KOOPMANN, BIRGER; NOLLENBURG, MATTHIAS and RUDOLPH, KLAUS

Institut fUr Pflanzenpathologie und Pflanzenschutz, Universitat Gottingen, Grisebachstr.6, 0-37077

Gotlingen

ISOLATION AND CHARACTERIZATION OF THE algD GENE OF

PSEUDOMONAS SYRINGAE PV. PHASEOLICOLA S2-1

ABSTRACT

The a/gO gene was amplified from total DNA of P. aeruginosa by the peR technique.
The gene was cloned and used as a probe in hybridization studies. We successfully
screened a Pseudomonas syringae pv. phaseo/ico/a (PSP) genomic library for the
corresponding gene which was sequenced. When the amino acid sequence of a/gO
from PSP was deduced and aligned with the deduced sequence of the
corresponding gene in P. aeruginosa, a high degree of similarity (92%) and identity
(78%) was found.

INTRODUCTION

Like most fluorescent pseudomonads Pseudomonas syringae pv. phaseo/ico/a (PSP)

cells are covered by loose slime consisting mainly of the extracellular
polysaccharides (EPS) levan (see HETTWER et a!., this volume) and alginate. It is
assumed that alginate produced by phytopathogenic pseudomonads is a virulence
factor providing the encapsulated bacteria with a protective environment and being
responsible for the water-soaked symptoms in infected plant tissue (GROSS &
RUDOLPH 1987, RUDOLPH et aL 1994). Alginate synthesis in P. aeruginosa (PAE)
involves at least seven enzymatic steps (DERETIC et aL 1987b) and, based on
hybridization studies, is likely to follow a similar pathway in P. syringae pathovars
known to produce this compound (FIALHO et a!. 1990). GDP-mannose
dehydrogenase (GMD) catalyzing the fourth biosynthetic step, i.e. the oxidation of
GDP-mannose to GDP-mannuronic acid, and encoded by a/gO is thought to be
specific for alginate biosynthesis (DERETIC et a!. 1987b). It was the goal of this study
to isolate the respective gene from the bean pathogen PSP and to investigate its
presence in other plant pathogenic P. syringae pathovars and fluorescent
pseudomonads not examined so far.
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K. Rudolph et al. (eds.), Pseudomonas Syringae Pathovars and Related Pathogens
© Kluwer Academic Publishers 1997
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MATERIAL AND METHODS

Bacterial strains originated from the collection "Gettinger Sammlung

phytopathogener Bakterien" (GSPB). Plasm ids were amplified in E coli recipient
strain DH5a. Cultivation of bacteria. E coli was grown in Terrific Broth at 37°C
OIN with the appropriate antibiotics (SAMBROOK et al. 1989). Pseudomonads and
other bacteria were grown in Rhodes medium (RHODES 1959) at 25°C for up to two
days. Isolation of DNA. Total DNA was isolated as described by AUSUBEL et al.
(1988). Plasmid DNA was prepared according to BIRNBOIM & DOL Y (1979). DNA from
agarose gels was prepared with the "Gene clean" kit (Bi0101, La Jolla, Ca., USA).
Construction of a gene library. Total DNA of PSP Sr1 was partially digested by
Hind"!. Fragments were separated in a sucrose gradient and those> 14 kb were
pooled and cloned into the cosmid vector pVK102 (KNAUF & NESTER 1982).
Generation of a DNA-probe. PCR primers were deduced from the a/gO DNA
sequence data published by DERETIC et al. (1987a). The a/gO sequence was
amplified from total DNA of PAE using a "touch down" protocol. The fragment was
cloned into pBluescript SK- (Stratagene, San Diego, USA). A probe was generated
with DIG-dUTP in a vector PCR procedure as previously described (KOOPMANN et al.
1994). Enzymatic DNA manipulations. DNA was digested according to the
manufacturer's recommendations (Fermentas, St. Leon Roth, Germany) with 5 fold
excess of enzyme. Creation of blunt ends, dephosphorylation and ligations were
done according to SAMBROOK et al. (1989). DNA was ligated into pBluescript-SK-.
Electrophoresis and blotting. DNA was separated in agarose gels run in TAE
buffer at 1V/cm, transferred by vacuum or capillary blotting (SAMBROOK et al. 1989) to
Hybond N+ membranes (Amersham Buchler, Braunschweig, Germany).
Hybridization and detection. Blots were hybridized at 42°C with 50% formamide
using the DIG-system of Boehringer Mannheim (Germany). Hybridizations and
detections were done according to the manufacturer's recommendations.
Insertional mutagenesis and DNA sequencing. Plasmid DNA was randomly
linearised with DNAsel in the presence of 1,5 mM Mn2+. Blunt ends were generated
by DNA polymerase!. The interposon Q (PRENTKI & KRISCH 1984) was ligated in the
presence of 15% PEG. After digestion with EstEll the monomers were circularised
and transformed to E. coli DH5a. The insertion locations were screened. Clones
were sequenced with the T7 -DNA-Sequencing kit of Pharmacia using labelled 35 S_
adATP (Hartmann, Braunschweig, Germany) using M13 (-20, reverse) and Q
primers.

RESULTS

The a/gO DNA probe was generated from total DNA of Pseudomonas aeruginosa by
PCR amplification. Oligonucleotides were designed according to the a/gO sequence
data of Pseudomonas aeruginosa (DERETIC et al. 1987a). The "touch down" PCR
protocol resulted in a fragment of the expected size (1.3 kb) which was cloned into
pBluescript SK-; its identity was confirmed by partial sequencing. We found 98%
identity between the 200 basepairs of the 5' end determined and the a/gO sequence