# Presentation: 650007

## Signature-tagged mutagenesis for identification of Desulfovibrio alaskensis G20 sediment survival genes

- Qingwei Luo, Xiangkai Li, Jennifer Groh and Lee R. Krumholz
**The University of Oklahoma**
- Norman, Oklahoma

- In 1995, Hensel, M. & Holden, D. W. et. al. published the first signature-tagged mutagenesis (STM) paper in *Science*.
- Find virulence genes of *Salmonella typhimurium* in vivo.
- STM has been used on more than 15 different pathogens, such as *Salmonella enterica**, **Staphylococcus aureus, Listeria monocytogenes*
- All of these pathogenesis studies focused on searching for genes related to survival, colonization or virulence in the mouse model.

## Goals

- Identify genes critical for survival in natural systems.

*Shewanella oneidensis* MR-1

- •Isolated from Lake Oneida in New York by K. Nealson, 1987
- •Gram-negative rod
- •-Proteobacteria
- • Can reduce O2, fumarate, nitrate, nitrite, thiosulfate, elemental sulfur, iron, manganese, trimethylamine *N*-oxide (TMAO), dimethyl sulfoxide (DMSO), U(VI)

## Desulfovibrio alaskensis G20

- Sulfate- Reducer
- Nalidixic acid resistant
- 4.1 mb, 3598 candidate genes
- Doubling time is ~4.6 hours

*Desulfovibrio alaskensis* G20

## Generate 96 random tags and ligate to transposon

- 5’-ctaggtacctacaacctcaagctt-[NK]20-aagcttggttagaatgggtaccatg-3’

- N = A,T,C or G
- K = T or G

**KpnI**

**KpnI**

## Overview of signature tagged mutagenesis (STM)

## Input pool

- Tag B11

- Missing tag B11

- Recovered pool

## Growth of potential non-survival mutants in sediment

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

## Sequencing

**Grow non-survivors from mutant library in lab medium**
**Extract genomic DNA **
**Amplify transposon insertion region by 2 rounds of PCR**
**Sequencing the products using internal Tn primer**

- External Tn primer

- Arbitrary  primer 1

- Internal  Tn primer

- Internal  primer 2

- Tn 10

## Slide 11

## Insertion sequences (IS) 

- 38 insertion sequences in G20 genome
- 23 of them belong to IS3 family
- Widely distributed in both gram-negative and gram-positive bacteria. sizes from 1.2 to 1.5 kb. Contain two consecutive and partially overlapping ORFs.  The functional transposase is proposed to be a fusion protein, OrfAB,
- 3 were identified in this study to be related to sediment survival (VIMSS393703, VIMSS392855(twice), VIMSS394084)

## 49.6% homolog to D. vulgaris ISD1.

- 49.6% homolog to *D. vulgaris* *ISD*1.
- It has 7 paralogs in G20 genome, and 100% identity to VIMSS392855.

- Belong to *ISL*3 family. Involved in the transposition of the insertion sequence. It has 12 paralogs in G20 genome, and 99% identities to VIMSS395038 and VIMSS395351.

## Potential Functions of IS3

- Control of neighboring gene expression
- Function as a mobile promoter in *E. coli*, *Shigella.*
- Cause high level vancomycin resistance in *Enterococcus faecium*
- Inactivate genes by inserting within them.
- One was shown to knock out *sac*B under sucrose stress in *D. vulgaris* (*ISD*1)

## Signal transduction modules

- http://www.asm.org/news/index.asp?bid=36163

## Histidine Kinase

- Three mutants in 3 independent histidine kinases.
- Cellular sensors extracellular or intracellular.
- Detect metabolites, light, ions, redox potential, etc.

## Mutant genes related to regulatory function

- Signal transduction histidine kinase
- 	Form part of a two-component regulatory system
- 	Involve in lesion formation, swarming & production of 	extracellular protease
- Response regulators consisting of a CheY-like receiver domain
- Transcriptional regulator
- 	Repressor for uxuRBA operon
- Predicted transcriptional regulators
- 	Transcriptional repressor of an arsenic resistance operon
- DNA-directed RNA polymerase, subunit K/omega
- Important for U(VI) reduction
- Transcriptional regulators containing PAS, AAA-type ATPase and DNA-binding domains
- 	Regulation of virulence factors (*E.coli* & *S. typhimurium*)

## Chemotaxis 

- Chemoattraction to nutrients.
- Allows cells to move away from toxins.
- Response has been shown to increase survival of denitrifiers in soils.
- Sense the oxygen concentration or redox potential of the environment in *D. vulgaris*

- Chemotaxis

## Chemotaxis proteins in G20

- There are 57 chemotaxis proteins in G20
- 31 methyl-accepting chemotaxis proteins (MCP)
- We identified 2 MCP & 1 CheD mutants as non-survivors
- CheD Glutamine deamidase
- Role in receptor maturation by deamidation of particular glutamine residues.
- Methyl-accepting chemotaxis proteins (MCPs).
- Receptors Binds chemoeffectors and transduces signal to CheA. Is methylated and demethylated on glutamate residues.
- One MCP has 30.95% similarity to DcrA (sensing oxygen or redox potential of environments) of *D. vulgaris**.*

## Response to mutagens

- Belongs to **UmuC/****impB/mucB/samB **family.
- It has 53.83% similarity to *D. vulgaris* umuC protein.

## Protection from a Severe Mutagenic Event

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

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

## Mutant B12(pF11)

- Mutagenesis is a survival issue for cells in aquifer sediment.**  **

## Peptide Methionine sulfoxide reductase MsrA

- Reverses the inactivation of proteins due to the oxidation of critical methionine residues.
- Acts by reducing methionine sulphoxide, Met(O), to methionine.
- Helps the cells deal with oxidative damage.

## Slide 24

## Mutants involved in Respiration

## Mechanisms of U(VI) reduction

## Mechanisms are Still not clear

- Additional pathways can bypass Cytochrome C3 in *D.* *desulfovibrio* strain G20 (R. Payne et. al. 2002)

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

- Screen mutants for loss of the ability to tolerate U(VI).
- 5760 STM mutants were screened. Each mutant was grown in individual wells of a 96-well microtiter plate containing lactate-sulfate medium with 2mM U(VI)-acetate.
- Non-growers were transferred into serum tubes and lack of growth was confirmed.
- 24 STM mutants were identified.

## 24 mutants were divided into four categories based on their sensitivity to U(VI)

## 24 mutants were divided into four categories based on their sensitivity to U(VI)

## Mutant strains gene information

## Mutant strains gene information

- Summary:
- 22 different genes were disrupted. one transposon is located in a non-coding area and the other transposon is located in a non-coding region within one operon.
- Among the 22 genes, four genes are related to signal transduction, three are involved in DNA repair, one is involved in rRNA methylation, one is involved in RNA polymerase renaturation, four genes encode hypothetical proteins, and nine genes encode other proteins.

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

- The mutant strains were grown with lactate sulfate, washed twice with NaHCO3.
- Cell solution, about 8 X 109 cells, was added to 10ml buffer  (20mM lactate, 30mM NaHCO3, 1 mM U(VI)).
- U(VI) concentration was monitored.
- The genes disrupted in those mutants can’t reduce U(VI) might play important role in U(VI) reduction.

## Further study on mutant B11E9

- Picture was taken after 24 hours. B11E9 cells can not reduce U(VI) at either concentration.

- The gene disrupted is a cAMP binding protein, which is a catabolite gene activator and regulatory subunit of cAMP-dependent protein kinases
- The first down stream gene encodes a thioredoxin protein and the gene next to it encodes a thioredoxin reductase, trxB. Thioredoxin system is known to be important of keeping the oxidative level in the cytoplasm.

*trxB*

*CBP*

*thioredoxin*

## Loss of As(V) Tolerance.  

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

## Conclusions:

- We are in the opposite situation of most of you.  We have the mutants and we need to define a physiological function for genes.

## Genes involved in central intermediary metabolism

## Cell envelope biogenesis, outer membrane

## Nucleotide metabolism

- DNA replication recombination and repair

## Further study on mutant G11D5

- G11D5 is the other mutant lost partial U(VI) reduction test in the washed cell test
-  Thioredoxin gene 1 and 2 are the hypothetical protein with a thioredoxin domain gene separated by a transposase gene.
- Orthologs to this hypothetical protein are present in *D. vulgaris, Geobacter metallireducens* and *G. sulfurreducens* in Δ-proteo bacteria but they appear to be one gene except in G20.

*Thioredoxin1*

*Thioredoxin2*

*Transposase*

- 102bp                              30bp

## Screening mutant library

- _Screening mutant library_

## rRNA methylation investigation

## rRNA methylation investigation

## rRNA methylation investigation

- Real time PCR analysis -need an internal control to use as a reference.  Have been using 16s rRNA gene.
- Tested  *apsA* (Adenylyl-sulfate reductase, alpha subunit, VIMSS395577), *apsB *(Adenylyl-sulfate reductase, beta subunit, VIMSS395578) and also 16S rRNA.
- Total RNA isolated from U(VI) treated cultures as follows.  Before harvesting cells, culture must be centrifuged at 1,000g for 2 min and take the supernatant to get rid of U(IV). High speed, pellet must be washed twice with 30mM NaHCO3. Without doing so, will significantly reduce the total RNA isolation amount.

## rRNA methylation investigation

- Mutants G11F6 which can not grow with 2 mM U(VI) condition has one gene directly involved in rRNA methylation (dimethyladenosine transferase) (Ddes02002729) disrupted.
- Also mutant E11H7 which can not grow well has one RNA methyltranferase (Ddes02003228) related gene disrupted.
- So the question was raised as to the whether RNAs are methylated during U(VI) stress.