Signaturetagged mutagenesis for identification of Desulfovibrio alaskensis G20 sediment survival gen

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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

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Background
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.
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Goals

• Identify genes critical for survival in natural
  systems.

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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)
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Desulfovibrio alaskensis G20
Sulfate- Reducer Nalidixic acid resistant 4.1
mb, 3598 candidate genes Doubling time is 4.6
hours
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Generate 96 random tags and ligate to transposon
5-ctaggtacctacaacctcaagctt-NK20-aagcttggttagaat
gggtaccatg-3
KpnI
KpnI
N A,T,C or G K T or G
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Overview of signature tagged mutagenesis (STM)
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Input pool
Tag B11
Recovered pool
Missing tag B11
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Growth of potential non-survival mutants in
sediment
Time (day)
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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

Arbitrary primer 1
Tn 10
External Tn primer
Internal primer 2
Internal Tn primer
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(No Transcript)
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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)

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Belong to ISL3 family. Involved in the
transposition of the insertion sequence. It has
12 paralogs in G20 genome, and 99 identities to
VIMSS395038 and VIMSS395351.
49.6 homolog to D. vulgaris ISD1. It has 7
paralogs in G20 genome, and 100 identity to
VIMSS392855.
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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 sacB under sucrose
  stress in D. vulgaris (ISD1)

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Signal transduction modules
http//www.asm.org/news/index.asp?bid36163
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Histidine Kinase

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

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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)

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Chemotaxis

• Involves a cascade of events starting with the
  MCP and leading to motility towards or away from
  something.
• 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

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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.

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Response to mutagens

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

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Protection from a Severe Mutagenic Event The SOS
response
Cited from Genetics 148 15991610 (April, 1998)
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Mutant B12(pF11)

• Mutagenesis is a survival issue for cells in
  aquifer sediment.

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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.

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Mutants involved in Respiration
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Mechanisms of U(VI) reduction
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Mechanisms are Still not clear

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

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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.

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24 mutants were divided into four categories
based on their sensitivity to U(VI)
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24 mutants were divided into four categories
based on their sensitivity to U(VI)
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Mutant strains gene information
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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.

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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 cant reduce
  U(VI) might play important role in U(VI)
  reduction.

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Further study on mutant B11E9

• 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.

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

trxB
CBP
thioredoxin
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Loss of As(V) Tolerance.

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

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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.

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Genes involved in central intermediary metabolism
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Cell envelope biogenesis, outer membrane
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Nucleotide metabolism
DNA replication recombination and repair
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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 ?-proteo bacteria but
  they appear to be one gene except in G20.

102bp 30bp
Thioredoxin1
Thioredoxin2
Transposase
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Screening mutant library
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rRNA methylation investigation
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rRNA methylation investigation
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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.

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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.