evaluation of sinorhizobium meliloti retreat garcia.pdf · •castleton –chris villa, molly...

Evaluation of Sinorhizobium meliloti

Signaling, Metabolism & Genetics

with their Symbiotic Plant Host,

Alfalfa

Preston P. Garcia, Ph.D.

Sinorhizobium meliloti

□ Exists in free living state or symbiont inside plant

roots

□ Ability to fix atmospheric nitrogen

Sinorhizobium + Alfalfa N2-fixing nodule

Medical significance

□ Sinorhizobium is classified within the α2

subdivision of Proteobacteria

□ Mammalian pathogens Rickettsia &

Brucella

□ Sinorhizobium & Brucella are members

of the Rhizobiales order, both forming

chronic infections of eukaryotic cells

□ Exchange chemical signals with their

eukaryotic host to gain entry & prevent

their own destruction once inside.

Using catabolite repression to

study S. meliloti physiology and

genetics

Bacterial catabolite repression

□ When bacteria use one carbon source preferentially over another □ Exhibits diauxic growth.

□ First described by Jacques Monod as the “glucose effect” in E. coli in the 1940’s. □ Model system for understanding carbon

usage, gene regulation, and global gene expression.

Identification of mutants with

altered Succinate-Mediated

Catabolite Repression (SMCR)

phenotype

SMCR screen results

30,000 colonies

52 strains showed SMCR relief on plates

(blue)

Further tested in liquid with succinate and 2° carbon

sources: α-galactoside (raffinose)

β-galactoside (lactose)

α-glucoside (maltose)

One strain showed relieved SMCR with these

secondary carbon sources

Mutation of sma0113 affects SMCR on multiple 2° C-sources

Garcia, P. P. Bringhurst, R.M. and D. J. Gage (2010). J Bacteriol. 192: 5725

Two component systems

Pi Pi

Physiological signal

PAS

H670 D57

□ Regulatory circuits that mediate responses to

diverse environmental signals & play a central

role in bacterial physiology.

□ Excellent context in which to study cell signaling and biochemical circuits

Utilization of fluorescent reporter

plasmids to monitor S. meliloti

competition and succinate sensing in

vivo

Succinate Biosensor Plasmid □ Used to visually monitor specific carbon

sources in the rhizosphere.

□ GFP (green fluorescent protein) is

expressed by the dctA promoter of the

DiCarboxylate Transport system

□ Facilitates movement of succinate, fumarate,

malate and aspartate across cell membrane

□ Cells fluoresce when actively utilizing C4

dicarboxylic acids

Glass slide

Pour Nod3 Agar

Media Place sterile

germinated

seeds

Overlay with moistened

dialysis tubing S. meliloti strain

Visualization of S. meliloti on

Medicago sativa (alfalfa) root

5ml Nod3 Media

Growth chamber cycle:

16 hour light

8 hour dark

Prepare slide for growth

chamber

Glass Slide

Dialysis tubing

Larger Glass

Slide

Inverted

microscope

objective

Orientation on Microscope

S. meliloti growth in the

rhizosphere □ Actively grows on C4-dicarboxylic

acid root exudate

Rm1021/pPG12 (dctA::gfp)

S. meliloti growth in infection

thread □ Actively grows on C4-dicarboxylic acids

within root hairs

Rm1021/pPG12 (dctA::gfp)

Competitive nodulation assay

□ All S. meliloti deletion strains

are Fix+/Nod+

□ 10 distinct S. meliloti strains

(mutation in single gene of

TCS) transformed with

constitutive rfp or gfp plasmid □ Co-inoculated on a single sterile

germinated alfalfa seed.

Growth chamber

cycle:

16 hour light

8 hour dark

Constitutive gfp and rfp

plasmids

*Thank you

to Norwich U.

Future directions

□ Visually document interactions in the

rhizosphere with the biosensor strains

□ Monitor relationship of the environmental

sensing capabilities for motility / chemotaxis

□ Analyze competition data to assess

infection and nodulation ability.

□ Complete knockout mutations of

genetically similar two component

systems to counter redundancy

• VGN

– Jeanne Harris, Ph.D.

• Castleton – Chris Villa, Molly Leach, Kelsey McKay, Katelynn

Leavey

– American Society for Microbiology

– Faculty-Student Research Grants

– Advance Study grants

• UConn

– Daniel J. Gage, PhD.

– Charles Bridges

• St. Josephs University

– Dr. Catalina Arango

S. meliloti growth at root cap

□ Growing on sloughed off root cap cells

Rm1021/pPG12 (dctA::gfp) @ root cap