STUDIES ON PIGMENT PRODUCTION BY PSEUDOMONAS

STUDIES ON PIGMENT PRODUCTION BY
PSEUDOMONAS AERUGINOSA
by
RALPH C. PORTER, B.S.
-~A THESIS
IN
MICROBIOLOGY
Submitted to the Graduate Faculty
of Texas Tech University in
Partial Fulfillment of
the Requirements for
the Degree of
MASTER OF SCIENCE
Approved
J^
,\
(\i\\-%^^,
\
Op. o?
ACKNOWLEDGEMENTS
I would like to express my sincere appreciation to
Dr. Lyle Kuhnley without whose guidance and assistance
this study V70uld not have been possible.
In addition,
I would like to thank Dr. Robert Rekers for his technical
assistance and Dr. C. L. Baugh and Dr. John Anderson for
their attention as committee members.
11
X
CONTENTS
ACKNOWLEDGEMENTS
ii
LIST OF TABLES
iv
LIST OF FIGURES
I.
II.
III.
IV.
V
INTRODUCTION
1
Literature survey
1
Thesis objectives
11
MATERIALS AND METHODS
12
Bacterial strains
12
Culture media
12
Phosphate assay procedure
14
Procedures of pigment purification
14
Pigment characterization
14
Shikimic acid assay procedure
17
RESULTS
-""^
Characteristics of pyocyanin
18
Culture conditions affecting
pyocyanin production
23
Other pigments produced by WT
30
Conditions affecting pigment
production by WT
40
Shikimic acid accumulation by WT and P"
46
DISCUSSION
48
LITERATURE CITED
55
•
•
•
111
LIST OF TABLES
Table
1. Properties of major Pseudomonas aeruginosa
pigments
2.
3.
4.
5.
Page
9
Characteristics of pigments produced by
wild-type Pseudomonas aeruginosa
19
Aerobic pigment production by wild-type
Pseudomonas aeruginosa
41
Pigment production under microaerophilic
conditions
45
Shikimic acid accumulated in preparations of
WT and P
47
IV
LIST OF FIGURES
Figure
Page
1.
The aromatic biosynthetic pathway
2.
Methods of pigment purification
3.
Infrared spectra of pyocyanin and
aeruginosin
4.
Absorption spectra of pyocyanin
5.
Influence of phosphate concentration
on pyocyanin production by WT
Influence on pyocyanin production by WT
of phosphate supplementation during
the course of culture incubation
Influence of phosphate and aromatic amino
acid supplementation on pyocyanin
production by WT
6.
7.
8.
9.
10.
6
16
22
25
27
29
32
Infrared spectra of pyoverdine and
chlorodeuteros
35
Absorption spectra of P. aeruginosa
pigments
38
Total pigment production by WT in variously
supplemented PsP cultures
44
CHAPTER I
INTRODUCTION
The ability of strains of Pseudomonas aeruginosa to
elaborate pigmented substances into their growth medium has
been known for some time.
In 186 3, Fordos reported experi-
menting with the deep blue pigment produced by P_. aeruginosa
(44).
He named the compound pyocyanin.
In 1929, Wrede and
Strack postulated the structure of pyocyanin (48) . They
determined the pigment to be composed of two attached subunits of n-methyl-1-hydroxyphenazine.
Michaelis later pro-
posed that the compound should be represented as simply
n-methyl-1-hydroxyphenazine (44).
The disclosure of the structure of pyocyanin was encountered with considerable interest.
It led to investiga-
tions entailing surveillance of occurrence of natural
phenazines and to investigations concerning the biosynthesis
of the phenazine nucleus.
Since the elucidation of the structure of pyocyanin,
the natural occurrence of several phenazines has been reported.
Twenty-one naturally occurring phenazines have been
found (16), All twenty-one are from microbial sources.
Various substituted phenazines are produced by myxobacteria
of the genus Sorangium, by actinomycetes of the genera
Streptomyces, Waksmania, and Microbispora, by a novel group
of Nocardiaceae, and by bacteria belonging to the genera
Brevibacterium and Pseudomonas (9, 15, 16, 17, 18, 25, 26,
32, 33, 34, 43, 45). However it was not until 1964 that it
became apparent that organisms other than species of
Pseudomonas had the capacity to synthesize the phenazine
ring (17). By this time five Pseudomonas pigments had been
reported and characterized (9, 23, 25, 27, 32, 44).
The acquisition of information concerning the biosynthesis of the phenazine nucleus has almost exclusively been
through studies of pyocyanin production by Pseudomonas
aeruginosa.
The development of suitable culture media was
prerequisite to any work in this area.
Therefore, initial
studies were oriented toward determining the culture conditions affecting pigment production and developing synthetic
media allowing sufficient pyocyanin elaboration to allow
studies of pyocyanin biosynthesis.
Burton et al determined the cultural conditions which
appeared most favorable for pyocyanin production and devised
a synthetic medium supporting its synthesis (5, 6 ) , A number of amino acids and combinations of amino acids when
utilized in conjunction with glycerol supported pigment production.
The ions Mg^"^, SO^^",
K"*", P O ^ ^ " ,
reported essential for pigment formation.
and Fe "^ were
Modifications of
the basic medium presented by Burton have been utilized
almost exclusively, and only recently have Engledew and
Campbell devised a medium which differs markedly and which
is utilizeable in studies of pyocyanin biosynthesis (29),
From the work of Ingledew and Campbell the necessity of
3PO.
for pigment production becomes questionable.
Their
resuspension medium was devoid of phosphate and yet allowed
the production of large concentrations of pyocyanin.
Upon reviewing the work of Burton, Frank and DeMoss
concluded that those amino acid substrates which support
pigment synthesis would, without exception, support the
growth of £. aeruginosa (14). They found a close relationship between growth and pigment production.
This relation-
ship was manifest also in the ability of inhibitors of
growth to inhibit pyocyanin formation.
There have been two reports in which resting cells
have been utilized to study the production of pyocyanin.
Halpern et al reported that up to 200 ug/ml of chloramphenicol did not inhibit the production of pyocyanin by
a nonproliferating suspension of P. aeruginosa (22). In an
earlier article Grossowicz et al reported that glutamic
acid and some related compounds would support the production
of pigment in resting cultures (21). However, not more
than 2% of the glutamic acid was converted to pyocyanin.
Due to the low yield of final product realized in these
studies, it was apparent that resting cell studies would
reveal little useful information.
This inability to separate pyocyanin production from
growth of P^. aeruginosa was the major limitation in determining probable precursors to the phenazine nucleus.
Those
compounds shown to be involved in pyocyanin biosynthesis
have been excellent substrate for the growth of P^.
aeruginosa.
As a result, compounds added to the medium as
possible precursors of pyocyanin are used rapidly for energy
production and cell synthesis and are not channeled specifically toward pigment production.
Blackwood and Neish confronted the problem by performing isotopic label incorporation studies (3). Labelled
14
substrates were added to basal medium and
C label incor-
poration into cell mass, respiratory carbon dioxide, and
pyocyanin were determined.
By comparing the ratio of label
occurring in pyocyanin to that occurring in cell mass and
respiratory carbon dioxide they were able to determine which
substrates supported pyocyanin production.
Millican and MacDonald independently reported that two
intermediates along the aromatic amino acid biosynthetic
pathway probably were involved in pyocyanin biosynthesis
(36, 41). The proposed aromatic biosynthetic pathway is
shown in Fig. 1,
shikimic acid.
The compounds were quinic acid and
In order to evaluate the possible role of
these two compounds in pyocyanin production, a mutant strain
of P. aeruginosa which was unable to utilize shikimic acid
Fig. 1.—The aromatic biosynthetic pathway
PEP denotes phosphoenolpyruvate; E-4-P, erythrose-4-phosphate; DAHP, 3-deoxy-D-arabino-heptulasonate-7-phosphate; 5-DHQ, 5-dehyroquinic
acid; 5-DHS, 5-dehydro shikimic acid; 3,4-DHB,
3,4-dihydrobenzoic acid; S-5-P, 5-phosphoshikimic acid; 3-EPS-5-P, 3-enolpyruvyl-5-phosphoshikimic acid.
QUINIC
HO^^COO
COO"
(i^'''^^^
3,4
DBH
PEP
COO"
CO-P
CHg
V
CHO
CHOH
CHOH
CHO-P
2
E -4- P
DEGRADATION
COO"
c=o
CH-j
' HOCH^
CHOH •«
CHOH
CH 0-P
DAHP
COO"
COO"
COO"
CHORISMiC
pOOH
QHNHg
CHo
, * = * ^
<r
TRYPTOPHAN
ANTHRANILIC
PHEPHENATE
OH
HENt'LPYRU)/ftTE
OH
4-HYDROXY
PHENYLPYRUVATE
TYROSINE
or quinic acid for growth was isolated.
Through label in-
corporation studies performed with this mutant it was found
that 85-90% of the carbon atoms of pyocyanin could come
from shikimic acid, while quinic acid could provide as much
as 4 0% of the carbon in the dye molecule (28). Podojil and
Gerber reported experiments dealing with the production of
iodinin (1,6-phenazinediol-5, 10-dioxide) by Brevibacterium
iodinum which indicated that of eleven substrates tested,
shikimic acid appeared to be the most likely precursor to
the dye molecule (4 3).
Levitch and Stadtman also reported
the incorporation of labelled shikimic acid into phenazine1-carboxylic acid produced by Pseudomonas aureofaciens (34).
Other intermediates of aromatic biosynthesis may serve
as precursors of phenazine pigments.
Carter and Richards
have indicated that one ring, at least, of chloraraphin
(phenazine-1-carboxamide) appeared to originate from
anthranilic acid (7). Although this does indicate another
possible precursor, it is an additional case of aromatic
biosynthesis proceeding via the shikimic acid pathway.
The
possibility remains that the basic phenazine nucleus may be
produced from more than one precursor, the pigment being
produced depending on the precursor involved and ultimately
upon the culture conditions.
Complete understanding of the biosynthesis of the phenazine ring requires continued study.
Further investigations
8
are required on all organisms exhibiting the ability to produce phenazine pigments.
However, species of Pseudomonas
continue to be the preferred organisms of study due to the
ease with which they are cultured and the large quantities
of pigment produced.
Pseudomonas species are versatile in
respect to phenazine pigment production.
Four species of
Pseudomonas, P. chlororaphis, P. iodinum, P. aureofaciens,
and P. aeruginosa, have exhibited the ability to synthesize
fourteen of the twenty-one naturally occurring phenazines
(17, 18, 40). Half of these have been found to be produced
by strains of P_, aeruginosa (8, 25, 27, 40). The properties
of the major P. aeruginosa pigments are shov/n in Table 1.
An additional pigment reported by Meader and designated as
pyoverdine or fluoresin may be a phenazine (12, 31, 39).
The emperical formula of this fluorescent pigment has been
reported to be C.H O2N (46).
The simultaneous production of phenazine pigments by
a single strain of a species of Pseudomonas is known.
The
production of two phenazine derivatives, phenazine-1-carboxylic acid and another closely related compound, by a
strain of P. aureofaciens has been reported (32). Varying
proportions of each pigment could be obtained depending on
the composition of the growth medium and on external conditions.
Pyocyanin, phenazine-1-carboxylic acid, and oxy-
chlororaphine may be produced simultaneously by P,
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11
aeruginosa (8), Conditions favoring the production of
individual pigments in this case as yet have not been reported.
The objective of the present study is to investigate
the conditions affecting the production of a number of pigments produced by a single strain of Pseudomonas aeruginosa
and to determine the conditions most favorable for the production of each.
Such information may be of value in eval-
uating possible precursors of the phenazine nucleus.
CHAPTER II
MATERIALS AND METHODS
Two strains of Pseudomonas aeruginosa were utilized in
these studies.
The principal strain was a wild-type strain
isolated from contaminated eggs.
The second was a mutant
strain lacking the capacity to produce pyocyanin.
The
mutant was derived from the wild-type by a procedure
described by Glover, utilizing N-methyl-N'-:nitro-N-nitrosoguanidine to increase the frequency of mutation (19). Subquently WT and P" will be used to designate the wild-type
organism and the mutant strain respectively.
The basal medium used in these studies was a modification of Pseudomonas P medium (Difco).
the modified medium is as follows:
The composition of
0.2% DLi-alanine, 1.0%
sodium citrate, 0.86% potassium sulfate, 0.14% potassium
chloride, and 0.14% magnesium sulfate.
The designation PsP
will be used to indicate medium of this basic composition
and any addibions will be noted, i.e., PsP+<0.5% K^HPO. .
Washed cells from PsP cultures provided inocula.
Growth does not occur in unsupplemented PsP.
Sufficient
cells were obtained through initial growth in nutrient
broth followed by washing and resuspension in PsP.
Incuba-
tion at 37 C with aeration by shaking was ciontinued until
pyocyanine was obvious, usually 6-8 hr.
12
Cells were
13
reharvested, washed, and resuspended in physiological
saline.
Experimental media were inoculated to a final tur-
bidity of 15 Klett Units as determined by a Klett-Suramerson
colorimeter with a 54 filter.
Cultures were incubated under aerobic and microaerophilic conditions.
a rotary shaker.
Aerobic cultures were incubated on
Microaerophilic conditions were estab-
lished by growing cultures in dilution bottles which v/ere
filled to capacity and the caps then securely fitted.
Time course studies of pigment production were performed on some aerobically grown cultures.
At specific
time intervals during the course of incubation culture
aliquots were aseptically removed and centrifuged at 12,000
x g.
Pigment concentrations and phosphate concentrations
in the supernatant solutions were determined.
The cells
obtained from each culture by centrifugation were resuspended in the original sample volume of saline and turbidities were determined using the Klett-Sommerson colorimeter.
These turbidity readings were then translated to dry weight
determinations.
Microaerophilic cultures were allowed to incubate undisturbed for 7 days.
Bacterial cells were removed by cen-
trifugation and the concentration of individual pigments
was determined in the supernatant solutions.
Turbidimetric
determinations were not possible due to the production cf
slime by the organisms (4).
14
Phosphate concentrations were deternined by the method
described for low phosphate determinations in Standard
Methods for the Examination of Water and Vâstewater (1) .
The reagents utilized were identical, hov7ever, the volume
used in each case was reduced by a favor of 10.
Samples
for the determinations were prepared by diluting 0.2 ml of
culture supernatant to 10.0 ml with distilled water.
Dibasic potassium phosphate was used in the preparation of
the standard phosphate solution.
A Perkin-Elmer model 44
spectrophotometer was used in assaying phosphate concentrations .
Pigments were extracted from culture supernatants according to the procedure described in Fig. 2.
Pyocyanin
extraction and purification was based on techniques presented by Blackwood and Neish (3). Information provided by
Goswami and Majumdar was used in purification of the three
additional pigments (20) . Sephadex G-10 column chromatography was the means for final pigment purification.
Initial characterization of pigments included determinations of absorbance spectra (UV, vis, and IR), emission
spectra, and solubility characteristics.
Instrumentation
utilized include a Beckman DB recording spectrophotometer,
Perkin-Elmer Infracord spectrophotometer, and an AmincoBowman recording spectrofluorimeter,
15
Fig, 2.—Methods of pigment purification
16
Culture Supernatant (l volume)
CHCl^ (l volume)
CHCl^
Layer
Aqueous
Layer
0.5 N HCl
(l volume)
Aqueous
Layer
Neutralize
by addition
of solid
NaHCO
VJater-saturated
Phenol (l volume)
CHCl^
Aqueous
Layer
Layer
Ether
Water
(l volume
each)
Discard
Discard
Aqueous Solution of
Pyocyanin
Phenol
Layer
Aqueous
Layer
Ether-Phenol
Layer
Ether extraction
repeated twice,
ether removed
by vacuum
Discard
Aqueous Solution of
Aeruginosin
Pyoverdine
Chlorodeuteros
Sephadex G-10
Column Chromatography
Sephadex G-IO
Column Chromatography
I
Pyocyanin
(purified aqueous
solution)
\
I
Aeruginosin Pyoverdine Chlorodeuteros
(purified aqueous solutions)
17
Concentrations of individual pigments in culture supernatants were determined by spectrophotometric means.
Ex-
tinction values for individual pigments were determined.
Purified aqueous pigment solutions were prepared as previously described.
A known volume of each was dried in
vacuo and dry weights of pigments determined.
The concen-
tration of pigment was calculated for each purified solution.
Correlation between specific OD readings under assay
conditions and concentration of pigments were made.
Shikimic acid was assayed by a procedure described by
Millican (42) . Both intercellular and extracellular shikimic
acid were determined for variously supplemented PsP cultures
of WT and P~.
After 24 hr aerobic incubation, cultures were
centrifuged at 12,000 x g for 5 min.
A 50 ml aliquot of
each culture supernatant was evaporated in an 80 C water
bath to a final volume of 10 ml.
The cells harvested from
the cultures were resuspended in 10 ml volumes of distilled
water and then ruptured by sonication with a Bronwill
Biosonik sonicator.
tion.
Cell debris was removed by centrifuga-
Culture supernatants and sonicate supernatants were
partially purified prior to assay by the procedure of
Yoshida and Hasegawa (49),
CHAPTER III
RESULTS
Wild-type (WT) Pseudomonas aeruginosa in unsupplemented
basal medium (PsP) elaborated a deep blue pigmented substance into the culture broth.
The characteristics of this
pigment are presented in Table 2 and are identical to the
characteristics of pyocyanin as presented in Table 1.
The
«
IR spectrum of the pigment is reported in Fig. 3A.
indicates that the compound is pyocyanin.
It also
Absorbance in
the region of 3400, 3100, 1650, 1500, 1400, and 1300 wavenumbers (cm""^) indicates the presence of nitrogen in the
compound, either in the form of a tertiary amine or a substituted imine.
The absorbance between 1600 and 1400 wave-
numbers indicates the aromatic nature of the compound,
similating peaks as found in naphthalenes.
Peaks at 1250
and 1300 wavenumbers could be interpreted as indicating an
aromatic alcohol, while those at 2900, 1475, and 1400 wavenumbers indicate a methyl group attached to a carbon or
nitrogen (47) . These interpretations are consistent with
the structure of pyocyanin.
The ability to extract pyocyanin from aqueous solution
with chloroform facilitated quantitation of this pigment.
In chloroform solution, pyocyanin has a relatively sharp
absorbance peak at a wavelength of 314 nm.
18
This is
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Color and
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CHARACTERISTICS OF PIGMENTS PRODUC
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+j
X
•H
S
Bright red
crystals
o
S
e c3
CO
0

O
>.
P4
& 0
•H 13
Cu H
rH
x:
u
•p
Ti 0
• 0 -P
0 CO -P
CO c; CO - d
Cr>
C
•H
-P "d
C
0
MH
O
1
tr>
g MH
CO
•H
•H
Pl4+J >
0 1
MH C ^>
• tn 0 to
to
c; o ;:5 o fd f1 g - H
0 -H - m >
0 C Ci4 to 0
to rH
-P 4-> TJ }H a O l O
G r^ fd 0 0 to ' ^ 0
• H rH
JH
fd
g
0
O
C
0
U
to
0
JH
0
-H
4J
CM
ca
CQ
<
0
0 - ^ 4J rH f d
JH
q;
JH -P T i " d fd
O fd - H 0
0 to
to CO a
fd
o
^ 0 -H 0 •H u
u to V^
^
fd j3 0 •
•P -P JH o
fd rH 0
0 13 3 0 X fd fd
CO
O
U
I
I
to - H
X5 •P
O fd
-P
0
rH r-H :? 0
0 MH U
-H 0
f d to o 44 -P
to
^ a 0
0
u
o c
0
(d -H c: c; a c o
C CO r - 0 ^ 0 0 0 0
to
-H ^
Q-i
"H -H -H ' H 0
e CO 4-» - p - p u
O W
oj::0Ci^0c::to-Hp:3 0
> O - H " d rH rH ::i
:3 0 r3
rH > d" o fd ro g rrt 0 O H
P4 0 ( < ; - P ^ r o 0 < ; t o c o
MH
Ti
ffi -H
0 H-> JH 0
-P JH Ch CO
rH 0
fd fd
< g
CO
0
JH
O
rH
+
+
CM
•H
-p w
o u
(d
^
-P -P
X -H
JH
JH
Ci^
-P -P
O X
0
^
0
C
fd -H
rH
o
JH
U
XI
fd
•p
0
(d
JH
-p •P
0 X
^
JH
Cn
g
(d
CO
0 MH
fd
JH
t-{
O
fd 0
eU rH
CM
0
JH
0
0
JH CO
tPrH
0
0
fd r-i
fd
JH -P
O CO
0
0
rH
XJ
fd
4J
O
fd
•rH ro
X J <H
fd u
JH JH
u
a*
0 JH
JH
0
U
O
B ^g
CI4TI 0 4-1
-P U
C CO t-^ 0
• H JH fd a
CO
0
CO - H > .
JH -P rH JH
0 J3 X( 0
•H r-\ fd - H
+J 0 • H -P
fd CO
0
U
JH
•
CO -P
CO CO
(d
>i
u
JH
0
u
a^
21
^
Fig. 3.--Infrared spectra of pyocyanin (A) and
aeruginosin (B).
22
WAVENUMBERCCM-i)
1200
1000
4000
WAVENUMBER(CMi)
1200
1000
7
8
9
10
WAVELENGTH (MICRONS)
11
15
23
indicated in Fig. 4.
aqueous solution.
A similar absorbance peak is noted in
However, the extinction at X=314 nm is
enhanced in chloroform, allowing quantitation of smaller
concentrations of pigment. The assay based on OD-.^ .
^ ^
-^
314
pyocyanin in chloroform is relatively sensitive. As
as 0.2 ug of pyocyanin per ml of culture supernatant
0 1^
detected. At a wavelength
of
314
nm
the
E,'^^
^
1.0 cm for
cyanin in chloroform was determined to be 113.25.
of
nm
little
can be
pyo^
-^
The pH
of the culture supernatant had no affect on the assay.
The effect of phosphate concentration in pyocyanin production is shov;n in Fig. 5.
In unsupplemented PsP, the pro-
duction of pyocyanin is noted within the first three hours
j
post inoculation.
|
Those cultures supplemented with phos-
phate did not exhibit pigment production until the concen-
J
tration of phosphate available in the medium reached a level
»
t
that was not detectable by the analytical assay employed.
The effect on pyocyanin production of supplementing a
PsP culture of WT v/ith phosphate during the course of incubation is shown in Fig. 6.
The addition of phosphate did
not affect the total amount of pyocyanin produced as compared to the unsupplemented culture.
However, once phos-
phate was incorporated into the medium cell growth was
initiated and the concentration of pyocyanin per unit mass
of bacterial cells showed a rapid decrease at subsequent
sampling times as compared with the control culture.
;'9
J
24
"L^
Fig. 4.—Absorption spectra of pyocyanin. Solutions of identical pigment concentration. Symbols:
O-o / in 2.0 N HC1;A-A/ in tris-HCl buffer (pH 5.5);
Q_Q , in maleate-NaOH buffer (pH 9.0); «—• , in
chloroform.
25
1.5
1.0
u
o
z
<
(•
Q:
o
v>
m
<
0.5
d
I
0.0
200
250
300
WAVELENGTH (^^)
350
400
450
26
Fig. 5.—Influence of phosphate concentration on
pyocyanin production by WT. A, production in unsuppl
mented PsP; B, in PsP supplemented with 0.005% K2HPO.
C, in PsP supplemented with 0.01% K2HPO.. Symbols:
0-0 / pyocyanin in ug/mg dry weight of cells ;A-A/
phosphate concentration expressed as ug/ml.
27
HU.U
A
30.0
o
\J
20.0
10.0
0.0
!J
I
1
1
1
1
1
1
l._
<0
40.0
_ 30.0
_ 20.0
-
10.0
an
0.0
5
ui
<
40.0
CL
to
O
X
-
30.0
-
20.0
-
10.0
i J 0.0
9
12
15
T I M E (HOURS)
CL
3'
i
23
Fig. 6.--Influence on pyocyanin production of
phosphate supplementation during the course (15 hr
post inoculation) of culture incubation. Time
course determination of pyocyanin in culture supernatant of an unsupplemented and supplemented culture.
Pyocyanin concentrations expressed in A as ug/mg dry
weight of cells; in B as ug/ml culture supernatant.
Symbols: o—o , unsupplemented PsP culture;A—A /
PsP supplemented with 0.5% K2HPO,.
29
25.0
CO
lU
20.0-
o
b.
O
X
UJ
>Q:
o
S^
z
<
>
o
o
>CL
6.0
Iz
<
z
bJ
(L
(O
:|
z
z
<
>o
o
>a
9
12
TIME
15
(HOURS)
18
21
24
27
30
30
The effect of aromatic supplementation on pyocyanin
production is shown in Fig. 7.
Relatively higher concen-
trations of pigment are produced in basal medium supplemented with 50 ug per ml of each tryptophan, phenylalanine,
and tyrosine and 0.5 ug per ml of para-aminobenzoic acid
than in unsupplemented PsP.
However, as in the previous
investigation pyocyanin production was not initiated until
the available phosphate in the medium reached an undetectable level.
Under microaerophilic conditions WT lacked the ability
to synthesize pyocyanin.
No pyocyanin was detected in
seven day cultures of the following composition:
(i) un-
supplemented PsP, (ii) PsP + 0.5% K2HP0^, (iii) PsP + 1.0%
KNO^, and (iv) PsP + 0.5% K2HPO. + 1.0% KNO^.
Substantial
bacterial growth occurred only in culture iv.
The ability of WT to produce pigments other than pyocyanin became apparent.
Three additional pigments were
r
[
J
^
recovered from the supernatants of variously supplemented
jcultures.
The characteristics of these pigments and their
probable identity are presented in Table 2.
No report of
a bacterial pigment having the characteristics of that pigment designated as chlorodeuteros has been observed in the
literature.
The name chlorodeuteros is Greek for the second
green, implying that this pigment is the second green pigment
found to be produced by P. aeruginosa.
Chlororaphine i.'^ the
/ -
31
Fig. 7.--Influence of phosphate and aromatic
amino acid supplementation on pyocyanin production
by WT. A, production in aromatic supplemented PsP;
B, production in PsP supplemented with aromsatics and
0.01% K^HPO-. Symbols:0—o, pyocyanin concentration;
A-A f pnospfiate concentration.
32
V)
_J
llJ
o
UL
o
X
Ul
0.0
>Q:
o
35.0
70.0
B
60.0
_ 30.0
z
o
o
>-
-J 2 5 . 0
-z
- 20.0
^
>(L
H 15.0 I
CL
CO
O
10.0
i
TIME
(HOURS)
5.0
1 0.0
a.
y
1'
33
other green pigment synthesized by some strains of this organism (Table 1).
The appearance of chlorodeuteros in aqueous
solution is much like that described for chlororaphine.
How-
ever, the absorption spectra of the two compounds differ
markedly.
Chlororaphine is readily oxidized to the com-
pound called oxychlororaphine, whereas chlorodeuteros appears to be relatively stable under normal laboratory
conditions,
~~~"
The IR spectra of the three additional pigments are
presented in Fig. 3B and Fig. 8A and 8B.
The spectrum pre-
sented in Fig. 3B is consistent with the basic structure of
the aeruginosins.
The presence of aeruginosin B was not
definitely indicated as the ability to detect absorbance
due to SO-." was beyond the range of the spectrophotometer
used.
The spectrum of aeruginosin is basically the same as
that for pyocyanin.
The aromatic characters between 1300
and 1700 wavenumbers (cm""i) are retained.
The presence of
a carboxyl group is indicated by the broad absorbance peak
at 3000 wavenumbers, while the absorbance peaks at 1600,
1100, and 825 wavenumbers indicate an amino group.
The
presence of a methyl group attached to either a carbon or
a nitrogen is indicated by the absorbance at 2850 and 1450
wavenumbers.
The absorbance spectra of pyoverdine and chlorodeuteros are basically similar.
Pyoverdine appears to be
r."
34
Fig. 8.—Infrared spectra of pyoverdine (A) and
chlorodeuteros (B).
35
ACOQ 3000
IT M
0.0 • 1 1
2000
1
-r—t—1—1—
1
WAVENUMBER(CMM
1200
1000
1500
1
' —1
1
r—
1
1
1
000
-r——r—
800
I
11
700
1
1
A
.10
\r
u;.23
o
z
•t
g.30
d
i?.4C
\l\
i -P
/
f-
•
/
'
.SO
r -I
'^V-i
.60-
-
-H
-y—^
.80
1.0
1 1 t 1 11
I I
2000
I
I
T
8
9
10
WAVELENGTH (MICRONS)
1
1
1
I
1
1
1
11
WAVENUMBER(CM-<)
1200
1000
1500
.^_,.„,,
»
1
L
7
30C0
—U—
1 ,- ,^
1
4000
0.0
^
1
L.
12
1
—
13
L
14
800
eoo
15
700
--1
B
.10 -
r—
1
\i
V^^
ui.20|^• - V I
c>
<
£.30 ' — 1
o
vw
CO
S.40
.50
.60
-
=
J
I
1
'
-.._1
7
.__!
1
1
8
9
1(
WAVELENGTH(MICRONS)
1
11
11
1
1
12
1
.lUlJL
.sr.
1. 1.. - J .
13
14
IS
36
an aromatic compound.
It exhibits aromatic characters at
3100, 1600, 1500, and 750 wavenumbers.
Absorbance at 3300,
1675, 1375, and 925 wavenumbers possibly indicates the
presence of nitrogen, as an imine or as a primary amine.
The absorbance at 3300, 1675, 1375, and 925 possibly indicates a carboxyl group.
The spectrum of chlorodeuteros as
shown in Fig. 8B indicates that the basic structure of this
compound does not differ to any great extent from that of
pyoverdine.
These three pigments could not be separated from
aqueous solution by extraction with common organic solvents.
Therefore, it was necessary to quantitate each of these pigments in partially purified extracts containing any combination of the three.
Spectrophotometric techniques were used
to determine the pigment concentrations.
The UV-vis absorp-
tion spectra of the pigments are shown in Fig. 9.
Concentrations of aeruginosin in culture supernatants
were determined through measurement of the ODc2o
ture extracts.
of cul-
At this wavelength interference due to
absorbance of pyoverdine and chlorodeuteros was minimum.
0 1%
In 0.1 N HCl the E,*
of aeruginosin was found to be 6.25.
1.0 cm
Quantitation of pyoverdine was based on the absorbance
shift observed with pH change of the solution.
As indicated
in Fig. 9, pyoverdine in 0.1 N NaOH exhibits an absorbance
peak at 395 nm.
This peak shifts to a lower wavelength as
37
Fig. 9.—Absorption spectra of P. aeruginosa
pigments. A, aeruginosin; B, pyoverdine; C,
chlorodeuteros. Line designation:
, spectrum
in 0.1 N NaOH;
, spectrum in 0.1 N HCl.
H
lit
39
13
38
0.5
0.0
1.5 _
1.0
0.5 _
0.0 200
250
300
"350
400 450
WAVELENGTH W ^ )
500 550 600
650
39
the solution is made acidic.
The absorbance from 400 nm to
4 50 nm in 0.1 N HCl is very low under such a condition; a
0.1% solution exhibiting an extinction of 0.50 at X=420.
1°' of pyoverdine in 0.1 N NaOH found to be 20.00.
The E0
•;:°
1. u cm
^-^
The decrease in OD.^^
was used to calculate concentra4 20 nm
tions of pyoverdine in culture extracts.
This wavelength
minimizes interference due to absorbance of aeruginosin and
chlorodeuteros.
Chlorodeuteros exhibited no peculiar absorbance characteristics by which it could be quantitated.
Therefore it
was necessary to calculate concentrations of this pigment
through additive absorbance.
In order to minimize the inter-
ference of aeruginosin and pyoverdine, the wavelength chosen
was 325 nm.
From OD^-t-^
and OD.^^ ^^ determinations, it
550 nm
4 2U nm
was calculated what portion of the absorbance at 325 nm was
•
.•
the OD-,^^
for a solution of aeruginosin
in
0.1 N. _HCl is
contributed
32 5 nmby aeruginosin and pyoverdine. For instance.
1.23 times as great as its OD^^Q ^^. By multiplying the
OD,.^^
the factor 1.23, that portion of the
550 nm reading^ by
J.
OD-,^n
attributable to aeruginosin can be calculated. The
3 2 5 nm
same type of determination can be made for pyoverdine.
subtracting the OD ^^z
By
due to aeruginosin and pyoverdine
from the 00^25 nm ^^^diri^/ "the contribution due to chlorodeuteros can be determined.
as follows:
The complete calculations are
.1
40
A = (OD^^^
^^ attributable to
DDK) n m) (1.23) = OD-.-^
32b nm
aeruginosin
B = (OD,.^^
4 20 nm) (0.65) = OD....^
32 5 ^^
nm attributable to
pyoverdine
(ODJZD
.^^ nm )-(A+B) = OD.,^^
^^ attributable to
32b nm
chlorodeuteros
The extinction of a 0.1% solution of chlorodeuteros in 0.1
N HCl was determined to be 12.40.
The results of the aerobic studies designed to determine
the influence of media composition on pigment production by
WT are presented in Table 3.
Time course investigations of
the production of all four pigments are presented.
The in-
formation obtained indicates that only pyocyanin and
aeruginosin are produced in unsupplemented PsP, and of these
two pigments, pyocyanin appears to be produced in greater
quantity.
The addition of KNO-^ to the basal medium resulted
in the production of pyoverdine by WT.
However, the concen-
tration of pyoverdine produced in medium without phosphate
was small in comparison with the quantities produced in
medium supplemented with both KNO., and K2HPO. . Phosphate
supplementation also allowed the production of chlorodeuteros.
This was the only pigment synthesized in the culture supplemented with only K2HPO..
These findings indicated that the pigment or pigments
produced by WT was dependent upon media composition.
1 i
,1
41
00
•^
0)
•H
"^ r-« o
o
.
.
.
Cr> VD O
^
CN
.
O
in o o
o
M* rH cr>
o
CM o
ro
o
VD O
O
-^
o
o
o o o
o
in o
CM
o
C3^
in
o
00 O
O
VD
o]
c:' KO
^
o
ro o
o
in
o
o
o
r-
in
EH
TJ
<
w
o
IS
H
o
•rH
VD
ro
•H
O
.
.
(N o
^
CO
.
o
o
CNj o
O
O
o
O
VD
ro
o
CM
VD
<-{
0-)
cvi
ro
CM
P.
r ^ CN o
w
<
.
r-
in rH in
-<:}»
CN
-p
.
.
o
.
rH CM VD
O
rH o
in
o
rH O
O
VD
iH o
rH
r--
00
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O
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00
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ro o
o
"^
o
ro
in
rro
VD O
O
rH O
00
O
iH o
o
in
rH o
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o
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o
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ro o
o
CM
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O
CM
in
CN
in
.
r- CO o
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in
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to
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rH
Q)
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w
w
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ro
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CTi
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cr> CO o
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cr^
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CN i n
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ro
^:
00
H
rH m o
(N
t7>
o
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VD
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>i
EH
rH
CNl
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P^
Q
§
O
---
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Q
U
.
o o
CO
§
. . .
o
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•^
CU
O
o
r-
ro
rH
CO
"^
VD
EH
O
H
u
4J
H
CQ
O
m
o
to
O
o
JH
JH
JH
JH
JH
O
• H -P
tn :5
O
•H -P
to
o
O
-P
O
- H -P 0)
w 13
•H O
H
•H
•H
CM
to
O
o n
JH
•H
o
Q)
>
u ^
O
>i
04
O
O
13 O
JH
O
>i
(U Xi
fi<
i
CM
•H
T3
03 •H O
JH
•H
JH
O
rH
Q) Xi

O
PM
o
u
CM
x:
•H
o
m
CM
to
CM
td d
g H
4J
td
Q>
•H
td •H O JH
> i tn JH (U
u :3 O >
O
JH
>i
Q)
PM
o
X
i
CM
•H
•H
to
0)
•H
•H
JH
O
•H
o
13 O
JH rH
> i Q) X
CM 
O
O
rH
>
O
o ^
CM
CM <; U
CM
in
dP
O
Xi
CAP
in
O
o
CVKN
(U fc^ r H
CM
to
CM - p
to • H
04
(U
^
CM -P
to -H
CM ^
CM
X
-P
to
•H
ro
O
^
«
^
•
O
CM
.H
ro
O
t^
fd
42
Although there was a difference in pigments produced in various cultures, the rate of total pigment production appeared
to be quite similar.
This is indicated in Fig. 10.
Least
squares analysis was used to determine the slope of the line
corresponding to the most linear portion of the total pigment versus time plot for each culture.
The resulting lines
are nearly parallel, which indicated similar rates of total
pigment production.
Investigations of pigment production by WT indicated
that microaerophilic conditions are not suitable for the
production of pyocyanin.
However, as shown in Table 4, WT
exhibited the ability to produce aeruginosin, chlorodeuteros,
and pyoverdine under such conditions.
The supplementation
to basal medium determined which pigment would dominate.
A
large amount of aeruginosin was produced in PsP supplemented
with KNO-.
Chlorodeuteros predominated in phosphate supple-
mented PsP, v;hile pyoverdine was the major pigment produced
in PsP supplemented V7ith both K2HP0ând KNO^.
The mutant (P~) strain lacking the capacity to produce
pyocyanine proved to lack the ability to produce chlorodeuteros and aeruginosin.
It did, however, exhibit the capacity
to produce pyoverdine.
It was able to synthesize this pig-
ment under both aerobic and microaerophilic conditions.
results of the microaerophilic studies are presented in
Table 4.
A substantial amount of pyoverdine was produced
The
43
Fig. 10.--Total pigment production by WT in variously supplemented PsP cultures. Symbols: c>—o ^ unsupplemented PsP;^^W^ , PsP supplemented with 0.5% K2HPO.
at 12 hr; Q — n / PsP supplemented with 0.5% K^HPO^
prior to inoculation; •—t , PsP supplemented with 1.0%
KNO^;^—A , PsP supplemented with 0.5% K2HP0.and 1.0%
KNO^. Line designation:
, without phosphate;
,
with phosphate.
44
105.0 (100.0
90.0 to
_J
_i
80.0
o
u.
o
X
70.0
D
u
>-
60.0
^
50.0
K
O
D
A
z
lU
40.0
o
»-
A
30.0
o
20.0
10.0
0.0
0
4
•
8
12
16
—J
20
TIME
1
24
«
28
(HOURS)
1
32
'
36
4
40
_j
44
I—
48
45
H->
C
fd
+J
fd
JH
4J
fd
CI4
CO
O
3
to
H
(U
H
Q
-P
4->
I
0
0
0
0 0 0
o in o
0
0
0
0 0 0
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46
only in basal medium supplemented with both KNO^ and K2HPO..
Similar results were observed in aerobic cultures of P".
During a 24 hour period of incubation, P~ in PsP + KNO.^ +
K2HP0^ produced sufficient pyoverdine to yield 112.5 ug of
pigment/mg dry weight of cells.
No significant pigment pro-
duction was noted in any other culture.
The capacity of variously supplemented cultures of WT
and P" to accumulate shikimic acid is shown in Table 5.
The
information indicates that under the experimental conditions
neither WT nor P~ exhibited the capacity to accumulate shikimic acid within the bacterial cell.
Under those condi-
tions conducive to the formation of pyocyanin, WT accumulated
a concentration of 8.28 ug of shikimate per ml of culture
supernatant.
Under growing conditions WT did not produce
excessive extracellular shikimic acid.
in the case of P~.
The opposite appeared
Under phosphate limiting conditions lit-
tie shikimate was found in the culcure supernatant.
However,
in phosphate supplemented cultures, the levels of external
shikimic acid were the highest determined for P".
Supple-
mentation with KNO^ reduced extracellular shikimate in
cultures of WT and P~ as compared to the corresponding
culture not supplemented with nitrate.
47
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CHAPTER IV
DISCUSSION
Previous investigations concerning determinations of
culture conditions optimum for pyocyanin production resulted
in various conclusions concerning the effect of phosphate on
synthesis of the pigment. Burton, Eagles, and Campbell (5)
3concluded the PO.
was essential for pyocyanin formation by
Pseudomonas aeruginosa, 0.04% as K2HPO. found to be optimum
concentration.
Several later workers agreed that PO.
was
essential for pigment formation, the optimum concentration
varying from study to study (14, 24, 29, 36, 37). MacDonald
(37, 38) concluded that cultures supplemented with progressively higher concentrations of K2HP0^ produced lower concentrations of pyocyanin.
In a recent publication, Ingledew
and Campbell (29) concluded that phosphate was not required
for the production of pyocyanin.
The results obtained in
the present investigation show agreement with the later
conclusion.
Time-course studies monitoring available culture phosphate and pyocyanin concentration in culture supernatant
show that in all cases observed the available phosphate must
be near depletion before the ce'lls begin pyocyanin production.
In addition, phosphate supplementation of a station-
ary pyocyanin-producing culture results in renewed growth
48
49
of the culture and an apparent inability of new cells to
synthesize the pigment.
It does appear, however, that
those cells originally having the capacity to produce pyocyanin may retain this ability for some time after phosphate
3—
enrichment. This indicates that not only is PO.
unessential for pyocyanin biosynthesis but the contrary, that this
ion may actually inhibit production of this pigment.
The ability of £. aeruginosa to produce pyocyanin only
in stationary culture may indicate that the synthesis may be
3growth phase dependent rather than PO.
dep>endent. This
possibility was investigated by monitoring pyocyanin syn2thesis in a stationary culture in which SO.
rather than
PO.
was deleted.
During a 48 hr period thiis culture pro-
duced no pyocyanin, indicating that the phase of growth is
of less importance than the phosphate concemtration of the
medium.
This phosphate limiting effect does result in the
optimum production of pyocyanin occurring duiring the stationary phase of growth.
reports.
This is contradictory to previous
Frank and DeMoss (14) concluded thiat pyocyanin
was produced only under conditions consistent with cell
growth.
During a time-course study monitorilng culture bac-
terial protein and pyocyanin content, it was recorded that
pyocyanin production increased and became linear as the increase in bacterial protein approached a ma:ximum.
Only a
50
slight increase in bacterial protein was noted toward the
end of the 24 hr period.
Therefore, re-evaluation of the
Frank and DeMoss investigation may cause a reversal of the
original conclusion based upon the data provided herein.
In relation to the phosphate effect on pyocyanin production, one of the observations made is confusing.
In
all investigations with WT the washed cell inocula were
producing relatively large concentrations of pyocyanin prior
to their preparation for use as inocula.
From the results
obtained in phosphate supplementation of a culture during
the course of incubation (see Fig. 5) it would be expected
that all cultures, regardless of supplementation, would
produce comparable quantities of pyocyanin.
the case.
This was not
Cultures supplemented with phosphate prior to
inoculation did not exhibit the ability to produce measurable concentrations of pyocyanin until phosphate was depleted from the medium.
These findings can only be
justified if cell washing affected the capacity of cells
to produce the pigment or if growing cultures have the
capacity to reassimilate small quantities of dye molecule
for utilization as a carbon supply.
Key intermediates to the shikimic acid pathway have
been indicated as likely precursors to the phenazine nucleus
(7, 34, 36, 41, 43). The most convincing information indicates that shikimic acid is the precursor to pyocyanin (28).
51
Investigations of pyocyanin biosynthesis in cultures supplemented with the end products of the aromatic pathway
indicate that they elicit little effect on pyocyanin production.
The ability of pyocyanin production to act in a
capacity to regulate amino acid synthesis along this pathway was not realized.
It appeared that the amino acids
were utilizable as carbon sources for the phenazine ring;
however, they did not alter the point of initial pigment
production.
This, again, appeared to be dependent on the
depletion of culture phosphate.
The investigation concern-
ing the accumulation of shikimic acid by cultures of WT
and P~ is of little value in evaluating the role of shikimic
acid in pyocyanin.
The ability of WT and the inability of
P~ to accumulate shikimate under culture conditions favorable to pyocyanin production are inconsistent with expected
results.
As indicated in Fig. 1, the conversion of shiki-
mate in the aromatic biosynthetic pathway requires the
phosphorylation of this compound.
Depletion of culture
phosphate expectedly would result in shikimate accumulation,
provided the pathway remains operative under such conditions.
The ability to convert shikimic acid to pyocyanin should
reduce shikimate accumulation.
This was not observed.
The
mutant strain exhibited an inability to accumulate shikimate
under conditions favoring pyocyanin production, whereas WT
accumulated this compound under like cultural conditions.
52
Shikimate accumulation by WT in phosphate starved cultures
might indicate that the reaction sequence from shikimate to
pyocyanin has an equilibrium such that shikimate is not completely converted.
The results could indicate that the
presence of excess shikimate is a prerequisite for pyocyanin
production.
This is doubtful since accumulated shikimate is
observed in culture supernatant rather than within the cells.
The inability of P" to exhibit shikimate accumulation under
phosphate starved conditions indicates that the biosynthetic
pathway is relatively non-functional under such conditions.
The structural similarities between pyocyanin and
aeruginosin and the similarity of culture conditions favoring their production indicate that comments concerning the
production of one pigment may likely apply to the other.
The only significant difference related to their production
is in the requirement of heavy aeration for pyocyanin production, whereas aeruginosin production is favored under
low oxygen tension, the organism utilizing nitrate as the
terminal hydrogen acceptor (13).
The accumulation of shikimic by P~ under conditions
favoring the production of chlorodeuteros by WT exemplifies
the type of results expected if shikimic appears to be involved in the production of the pigment.
The lack of excess
shikimate in the WT culture indicates that excess of this
compound may be converted to chlorodeuteros.
The inability
53
of P" to convert excess shikimate to chlorodeuteros is consistent with shikimate accumulation.
The simultaneous loss in ability of P~ to produce pyocyanin, aeruginosin, and chlorodeuteros indicates that these
compounds are closely related.
The indication that shiki-
mate is involved in the synthesis of chlorodeuteros
strengthens the interpretation that this compound is related
to aeruginosin and pyocyanin.
It is, however difficult to
conclude the relatedness of pyoverdine to the other pigmented substances produced by WT.
The ability of P" to
produce this compound makes it appear that pyoverdine is
unrelated to these compounds.
However, other findings can
be interpreted to show that all four pigments appear to be
closely related.
The IR absorbance characteristics of pyo-
verdine are similar to those of chlorodeuteros, and indicate
an aromatic nitrogen containing compound.
The investigations
of rates of pigment production indicate the likelyhood of
all four compounds result from a common precursor, the rates
of total pigment production appearing very similar despite
which pigment or combination of pigments were produced.
Therefore, it is assumed that the four pigments are closely
related.
Pyoverdine and chlorodeuteros do not appear to be
phenazines.
Their indicated structures, however, are such
that it is likely that they could be derived from the same
54
aromatic precursor or precursors yielding phenazine compounds.
Since all reported P^. aeruginosa pigments, except
a melanin-like pigment reported by Liu, are phenazines it
is likely that newly described pigments will be either
phenzines or compounds of close relationship (10, 35).
In summary, a wild-type strain of Pseudomonas aeruginosa was found to produce four pigments, pyocyanin,
aeruginosin, pyoverdine, and chlorodeuteros.
That compound
designated as chlorodeuteros is a newly described pigment.
The phenazine nature of pyocyanin and aeroginosin was substantiated.
Pyoverdine and chlorodeuteros do not appear
to be phenazines, but are indicated to be aromatic nitrogen
containing compounds.
common precursor.
All four pigments appear to have a
The pigment or pigments produced by any
culture is dependent on the composition of the medium and
the conditions of incubation.
Phosphate depletion from
the culture medium appeared to be necessary for phenazine
production, whereas pyoverdine and chlorodeuteros are produced in phosphate-containing cultures.
LITERATURE CITED
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