view release on metacpan or  search on metacpan

t/data/utfs/10049847.utf8  view on Meta::CPAN

FIG. 5
FIG. 5
Relationship of the amino acid sequence deduced from ORF4 from
Pseudomonas sp. strain HR199 and four representative regulatory
proteins from different sources. The program and parameters for the
construction of the dendrogram were the same as for Fig. (more ...)
FIG. 6
FIG. 6
Relationship of the amino acid sequence deduced from ORF5 from
Pseudomonas sp. strain HR199 with 4-hydroxybenzoate 3-hydroxylases
(PobA) from other sources. The program and parameters for the
construction of the dendrogram were the same as for Fig. 
(more ...)
Analysis of mutant SK6169, which exhibited a defective 3,4-PCD but was
still able to grow on protocatechuate.
The results of the genetic analysis of mutant SK6169 revealed a defect
in the β subunit of 3,4-PCD. In consequence, this mutant lacked 3,4-PCD
activity (Table (Table2).2). Nevertheless, it was able to grow on
eugenol, ferulate, vanillate, or protocatechuate as the sole carbon
source. To investigate this obscure phenotype in more detail, the
mutant was grown in the presence of vanillin, protocatechuate, or
vanillin plus gluconate in liquid MM. These investigations revealed
that mutant SK6169 grew as well as the wild type on vanillin plus
gluconate and was still able to grow on vanillin without an additional
carbon source. However, in comparison to the wild type, the mutant grew
only after a long lag phase of about 20 h. In contrast to the analysis
on MM agar plates, the growth of the mutant in liquid MM with
protocatechuate as the sole carbon source was reduced compared to the
wild type. As revealed by HPLC analysis of culture supernatants,
vanillin was converted to vanillate and protocatechuate by this mutant,
which was further metabolized. In comparison to the wild type, the
occurrence of these intermediates was retarded. To exclude the
appearance of revertants, aliquots of the cultures were spread on
vanillin- or protocatechuate-containing MM agar plates, respectively.
No growth of revertants on vanillin agar plates could be observed, and
there was no difference in the growth of the wild type and the mutant
on protocatechuate-containing agar plates.
Crude extracts obtained from the cells of the aforementioned cultures
were investigated for 3,4-PCD, 4,5-PCD, and 2,3-PCD activities. None of
these activities were detectable in the extracts of the mutant, whereas
extracts of the wild type exhibited 3,4-PCD activity (Table
(Table2).2). The way in which mutant SK6169 metabolized
protocatechuate remained unknown.
Analysis of mutants SK6184 and SK6190, which were also impaired in the
catabolism of vanillin.
During the nitrosoguanidine mutagenesis, mutants SK6184 and SK6190,
which had lost their ability to grow on vanillin, vanillate, and
ferulate but were still able to grow on eugenol or protocatechuate,
were isolated. These mutants were not complemented by the hybrid
cosmids pV372 and pV801 after conjugative reception. When these mutants
were cultivated in MM with eugenol, vanillin, or protocatechuate as the
sole carbon source, the substrates were completely converted to an
unknown substance. To identify this product, we isolated it from the
culture supernatant of mutant SK6190 grown in MM with eugenol. The
light brownish crystals we obtained exhibited a blurred melting point
of 114 to 150°C. The isolated substance was soluble in HO, methanol,
ethyl acetate, and diethylether. Its mass spectrum showed a molecular
ion at m/z 186. From these results along with the data obtained from
1H NMR (four protons) and 13C NMR (seven carbon atoms) spectra and
the infrared spectrum (OH group[s], three carbonyl bands), the
molecular formula was found to be CHO. The 1H NMR spectrum of
the substance showed an ABXZ spin system (J[AB] = 16.6 Hz, J[AX] = 8.0
Hz, J[BX] = 3.2 Hz) at 2.680, 3.207, and 5.566 ppm, with an allylic
coupling (2.1 Hz) between the X proton and the olefinic proton at 6.678
ppm. These findings established the structure fragments shown in Fig.
Fig.7.7. The 13C NMR spectrum showed seven signals at 173.1,
172.6, 163.7, 160.1, 127.0 ( [pc-E00C.gif] CH) 80.6 (CH), and 37.9
(CH) ppm. Correlations between 1H and 13C extracted from the HMQC
spectrum are shown in Fig. Fig.7.7. Because the protons at C-3
are diastereotopic, an asymmetrically substituted carbon atom must be
in the direct neighborhood. C-1 must be part of a double bond with a
quaternary carbon atom (160.1 ppm) as a partner. H-1 must be in the
β-position to a carbonyl function because of its high shift value. The
shift value of 80.6 ppm for C-2 indicates that it must be bonded to an
oxygen atom. From these data, the chemical structure of the isolated
substance could be assigned to 3-carboxy muconolactone (Fig.
(Fig.7).7). Furthermore, the obtained spectral data are in good
agreement with the values published by Kirby et al. (21).
FIG. 7
FIG. 7
Identification of the substance accumulated by mutants SK6184 and
SK6190 during growth on eugenol. (A) Structure fragments established by
NMR spectroscopy: 1H-NMR data (CD3OD), δ [ppm], J [Hz]. 13C NMR shift
values (more ...)
Thus, the mutants SK6184 and SK6190 accumulated 3-carboxy muconolactone
during growth on eugenol, which is most probably due to a lactonization
of 3-carboxy-cis,cis-muconate by a syn absolute stereochemical course.

DISCUSSION


A 5.8-kbp EcoRI fragment cloned from genomic DNA of Pseudomonas sp.
strain HR199 was found to encode proteins which are essentially
involved in the degradation of vanillin. A “vanillin-negative” mutant
which lacked 3,4-PCD activity was phenotypically complemented by the
structural gene pcaH, encoding the β subunit of 3,4-PCD. The structural
gene of the corresponding α subunit, pcaG, was localized downstream of
pcaH. The amino acid sequences deduced from pcaH and pcaG showed
extended homology to the 3,4-PCD from P. putida (11). pcaHG were
expressed in E. coli driven by the lac promoter of pBluescript SK-.
The 3,4-PCD activity was 13-fold higher than that obtained by the
expression of pcaHG of P. putida in E. coli (11). Since the G+C
contents and the codon bias of the pcaHG genes of Pseudomonas sp.
strain HR199 and P. putida were very similar, it is unlikely that the
weak expression of the P. putida genes in E. coli is due to these
properties, as concluded by Frazee et al. (11).
Upstream of pcaHG was an ORF which had the same orientation and whose
deduced amino acid sequence exhibited strong homology to the
transcriptional activator protein PcaQ from Agrobacterium tumefaciens
(27). The region of highest homology was located in the N-terminal
region as described by Viale et al. (47) for transcriptional
activator proteins belonging to the LysR family, comprising the
helix-turn-helix motif (20) for DNA binding.
In the immediate neighborhood of the pca genes were found two ORF that
exhibited high homologies to the pob genes responsible for
p-hydroxybenzoate metabolism. The first ORF exhibited high homology to
the PobR regulator protein of A. tumefaciens (28), which belongs
to transcriptional regulator proteins of the XylS/AraC type (14).
As also observed for the PobR regulator protein of A. tumefaciens
(28), no homology was obtained with the transcriptional regulator
proteins of the so-called PobR subfamily (8), which refers to PobR