Seveno, Nadine Antoinette (2000) Phenazine antibiotic production in liquid culture, on surface agar, and on plant roots by Pseudomonas aureofaciens PGS12. PhD thesis, University of Warwick.

Preview

Text WRAP_thesis_Seveno_2000.pdf - Submitted Version Download (19Mb) | Preview

Request Changes to record.

Abstract

The growth of P. aureofaciens PGS12 was followed in nutrient broth, on nutrient agar,
and on plant roots by monitoring cell numbers, the production of the auto inducer N-acyl-
L-hexanoyl homo serine lactone (HHL), and the antibiotic phenazine-l-carboxylic acid
(PCA). In nutrient broth, as the growth rate declined, HHL synthesis increased rapidly.
Up to 38% more HHL was produced compared to the increase in cell numbers indicating
that transcription of the phenazine operon was auto induced. As the bacterial culture
entered stationary phase, HHL concentration declined rapidly while PCA concentration
continued to accumulate at a high rate. In stationary phase, HHL concentration continued
to decline while PCA accumulated slowly. A promoterless mini-Tn5-luxAB transposon
was used to generate isogenic strains of P. aureofaciens PGSI2. Strain BI03 was shown
to have the luxAB reporter gene inserted in the phzB gene within the phenazine operon.
Phenazine transcriptional activity (bioluminescence) was compared with the light output
from a constitutive reporter, strain 117. Levels and pattern of bioluminescence from strain
B 1 03 followed closely HHL production and indicated that gene expression was maximal
in transition phase and silenced in stationary phase. PCA production continued in
stationary phase suggesting that the protein products of the phenazine operon were
maintained in the cell after down regulation. The induction of the phenazine operon
started in nutrient broth when cell density was ca. 2x 108 cells mrl and HHL had
accumulated to a threshold concentration of 0.63x 1 0-
6
± O.3x 1 0-
6
ng celrl.
± On NA, cells were in a transition phase of growth for at least 9 h. The cell density
was 55 to 75 times higher within a colony than in liquid culture. The maximal production
of HHL and light output per calculated equivalent volumes were also between 50 and 65
times higher on NA than in broth. The maximal light output and maximal HHL
accumulation per cell were similar on both media. Therefore, the increased levels on NA
may be mainly due to a higher cell number in the colony. The production of the antibiotic
PCA per cell was ca. 7 times higher in a colony than in NB, and the production in an
equivalent volume was ca. 360 times higher in a colony. Therefore, the higher PCA
concentration in the colony cannot be explained solely on the basis of an increase in cell
density. The auto inducer concentration remained high within a colony for a prolonged
period of time compared to the burst seen in NB. A high concentration of HHL per cell
for a longer period of time may have sustained the greater production and accumulation
of PCA in the colony. Similarly the transcriptional activity of the phenazine operon, as
reflected by phzB::luxAB expression in strain BI03, remained maximal during this time.
In contrast to laboratory culture studies, in all the experiments where P.
aureofaciens PGS 12 was inoculated onto roots, neither HHL nor PCA was detected,
although the bacteria colonised the bean roots and wheat seedlings efficiently. On wheat
seedlings, the transcriptional activity from both reporter strains decreased during the
experiment. On bean roots, bioluminescence per cell from strains 117 and B 1 03
increased 25-fold during the first 3 days and the ratio of bioluminescence from strains
B 1 03 over 117 indicated an up to 5 times greater transcriptional activity from strain B 1 03
than from strain 117. The minimum levels for the detection of HHL and PCA were low.
Therefore, either these compounds were produced in minute amounts, or HHL and
phenazine were degraded or adsorbed onto the plant material lowering their levels below
the detection limit.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QH Natural history > QH301 Biology
Library of Congress Subject Headings (LCSH):	Phenazine , Antibiotics, Pseudomonas , Pseudomonas aureofaciens
Official Date:	September 2000
Dates:	Date Event September 2000 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Wellington, E. M. H. (Elizabeth M. H.), 1954- ; Morgan, J. Alun W.
Extent:	xii, 195 leaves : illustrations
Language:	eng

Request changes or add full text files to a record

Repository staff actions (login required)

View Item

Downloads