Jonas Weissmann Gaiarsa

1
Analysis of toxins of the hemagglutinin, RTX and
killer/antikiller type present in different
strains of the phytopathogenic bacteria Xylella
fastidiosa.
Rua do Matão, 277, lab. 153Depto. de Botânica,
Instituto de Biociências Universidade de São
PauloCaixa Postal 11461, CEP 05422-970, São
Paulo, SPPhone/FAX [phone]
Jonas Weissmann Gaiarsa jonaswg_at_gmail.com
Advisor Marie-Anne Van Sluys mavsluys_at_ib.usp.br
Project objectives The objective of this project
is to conduct a comparative analysis of toxins
present in the genome of different strains of the
bacteria Xylella fastidiosa. Specifically the
hemagglutinins, hemolysins/RTX and phage related
toxins. In silico studies as well as plant
systemic effects experiments will be carried out.
The following are guideline steps for this
undergraduation project. A. Identification of
number and type of toxins present in the genome
of the strains 9a5c (CVC) and Temecula1 (PD).
(Archieved in the 1st semester of 2005.) B. Study
of repetitive patterns between toxins identified
in each class. (Archieved in the 1st semester of
2005.) C. Comparative analisys of the genomes of
plant associated bacteria, using the databases
available, aiming to identify which genomes
contains similar toxins and if the genomic
context of these toxins are the same. This step
will be archieved with the help of comparative
genomics tools such as the System for Automated
Bacterial Integrated Annotation
(S.A.B.I.A).(Postponed) D. These information will
be used in the selection of candidates for
construction of plasmids appropriate to
heterologus expression. With the obtention of
these toxins a plant systemic effect experiment
will be done. (This step was initiated in the
1stº semester of 2006.)
Hemagglutinin repetitive patterns
Future work
Agroinfiltration
ORFs
ORFs Promoter
Growth measurment
XUUT1491B09
Saturated E.coli cultures containing Xf. toxins
display reduced growth in overnight cultures
PD0986 Coords Chromosome (1182764-1181583) (1185
bp / 395 aa) hemagglutinin-like secreted protein
(biofilm) pspA
XUUT1678A02
Clone retrieval preparations
pUC18
...
in
Inoculation with E.coli clones (Nt.)
7 days 12 days 21 days
7 days 12 days 21 days

• References
• Simpson A.J. et al. The genome sequence of the
  plant pathogen Xylella fastidiosa Nature, Vol.
  406, 13 July 2000
• Van Sluys M.A. et al Comparative Genomic
  Analysis of Plant-Associated Bacteria Annual
  Review Phytopathology 2002. 40169-89
• Van Sluys M.A. et al. Comparative Analyses of
  the Complete Genome Sequences of Pierce's Disease
  and Citrus Variegated Chlorosis Strains of
  Xylella fastidiosa Journal of Bacteriology, Feb.
  2003, p. 1018-1026
• Ishibashi Y. et al Invasion of human respiratory
  epithelial cells by Bordetella pertussis
  Possible role for a filamentous hemagglutinin
  Arg-Gly-Asp sequence and alpha5beta1 integrin
  Microbial Pathogenesis 2001 30 279-288
• Klee S.R. et al Molecular and Biological
  Analysis of Eight Genetic Islands That
  Distinguish Neisseria meningitidis from the
  Closely Related Pathogen Neisseria gonorrhoeae
  Infection and Immunity, Apr. 2000, p. 2082-2095
• Rojas C. M. et al HecA, a member of a class of
  adhesins produced by diverse pathogenic bacteria,
  contributes to the attachment, aggregation,
  epidermal cell killing, and virulence phenotypes
  of Erwinia chrysanthemi EC16 on Nicotiniana
  clevelandii seedlings PNAS October 1, 2002, Vol.
  99, no. 20, 13142-13147
• I.Jonassen, J.F.Collins, D.G.Higgins.
• Finding flexible patterns in unaligned protein
  sequences Protein Science 4, 1587-1595 (1995)I.
  Jonassen
• Efficient discovery of conserved patterns using a
  pattern graph CABIOS 13, 509-522 (1997).
• Higgins D., Thompson J., Gibson T.Thompson J.D.,
  Higgins D.G., Gibson T.J. (1994). CLUSTAL W
  improving the sensitivity of progressive multiple
  sequence alignment through sequence weighting,
  position-specific gap penalties and weight matrix
  choice. Nucleic Acids Res. 224673-4680.
• J. Sambrook, E.F. Fritsch, T. Maniatis Molecular
  Cloning a laboratory manual 2nd edition 1987
  Cold Spring Harbor Laboratory Press
• Edgar, Robert C. (2004), MUSCLE multiple
  sequence alignment with high accuracy and high
  throughput, Nucleic Acids Research 32(5),
  1792-97.
• "A Contig Assembly Program Based on Sensitive
  Detection of Fragment Overlaps" (submitted to
  Genomics, 1991). Xiaoqiu Huang. Department of
  Computer Science. Michigan Technological
  University. Houghton, MI 49931. E-mail
  huang_at_cs.mtu.edu
• Ewing B, Green P Basecalling of automated
  sequencer traces using phred. II. Error
  probabilities. Genome Research 8186-194 (1998).
• Ewing B, Hillier L, Wendl M, and Green P
  Basecalling of automated sequencer traces using
  phred. I. Accuracy assessment. Genome Research
  8175-185 (1998).
• Gordon, David. "Viewing and Editing Assembled
  Sequences Using Consed", in Current Protocols in
  Bioinformatics, A. D. Baxevanis and D. B.
  Davison, eds, New York John Wiley Co., 2004,
  11.2.1-11.2.43.
• Gordon D, Desmarais C, and Green P Automated
  finishing with Autofinish. Genome Res 11614-625
  (2001).
• Gordon D, Abajian C, and Green P Consed a
  graphical tool for sequence finishing. Genome
  Research 8195-202 (1998).

• Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J.
  Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and
  H. A. Erlich. "Primer-Directed Enzymatic
  Amplification of DNA with a Thermostable DNA
  Polymerase." Science 239 (1988) 487-491.
• A new plasmid-encoded proteic killer gene system
  cloning, sequencing, and analyzing hig locus of
  plasmid Rts1. Tian QB, Ohnishi M, Tabuchi A,
  Terawaki Y Biochem. Biophys. Res. Commun.
  1996220280-284.
• Steve Rozen, Helen J. Skaletsky (1996, 1997,
  1998) Primer3. Code available athttp//www-genom
  e.wi.mit.edu/genome_software/other/primer3.html
  http//www.idtdna.com/Scitools/Applications/Primer
  quest/
• http//www.cshprotocols.org/
• http//www.ncbi.nlm.nih.gov/Taxonomy/Browser/
• Altschul, S.F., Gish, W., Miller, W., Myers, E.W.
  Lipman, D.J. (1990) "Basic local alignment
  search tool." J. Mol. Biol. 215403-410.
• Alkema, W. B. L., Lenhard, B. Wasserman, W. W.
  (2004). Regulog analysis detection of conserved
  regulatory networks across bacteria application
  to Staphylococcus aureus. Genome Res. 14,
  1362-1373.
• Schneider, T. D., Stormo, G. D. Gold, L.
  (1986). Information content of binding sites on
  nucleotide sequences. J. Mol. Biol. 188, 415-431.
• Schreiber, M. Brown C. (2002). Compensation for
  nucleotide bias in a genome by representation as
  a discrete channel with noise. Bioinformatics 18,
  507-512.
• Guilhabert MR, Kirkpatrick BC. Identification of
  Xylella fastidiosa antivirulence genes
  hemagglutinin adhesins contribute a biofilm
  maturation to X. fastidios and colonization and
  attenuate virulence. Mol Plant Microbe Interact.
  2005 Aug18(8)856-68.
• Alouf, J., Popoff, M. The comprehensive
  sourcebook of bacterial protein toxins, 2 Ed.