaker Flasks	Greiner Bio-One	679502
40X-1000X Lab Binocular Biological Compound Microscope	Omax	M82EZ
50 mL Bio-Reaction Tubes, Sterile	VWR	76211-286
50 mL High-Performance Centrifuge Tubes with Flag Caps	VWR	89039-656
Alcojet Low-Foaming Powdered Detergent	Alconox	1404
Ammonium iron (III) citrate, brown	Thermo Scientific	AAA1119930	MW: 265 g/mol, for 1M solution add 1.325 g to 5 mL ultrapure water; store protected from light at 4 C
Bovine serum albumin	Thermo Scientific	J10857-A1
Calcium D-Pantothenate	Thermo Scientific	AC243300050
Catalase	Sigma-Aldrich	C1345-10G
Citric acid anhydrous, crystalline	Fisher Scientific	BP339-500
Dextrose anhydrous	Fisher Scientific	D14-500
Difco Middlebrook 7H9 Broth	BD Diagnostics	271310
DiluPhotometer OD600 for the Determination of Cell Density and Bradford Assays	Implen	OD600-10
GENios FL Microplate Reader	Tecan		Discontinued
Glycerol	Fisher Scientific	G33-4
Infinite M200 PRO Multimode Microplate Reader	Tecan	LGE140213-20BL
L-Asparagine	Thermo Scientific	AAB2147336
L-Leucine	Thermo Scientific	A1231122
Macrofire SP CCD camera	Optronics		For use with Olympus microscope
Magnesium sulfate heptahydrate	VWR	97062-134
Milli-Q Direct 8 Water Purification System	Millipore	C85358
Moticam X3	Motic		Discontinued, replaced by Moticam X5 Plus; for use with compound microscope
Oleic acid	Thermo Scientific	031997-14
Olympus BX51 upright microscope	Olympus		Discontinued, replaced by BX53M
Potassium phosphate, monobasic	Fisher Scientific	BP362-500
Semi-Micro Two-Sided Disposable Plastic Cuvettes	VWR	97000-590
Sodium chloride, crystal	VWR	VW1494-01
Sodium hydroxide	Fisher Scientific	S318-500
Tween 80	MP Biomedicals	103170
Zinc sulfide heptahydrate, crystalline/ACS	MP Biomedicals	219145290