# Presentation: 528816

## Mechanisms of U(VI) Reduction and Sediment Growth in Desulfovibrio 

- Mechanisms of U(VI) Reduction and Sediment Growth in *Desulfovibrio*

- Lee Krumholz
**Department of Botany and Microbiology**

## Goals

- Identify mechanisms of survival of sediment bacteria in U(VI) contaminated sediments.
- This was further refined to
- Identification of mechanisms of dealing with U(VI)
- Identification of mechanisms of survival in Anoxic sediments.

## Desulfovibrio  G20

- Isolated from an oil producing reservoir.
- 4.1 Mb, 3598 candidate genes
- Doubling time is ~4.6 hours

*Desulfovibrio * G20

## Mechanisms of U(VI) reduction

## Identification of Genes involved in U(VI) Response

## 10 mutants

## Potential Functions of mutated genes in U(VI) Sensitive Mutants

- DNA repair
- rRNA methylation
- Protein Renaturation

## Washed Cell U(VI) reduction test by 24 mutants

- Growth Experiment with B11E9

## The Region surrounding the mutation in B11E9

- D. vulgaris

## Mechanism of As(VI) Reduction

## Loss of As(V) Tolerance.  

- B11E9 also lost As(V) resistance when grown in lactate sulfate media with 20mM As(V)

## Growth and U(VI) reduction of G20 with Cd

## Mechanism of U(VI) Reduction

## U(VI) Reduction by E. coli transformant

## Signature Tagged Mutagenesis

- First developed to identify virulence genes in pathogens.
- Use the technique to identify functions involved in sediment growth.  Different from functions needed for lab growth.

## Overview of signature tagged mutagenesis (STM)

## Growth of potential non-survival mutants of Desulfovibrio in sediment

**Growth of potential non-survival mutants of *****Desulfovibrio***** in sediment**

## Recurring Themes

- DNA repair
- Transcriptional regulators
- Phage-related proteins
- Transport proteins (multidrug efflux)
- Conserved and hypothetical proteins

**Recurring Themes**

## DNA repair

- •sediment conditions may be mutagenic
- •these genes may correct DNA defects resulting from sediment mutagens (e.g., organic acid fermentation products)

**Notes:**
Competitive index = output of mutant divided by output of parent/ input of mutant divided by input of parent
•increased resistance to several weak organic acids, such as acetate, was conferred on Escherichia coli by overexpression of the ATP-dependent helicase RecG (Steiner et al, 2003) 

## Response to mutagens

- One mutant B12(pF11) belongs to **UmuC **family.
- It has 53.83% similarity to *D. vulgaris* umuC protein.

**Notes:**
SOS induction occurs when RecA protein binds to regions of single-stranded DNA that are produced as a consequence of DNA replication blockage, for example when DNA polymerase III, which replicates the bacterial chromosome, stalls at the sites of DNA lesions. RecA then undergoes a conformation change that leads to activation of its latent coprotease activity. The RecA coprotease then promotes cleavage of LexA, the repressor of the SOS regulon. On LexA inactivation, some 20-30 proteins are induced, including the genes of the umuDC operon, which are essential for SOS mutagenesis (1, 2). RecA also facilitates cleavage of UmuD to produce the mutationally active form, UmuD'. The UmuD'2C complex then acts jointly with RecA protein, which also has a direct role in the process, to create mutations at the sites of DNA lesions (targeted mutagenesis). The UmuD'2C complex has recently been demonstrated to possess a polymerase activity (pol V) (3-6), which likely plays a critical role in producing the mutation. UmuC is an example of a specialized polymerase, a number of which have been reported from a variety of organisms and which are generally thought to function in mutagenesis and DNA damage processing (for reviews, see refs. 7 and 8). 

## Protection from a Severe Mutagenic Event

- Cited from:  Genetics 148: 1599–1610 (April, 1998)

**Protection from a Severe Mutagenic Event**
**The SOS response**

**Notes:**
SOS induction occurs when RecA protein binds to regions of single-stranded DNA that are produced as a consequence of DNA replication blockage, for example when DNA polymerase III, which replicates the bacterial chromosome, stalls at the sites of DNA lesions. RecA then undergoes a conformation change that leads to activation of its latent coprotease activity. The RecA coprotease then promotes cleavage of LexA, the repressor of the SOS regulon. On LexA inactivation, some 20-30 proteins are induced, including the genes of the umuDC operon, which are essential for SOS mutagenesis (1, 2). RecA also facilitates cleavage of UmuD to produce the mutationally active form, UmuD'. The UmuD'2C complex then acts jointly with RecA protein, which also has a direct role in the process, to create mutations at the sites of DNA lesions (targeted mutagenesis). The UmuD'2C complex has recently been demonstrated to possess a polymerase activity (pol V) (3-6), which likely plays a critical role in producing the mutation. UmuC is an example of a specialized polymerase, a number of which have been reported from a variety of organisms and which are generally thought to function in mutagenesis and DNA damage processing (for reviews, see refs. 7 and 8). 

## STM mutants in MR-1 with potential role in drug efflux

**COG**

**Multidrug resistance efflux pump **

**Multidrug resistance efflux pump **

**Cation/Multidrug efflux pump **

**Transcriptional regulator **

**STM mutants in MR-1 with potential role in drug efflux**

## mexF deletion cannot grow in the presence of 5 g/ml chloramphenicol

***mexF***** deletion cannot grow in the presence of 5 ********g/ml chloramphenicol**

- WT MR-1

- ∆*mexF*

- WT MR-1 & ∆*mexF* w/o antibiotic

- + antibiotic

**Notes:**
AS with Pseudomonas MexF, it confers resistance to chloramphenicol

## Tripartite efflux pump system

- Outer membrane

- Inner membrane

- Periplasmic space

- Outer membrane protein

- Membrane fusion protein
- (MexE)

- RND Exporter (MexF)

- antibiotic

- RND=resistance-nodulation-division

- Adapted from Aeschlimann, 2004

**Notes:**
RND is one of 5 families of MDR proteins

## -proteobacteria

- -proteobacteria

- -proteobacteria

- -proteobacteria

- /-proteobacteria

- Other

- Other environmental bacteria proteins similar to MR-1 MexF from NCBI pblast; Gaps range from 0-3%

## What have we learned from STM

- Identified a number of genes and cell functions that are required for life in the real world but not needed for growth in culture.
- Demonstrated for the first time the importance of specific genes as cells are growing in natural environments.
- Raised many questions regarding function of specific genes.  Regulatory genes.  Protection from toxins.  DNA repair, etc.

## Acknowledgments

- Funding from DOE ERSP program.
- Qingwei Luo, Jennifer Groh, Xiangkai Li and Nydia Castaneda.
- EMSL