Analyzing the relationship between gene expression and cell metabolism in Streptomyces tenjimariensi

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Analyzing the relationship between gene
expression and cell metabolism in Streptomyces
tenjimariensis

• University of Hawaii

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Objective

• To correlate changes in the metabolism of the
  marine bacterium Streptomyces tenjimariensis to
  corresponding changes in genetic regulation and
  control

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Metabolism and Gene Expression

• Metabolism represents the ultimate end product of
  gene expression

Sumner et al. 2003
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Streptomyces tenjimariensis

• Gram-positive bacteria
• Isolated from sea mud samples near Sagami Bay,
  Japan in 1979.
• Complex biochemical pathways
• Breakdown of organics
• Antibiotic production
• Genetically uncharacterized
• Only one gene has been sequenced

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Acid/base consumption, cell mass, and bioactivity
acid
cell mass
Bioactivity diameter zone of inhibition (cm)
0.90 g/L
base
bioactivity
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Metabolic Pathways of Interest

• Target genes
• Antibiotic biosynthetic genes
• 2-deoxy-scyllo-inosose synthase (DOI)
• L-glutamine aminotransferase (AMT)
• Alpha amylase
• Starch breakdown
• Possible housekeeping genes
• 16S rRNA
• tryptophan synthase (trpB)
• NADH dehydrogenase

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Hypothesis

• Changes in metabolism will correlate with changes
  in gene expression of alpha amylase, AMT, and DOI.

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Methods
PCR/ Sequencing
DNA
Microarrays/ RT-PCR
RNA
2D gel/LC-MS
Proteins
Metabolite profiling
Metabolites
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Primer Design

• Compared annotated sequences from other
  Streptomyces species using the Genbank database
  and Vector NTI bioinformatic software
• Alpha amylase
• trpB
• NADH
• Primers used with other Streptomyces species
  gathered from published papers.
• DOI (Kharel et al. 2004)
• AMT (Kharel et al. 2004)
• 16S (Lane et al. 1991)

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Primer Testing Results
16S

• PCR reaction Optimized
• DNA concentration
• Anneal Temp
• Cycle length and number
• Additives

DOI
a. amylase
1500 bp
AMT
500 bp
345 bp
264 bp
130 bp
100 bp
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PCR Product Sequencing DOI
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AMT gene of interest
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Alpha amylase gene of interest
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16S gene of interest
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Reverse Transcription PCR (RT-PCR)

• Most sensitive method for mRNA detection (Bustin
  et al. 2000)
• Reverse transcriptase
• mRNA cDNA PCR amp.
• Tested gene expression at various time points
  (24h, 36h, 60h) using designed primers

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RT-PCR Results
24 24 hr 36 36 hr 60 60 hr
D DOI , T AMT, a1 a. aml 1, a2 a. aml 2, N
NADH
D D D 24 36 60
T T T 24 36 60
a1 a1 a1 24 36 60
a2 a2 a2 24 36 60
N N N 24 36 60
1500 bp
500 bp
100 bp
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RT-PCR 16S control
16S 16S 16S 24 36 60
C C C 24 36 60
1500 bp
16S 16S rRNA C control (16S primers with no
RT step)
500 bp
24 24 hr 36 36 hr 60 60 hr
100 bp
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Quantitative RT- PCR

• Analyze amplification during exponential PCR
  amplification phase
• Standard curve method of quantification
• cDNA samples for each primer set
• Obtain relative changes in gene expression

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Conclusions

• Obtained partial sequence information for
  previously uncharacterized S. tenjimariensis
  genes
• Gene expression was observed across all time
  points
• Quantification of gene expression will be carried
  out in future experiments using quantitative
  RT-PCR
• Further primer optimization may be necessary

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Potential applications

• Determine how environmental factors influence
  secondary metabolite production
• Metabolic engineering of antimicrobials
• Method could be used to describe other
  uncharacterized species
• Based on metabolic analysis

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Acknowledgements

• Support for this project was from NSF grant
  0243600