# Presentation: 485473

## Computational Core Milestones and Directions

- Eric Alm
- Lawrence Berkeley National Laboratory
**The Virtual Institute for Microbial Stress and Survival**
- http://microbesonline.org

## Computational Core

- We are applying comparative genomics and fundamentals of phylogenetic reasoning to data being generated by the VIMSS pipeline:

- Operon prediction
- Evolution of gene expression and genome structure (operons)
- Use of these predictions to QC functional genomics
- Evolutionary relationships highlight new targets for detailed characterization/genetics
- Evolution of signaling proteins holds evidence of niche adaptation of “our” bugs

## Methods: Predicting Operons

- (MN Price *et al.*, Nuc. Acids Res. 2005)

## Methods: Phylogenetic Inference - Genes

## Methods: Phylogenetic Inference - Operons

## Birth of an Operon Pair: Mechanisms

## Types of Genes in New Operons

- Large proportion of ORFan genes in new operons.  Did these operons form at the time of appearance of the ORFan gene?

## Birth of an Operon: Native-ORFan Pairs

## Evolution of an Operon: Compaction

## Highly Expressed Operons Contain Wide Intergenic Spacings

## Known Alternative Transcripts Are Similar to ‘Wide’ Operons

- 47/156 ‘wide’ operons have conserved intergenic footprints
**13/15 manually inspected footprints were reported previously in the literature**

## Evolution of an Operon: Function and Expression

- New operons tend not to have functionally coherent annotations among genes
- Niche-specific adaptations?
- Neutral evolution of expression patterns?
- We can’t study gene expression in ancestor, but we **can** study expression in outgroup of similar age to new operons - *Shewanella oneidensis*

*Shewanella*

*E. coli, *K12

## Death of an Operon

- ‘Death’ rates of **new** *vs*. **native** operons

- Fraction of dead ancestral operons

- Last outgroup containing ‘live’ operon

## ‘Dead’ vs. ‘Unborn’ Operons

- Dead operons are still largely coexpressed, unlike ‘unborn’ operons.
- Is operon death adaptive (small changes in regulation) or largely neutral?

## Life Cycle of an Operon

## What Did We Learn of Relevance to GTL?

- Operons can give important functional clues about uncharacterized genes
- But **only** if those genes have been maintained by natural selection for an extended period of time
- Genome structure (intergenic spacing) can alert us to alternative regulatory elements
- The data we are generating allows us to test key questions in the evolution of genome structure
- Knowledge of operon structure can lead to better models of significance in functional genomic data (next...)

## OpWise: A New Method for Determining Significance in Gene Expression Studies

- Experimental measurements can contain **noise** as well as **systematic biases**
- Systematic bias is not reduced when replicate experiments are performed
- We can use our prior knowledge of operon structure to estimate this systematic bias, and avoid reporting inflated confidence levels: OpWise
- We can also use multiple changers in the same operon to construct a more powerful test of significance

## Comparison With Existing Methods

- Systematic bias not
- reduced by replication
- can be estimated
- using operons

- Standard approaches
- including FDR can be
- overconfident

## Evolution of Sulfate Reduction and ‘Signature’ Genes

## Biology of (Dissimilative) Sulfate Reduction

- 8e- + 8H+

- SO42 -

- APS

- HSO3 -

- HS-

**ATP**

**PP****i**

**2 P****i**

- 2e- + 2H+

**AMP**

- 6e- + 6H+

- ?

- ?

## Biology of (Dissimilative) Sulfate Reduction

- 8e- + 8H+

- 2 lactate

- 2 acetate
- + 2 CO2

**2 ATP**

**2 ADP**

- SO42 -

- APS

- HSO3 -

- HS-

**ATP**

**PP****i**

**2 P****i**

- 2e- + 2H+

**+ 2 P****i**

**AMP**

- 6e- + 6H+

- ?

- ?

## Biology of (Dissimilative) Sulfate Reduction

- 8e- + 8H+

- 2 lactate

- 2 acetate
- + 2 CO2

**2 ATP**

**2 ADP**

- SO42 -

- APS

- HSO3 -

- HS-

**ATP**

**PP****i**

**2 P****i**

- 2e- + 2H+

**+ 2 P****i**

**AMP**

- 6e- + 6H+

- ?

- ?

- ATP    AMP + 2Pi
- 2ADP + 2Pi    2ATP
- 2ADP    AMP + ATP

## ‘Hydrogen Cycling’ Model

**periplasm**

**cytoplasm**

**6e****-**

**2e****-**

**2.7 ATP**

**8H****+**** + 8e****-**

**4H****2**

**2.7 ADP + P****i**

**4H****2**

**8e****-**** + 8H****+**

- Trans-
- membrane
- Complexes

- 2 lactate

- 2 acetate
- + 2 CO2

**2 ATP**

**2 ADP**

- SO42 -

- APS

- HSO3 -

- HS-

**ATP**

**PP****i**

**2 P****i**

**AMP**

- 2H+

- 6H+

**+ 2 P****i**

- Cytoplasmic
- Hydrogenase

- (Odom and Peck, 1981)

- Figure adapted from Haveman, 2004

## Molecular Basis for Hydrogen Cycling

**periplasm**

**cytoplasm**

**2.7 ATP**

**8H****+**** + 8e****-**

**4H****2**

**2.7 ADP + P****i**

**4H****2**

**8e****-**** + 8H****+**

- Trans-
- membrane
- Complexes

- Cytoplasmic
- Hydrogenase

- DsrMKJOP

- Dhc

- Ohc

- Tmc

- Hmc

- Nqr

- Qmo

- ?

- Ech

- CO de-H2ase

- ?

***Desulfovibrio vulgaris***

- (Heidelberg, *et al.*, 2004)

- (Dahl, *et al*., 2005)

- (Haveman *et al*., 2004)

- (Higuchi *et al*., 1987)

**other redox proteins**

- ?

## Comparative Approach to Understanding Sulfate Reduction

- Dv

- Af

- Dp

- Da

*Desulfovibrio vulgaris*
*Desulfovibrio alaskensis *G20
*Desulfotalea psychrophila*
*Archaeoglobus fulgidis*

- Bacteria

- Archaea

- What genes are shared among all SRB?

## Comparative Approach to Understanding Sulfate Reduction

- Dv

- Af

- Dp

- Da

*Desulfovibrio vulgaris*
*Desulfovibrio alaskensis *G20
*Desulfotalea psychrophila*
*Archaeoglobus fulgidis*

- Bacteria

- Archaea

- What genes are shared among all SRB?

- Phylogenetic profiles
- Doesn’t pick up subtle differences/shared traits among a group of proteins
- Some key proteins may be shared with similar organisms outside the set (S-reducing, or S-oxidizing bacteria)

## Comparative Approach to Understanding Sulfate Reduction

- Dv

- Af

- Dp

- Da

*Desulfovibrio vulgaris*
*Desulfovibrio alaskensis *G20
*Desulfotalea psychrophila*
*Archaeoglobus fulgidis*

- Bacteria

- Archaea

- What genes are shared among all SRB?

- Phylogenetic profiles
- Doesn’t pick up subtle differences/shared traits among a group of proteins
- Some key proteins may be shared with similar organisms outside the set (S-reducing, or S-oxidizing bacteria)
- Simple homology-based approach
- Find genes for which orthologs in the chosen set of bacteria are within top (ten) BLASTp hits for all 200+ genomes

## Molecular Basis for Hydrogen Cycling

**periplasm**

**cytoplasm**

**2.7 ATP**

**8H****+**** + 8e****-**

**4H****2**

**2.7 ADP + P****i**

**4H****2**

**8e****-**** + 8H****+**

- Cytoplasmic
- Hydrogenase

- SO42 -

- APS

- HSO3 -

- HS-

**ATP**

**PP****i**

- 2H+

- 6H+

- Qmo

- Dsr

- MKJ
- OP

**2e****-**

**6e****-**

- Ech
- Coo

- Dv

- Da

- Fdh

- Dp

- Fdh
- F420

- Ppa

- Aps

- Dsr

**2 P****i**

**AMP**

## Genomic Plasticity

- Niche-specific organic e- donors

- cyt-H2ase

- H2

- per-H2ase

- H+

- e-

- Genome-specific TM complexes/ e- acceptors

- conserved SO42-
- pathway

- specialized
- e- acceptors

## New SRB Signature Genes

- Known (or suspected) SO42- reduction pathway ( genes)
- ABC transporters
- Gln (sulfate?) transporter
- Enzymes
- CarAB (carbamoyl-P)
- PflA
- CobB (Dsr operon)
- Molybdopterin biosynthesis
- DapF
- Redox (14 genes)
- Ferredoxins (3)
- Glutaredoxin/ ferredoxin-thioredoxin reductase operon (2)
- Rubredoxin operon (3)
- Ferritin
- Other (5)
- Unknown (5)
- Many new targets for genetic characterization!
- Are these genes co-regulated with known SO42- reduction genes?

## Co-expression of ‘Signature’ Genes

- heat

- log

- -

- NO32 /NO2-

- NaCl

- cold

- O2

**DsrMKJO** **complex** + ABC transporter

**Aps**, **Qmo**, **DsrAB**, **PpaC** + ferredoxin II, glutaredoxin, ferritin, + 2 unknown

- Difference mainly in nitrate/nitrite response

- up-regulated

- down-regulated

- Expression level

## Genomic ‘Signature’ Summary

- Simple homology method for identifying physiologically relevant genes
- Nearly 50 genes identified as shared among SRB
- Including most known SO42- reduction genes
- Some new genes are co-expressed with known genes
- Core cytoplasmic SO42- reduction machinery conserved, but not H2 intermediate generation/consumption
- Genomic plasticity facilitates niche adaptation?

## Environmental Sensing in SRB

- Geobacter sulfurreducencs

- Geobacter metallireducencs

- Dechloromonas aromatica

- Nostoc sp.

- Wollinela succinogens

- Desulfovibrio vulgaris

- Desulfovibrio alaskensis G20

- Desulforomonas spp.

## Evolution of HPK Signaling Proteins

- genomes

- 200+

- Identify HPK domain proteins

## Evolution of HPK Signaling Proteins

- 4000+ HPKs

- Identify HPK domain proteins

- HPK domains from different genomes

- HPK

- Other signaling domains

- genomes

- 200+

## Evolution of HPK Signaling Proteins

- 4000+ HPKs

- Identify HPK domain proteins

- Isolate HPK domains

- genomes

- 200+

## Evolution of HPK Signaling Proteins

- 4000+ HPKs

- Identify HPK domain proteins

- Isolate HPK domains

- Cluster domains of high sequence similarity (BLASTclust)

- Lineage-specific expansions

- Ancient gene family expansions

- ?

**HPK1b**

- genomes

- 200+

## Gamma

- Dechloromonas
- expansion

- Bdellovibrio

- D. Vulgaris

- Caulobacter

- Vibrio

- NJ tree of HPK1b,
- Cluster1 of 213 HPKs

## Age of HPKs in Geobacter sulfurreducens

- Number of HPKs

*Geobacter sulfurreducens*
*Geobacter metallireducens*
- other S-reducers
- SO42- reducers

- Time since expansion

**1**

**2**

**3**

**4**

**1**

**2**

**3**

**4**

## Branch Length

- Cumulative counts

## Pseudomonas

- TM

- HAMP

- PAS

- HPK

- cheY

## Slide 42

## Rapid Evolution of Signaling Proteins

- SRB have some of the highest numbers of HPK proteins
- Many of these proteins are the result of lineage specific expansions (niche adaptation?)
- Extremely high rates of domain shuffling established for prokaryotes
- N-terminal domains (distant from HPK) appear to evolve more rapidly
- Do these duplications have different functions in the cell?

## Other (Future) Projects

-                            functional genomics website
- Comparative data from SMD, GEO, ... (soon)
- New links in genome/regulon browser
- Phenotype
- Regulatory elements
- Protein-protein interactions
- New tools for shopping cart
- Motif-finding/validation
- Metagenome analysis (Desulfotomaculum-like)
- Research on genome structure (posters)

## Acknowledgements

- Computational Core
- Adam Arkin
- Katherine Huang
- Morgan Price
- Keith Keller
- Janet Jacobsen
- Functional Genomics Core
- Keasling Lab
- Zhou Lab
- Microbiology Core
- Hazen lab
- Wall lab
- Stahl lab