# Presentation: 474173

## DOE Genomics: GTL

**Annual Retreat 2005**
**Sequence-Based Discovery**
**Carl Abulencia**
**Diversa Corporation**
**San Diego, California**

## Sequence-Based Discovery

**Goals**
- Gain a better understanding of how organisms live in contaminated environments, and how they have adapted to external stressors.
- Obtain sequence data by accessing the genomes from the organisms living in contaminated environments.
- Analyze the genes, operons, and stress response pathways from the natural diversity to study evolutionary changes, selections, expansions and gene transfers.

## Technology Flow for Sequence-Based Discovery

- Pathways analyzed
- by computational core

- Extract
- large-
- fragment
- DNA

- MDA
- amplify
- & clone

- Screen for genes involved in stress response

- Form Genomic Libraries

- Microbial DNA

- Contaminated Soil

- Genes and Pathways Analyzed by Functional Genomics

## Limitations of sampling from contaminated soil sites:
Very low biomass.
Very little extracted DNA.
DNA concentrations too low for:
16S rRNA gene PCR
DNA Library construction

- Limitations of sampling from contaminated soil sites:
- Very low biomass.
- Very little extracted DNA.
- DNA concentrations too low for:
- 16S rRNA gene PCR
- DNA Library construction

- Samples From the NABIR FRC SiteOak Ridge, Tenn.

- FRC Sampling, November 2004
- 9 uranium contaminated soil samples and one background sample.
- 12 Large fragment DNA extractions completed on 9 samples.

## Amplification of Sample DNAWhole Genome Amplification

- Isothermal amplification method.
- phi29 polymerase.
- Processive (up to 70kb).
- Strand-displacing.
- 3’-5’ exonuclease activity (proofreading activity).
- Random hexamer primers.
- 3 hour – 16 hour reaction time.

- 12kb

- 49kb

- 97kb

- Multiple Displacement Amplification (MDA)

## MDA Genome Coverage Analysis

- Comparison of Unamplified to Amplified gDNA
- Positive Control is gDNA from an overnight culture (unamplified).
- Dilute culture to 5000 cells, extract gDNA, MDA amplify.
- Dilute culture to 5 cells, extract gDNA, MDA amplify .

- 7123 probe sets on the chip that represent the full genome.
- Presence or absence of ~700 bp regions of sample DNA are detected by DNA hybridization.

- Affymetrix *E.coli* Genome GeneChip Array

|   |  +Control gDNA | MDA    5000 cells | MDA       5 cells |
| --- | --- | --- | --- |
| Number of probe sets detected | 7123 | 7119 | 7066 |
| Percent of probe sets detected | 100 | 99.94 | 99.2 |
| Number of probe sets overamplified |   | 26 | 41 |
| Number of probe sets underamplified |   | 62 | 327 |

## MDA Bias Analysis

- Mix the isolate gDNAs.
- Create a library from unamplified,mixed DNA.
- (66 μl of 60ng/ μl = 4μg)
- Create two libraries from MDA amplified, mixed DNA.
- amplified from 1/100 dilution of mixed DNA. (600pg)
- amplified from 1/10,000 dilution of mixed DNA. (6pg)

| Isolate | Size (Mb) | G/C content (%) |
| --- | --- | --- |
| Deinococcus radiodurans | 3.3 | 66 |
| Desulfovibrio vulgaris | 3.8 | 60 |
| Geobacter sulfurreducens | 3.8 | 61 |
| Mesorhizobium loti | 7.6 | 62 |
| Nitrosomonas europaea | 2.8 | 50 |
| Shewanella oneidensis | 5.1 | 45 |
| Staphylococcus epidermidis | 2.5 | 32 |
| Streptomyces coelicolor | 8.7 | 72 |

## Sequence Analysis of Random Library Clones

| Isolate | Size (Mb) | G/C content (%) | Unamplified | MDA 1/100 (600pg) | MDA 1/10,000 (6pg) |
| --- | --- | --- | --- | --- | --- |
| Deinococcus radiodurans | 3.3 | 66 | 9.2% | 1.2% | 3.3% |
| Desulfovibrio vulgaris | 3.8 | 60 | 8.6% | 1.2% | 2.2% |
| Geobacter sulfurreducens | 3.8 | 61 | 5.7% | 12.8% | 16.2% |
| Mesorhizobium loti | 7.6 | 62 | 24.1% | 3.6% | 3.3% |
| Nitrosomonas europaea | 2.8 | 50 | 0.8% | 10.0% | 12.8% |
| Shewanella oneidensis | 5.1 | 45 | 19.0% | 65.8% | 53.5% |
| Staphylococcus epidermidis | 2.5 | 32 | 5.1% | 5.0% | 8.4% |
| Streptomyces coelicolor | 8.7 | 72 | 27.5% | 0.5% | 0.3% |
| Total number of sequences |   |   | 510 | 421 | 359 |

## From Soil to Sequence

- MDA
- 16S rDNA Libraries
- DNA Libraries

| Sample # | Sample ID | Location Group | Sampling Date | Interval Cored (in.) | Core Segment (in.) | Extraction Date |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | FB075-04-07 | Area 3 | 11/8/2004 | 324-360 | 7-31 | 11/10/04 |
| 2 | FB077-01-20 | Area 4 | 11/8/2004 | 240-276 | 0-23 | 11/10/04 |
| 3 | FB-076-01-10 | Area 3 | 11/8/2004 | 144-180 | 10-34 | 11/10/04 |
| 4 | FB-075-01-04 | Area 3 | 11/8/2004 | 216-252 | 4-28 | 11/10/04 |
| 5 | FB-078-01-26 | Area 2 | 11/9/2004 | 216-252 | 26-38 | 11/11/04 |
| 6 | FB080-01-00 | Area 5 | 11/9/2004 | 264-286 | 0-12 | 11/11/04 |
| 7 | FB-078-01-02 | Area 2 | 11/9/2004 | 216-252 | 2-26 | 11/11/04 |
| 8 | FB-075-02-19 | Area 3 | 11/8/2004 | 252-288 | 19-38 | 11/11/04 |
| 9 | FB-079-01-16 | Area 1 | 11/9/2004 | 216-252 | 16-32 | 11/11/04 |
| B | FB-62304 | Bkgrnd | 11/9/2004 | 180-216 | 0-24 | 11/11/04 |

## 16S rDNA Library Sequence Data

- 16S rDNA Library Sequence Data

## Library Clone QC Sequencing Data

- Library 3868, Sample 1, Area 3, FB-075-04-07
- Depth: 324-360 inches

- Library 3875, Sample 3, Area 3, FB-075-04-07
- Depth: 144-180 inches

| Total Clone- ends Sequenced | 1270 |   |
| --- | --- | --- |
| Identical Clones | 52 | 4.1% |
| Clones with no Blast hits | 155 | 12.2% |

| Total Clone- ends Sequenced | 1119 |   |
| --- | --- | --- |
| Identical Clones | 8 | 0.7% |
| Clones with no Blast hits | 206 | 16% |

## Interesting Library QC Sequences

| E value | Top Blast hit |
| --- | --- |
| 3.00E-59 | gi|47059343| heavy metal transporting ATPase [Ornithobacterium rhinotracheale] |
| 3.00E-46 | gi|39996434| heavy metal efflux pump, CzcA family [Geobacter sulfurreducens PCA] |
| 1.00E-17 | gi|48846102| COG1235: Metal-dependent hydrolases of the beta-lactamase superfamily I [Geobacter metallireducens GS-15] |
| 1.00E-11 | gi|18977112|ref|NP_578469.1| heavy-metal transporting cpx-type atpase [Pyrococcus furiosus DSM 3638] |
| 2.00E-87 | gi|56460133|ref|YP_155414.1| Predicted extracellular metal-dependent peptidase [Idiomarina loihiensis L2TR] |
| 1.00E-45 | gi|53760196| COG0347: Nitrogen regulatory protein PII [Methylobacillus flagellatus KT] |
| 3.00E-24 | gi|46131534| COG0715: ABC-type nitrate/sulfonate/bicarbonate transport systems, periplasmic components [Ralstonia eutropha JMP134] |
| 1.00E-10 | gi|62484831| predicted cytotoxic translational repressor of toxic-antitoxic stability system [uncultured bacterium] |
| 2.00E-56 | gi|46579304| aminotransferase, classes I and II [Desulfovibrio vulgaris           subsp. vulgaris str. Hildenborough] |

## Library Screening for Histidine Kinase Genes

- Histidine kinase superfamily chosen for sequence-based discovery from environmental libraries.
- Cells sense and respond to extracellular stimuli through phosphotransfer-mediated signaling pathways controlled by HKs and response regulators.
- Library clones containing HKs retrieved by DNA hybridization using  degenerate probes designed from a subfamily of HKs from *D. vulgaris.*

- Sense Primer
- 5’-GGCSCAYGARATSAACAACCC-3’

- 5’-GGTSGTGAAGAASGGYTCGAA-3’
- Antisense Primer

## A Histidine Kinase Library Clone

- Library Clone 3870PT9
- Sample 1, Area 3
- 3890bp

- 5’

- 3’

- ORF 1

- Partial ORF

- ORF 3

- ORF 2

- _two-component system sensor histidine kinase_

- two-component system response regulator

- malate dehydrogenase

- outer membrane lipoprotein

## Histidine Kinase Discovery Progress

- Sequence-Based Screening
- 12 Libraries Screened, 50,000 clones/ library
- 26 HK clones in Sequencing

- Library QC sequencing
- 34 HK clones fully sequenced

## Summary

- gDNA extracted from 9 contaminated soil samples
- MDA used to amplify gDNA from low-yield samples
- 16S rDNA libraries constructed from each sample
- DNA libraries constructed from each sample
- Library diversity verified by random QC sequencing
- Histidine kinases discovered in libraries by random sequencing
*Desulfovibrio*-like histidine kinases discovered in libraries by sequence-based hybridization
- 16S rDNA sequences, library QC sequences, and full length HK clone sequences all uploaded to the VIMSS database
- A manuscript for publication is near completion detailing analysis of DNA libraries constructed from contaminated environmental soil samples

## Acknowledgments

- LBNL – Dominique Joyner, Sharon Borglin, Eoin Brodie, Terry Hazen
- LBNL – Eric Alm, Adam Arkin
- LBNL – Tamas Torok
- ORNL – David Watson
- Diversa – Denise Wyborski, Mircea Podar, Joe Garcia, Cathy Chang, Sequencing Group, Keiko Obokata, Toby Richardson, Sherman Chang, Karsten Zengler, Martin Keller