Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

5A/2004G140

Structure of transcription factors responsible for Pseudomonas aeruginosa
pathogenicity
Yasuo SHIRAKIHARA* 1, Aya SRATORI 1, Hiroshi ITO 1, Hironori ARAMAKI2
1National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
2
Daiichi College of Pharmaceutical Sciences, Minami-ku, Fukoka, 815-8511, Japan
Introduction
In bacteria, transcription regulation factors work, forexample, as many as 300 in E. coli, depending upon thecell growth phase and the living environment byregulating transcription of its target gene(s). The
transcription factors contribute to a complicated networkof regulation of transcription activity in a cell together
with various RNA polymerase +-subunits.

was an exception, ands was purified with Q-sepharosehigh performance, Butyl-toyopearl and hydroxyapatitecolumns, because the protein did not bind to a Ni-NTA
column. A gel filtration analysis and the subsequent xray crystal analysis showed that the PhzR molecules forma tetramer, suggesting that the His-tag attached Cterminus of the molecule may be buried in the tetramer
thus explaining no attachment of PhzR to Ni-NTA.

We have tried to do a sweeping structural determinationof E. coli transcription factors by x-ray crystallography,
combining the recent progress in x-ray structure analysis
tools and a recent collection of over 65 purified E. colitranscription factors. The study aims to reveal the overall picture of transcription factors in structural terms.
However, we encountered difficulties in this approach.
The problem was their inability to crystallize. This may
be probably due to their structural flexibility or
unsatisfactory quality of preparations obtained by a single
step Ni-NTA column chromatography.

The preparations were then subjected to crystallization
study. All proteins gave signs of crystals in the screeningstage, however, the diffraction grade crystals wereobtained only from PhzR so far. PhzR crystals wereobtained from polyethylene glycol at pH 4.7, and with atypical size of 0.2 x 0.2 x 0.05 mm. The previous MAD
data collected from HgCl2 derivative elsewhere were notgood enough for structure determination.
In theexperiment at BL5A, Mosflm strategy option was
successfully applied, which, we believe, allowed muchbetter data (less prone to radiation damage) than before tobe collected. MAD data were collected from HgCl2
derivative. The effective resolution of the data set wasabout 3.5 A resolution. The native data for PhrzR werealso collected. Those data sets were processed with
MOSFLM and CCP4 program suite. The unit cell is
a=74.4A b=242.9 A c=129.3 A (space group C222), andits asymmetric unit contains 4 molecules of PhzR. MAD
analysis using programs SOLVE and Sharp revealed anovel mode of tetramer assembly of the PhzR, andshowed an electron density map which showed clear
helical densities in some part.

As a move to a differerent line of study, we have recently
included in the structural study transcription factors (PtxR,PtxS, PhzR, and PA3547) that are responsible for thepathogenicity of Pseudomonas aeruginosa. Pseudomonas
aeruginosa has nearly twice as many transcription
regulation factors as E. coli in spite of similar genome
sizes, which benefits the bacterium living inenvironments varying from wild water to human body.
The well known opportunistic pathogenecity of
Pseudomonas aeruginosa may come out of thisbackground. Understanding of functional mechanisms ofthese pathogen-related transcription factors in terms ofstructure may lead to a better guard against the pathogens.
We have started cloning, expressing, and crystallization
studies of these Pseudomonas aeruginosa transcriptionfactors.
Results
An Over expression system was constructed byincorporating the provided expression plasmids, whichwere designed to have 6 His-tags at The C-terminus, into
host cells (BL21 (DE3), B834 (DE3), JM109 (DE3)) andby examining which conditions, characterized bytemperature (37°C, 25°C) and inducer presence (plus,minus), produce the target protein in a soluble fraction.
Fortunately good conditions were found for all theproteins. Three proteins out of the four were purified by
the Ni-NTA column and an extra column, of which resin
was chosen empirically depending upon the protein. PhzR

After the diffraction experiment, we had a chance tocheck the origin of the expressed PhzR. This was led by alesson from a failing expression experiment of a plantcell-death related protein SAG12 (molecular mass ofabout 30k) where SAG12 was not expressed but
lactamase from a vector was instead expressed. Cterminus amino acid sequence of PhzR (26K) expressedin our hands was examined. The sequence corresponded
to that of C terminus residues of lactamase from
Staphylococcus aureus, not to that of PhzR. There is astrong suspect that our PhzR crystals were not genuine.
Diffraction data have been re-examined in this respect.
* yshiraki@lab.nig.ac.jp

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