Microbes Environ. Vol. 19, No.

3, 236–240, 20046
http://wwwsoc.nii.ac.jp/jsme2/

Temperature-Dependent Bacteriostatic Activity of Serratia marcescens

YUKO TANAKA1, JUNKO YUASA1, MASAHIRO BABA1, TAICHIRO TANIKAWA1, YOJI NAKAGAWA1 and
TOHEY MATSUYAMA2*

1 Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Niigata 950–2181,
Japan
2 Department of Infectious Disease Control and International Medicine, Niigata University Graduate School

of Medical and Dental Sciences, Niigata 951–8510, Japan

(Received February 6, 2004—Accepted June 4, 2004)

Serratia marcescens produces the red pigment prodigiosin and biosurfactant serrawettin at 30°C but not at
37°C. In an analytic study of the thermoregulation of S. marcescens 274, we noticed another temperature-depen-
dent activity that was bacteriostatic to gram-negative and -positive bacterial species, at 37°C but not at 30°C. The
thermo-insensitive isogenic mutant strain N0075 (a producer of prodigiosin and serrawettin even at 37°C) was
devoid of such growth inhibitory activity. The revertant of strain N0075 made by transformation again exhibited
the temperature-dependent mode of prodigiosin and serrawettin production, and bacteriostatic activity. This nov-
el activity was correlated to the thermo-responsive acidification of the medium by S. marcescens. In response to
higher environmental temperatures, S. marcescens seems to suppress its own growth and the growth of other
bacteria.

Key words: Thermo-response, bacteriostatic activity, Serratia marcescens

Serratia marcescens is a bacterium found in water, soil,
plant and animals. It was once considered such a relatively
harmless organism that pigmented strains were used as a
tracer of an aerosol in field experiments2,13). Now it is infa-
mous for its pathogenicity to hospitalized patients. There
are many reports on the contamination of medical devices
and nosocomial infections with this bacterium4,5). Recently
in Japan, outbreaks of fatal nosocomial infections due to S.
marcescens have been sporadically occurring: 5 deaths in
Tokyo in 1999, 8 deaths in Sakai in 200012), and 7 deaths in
Tokyo in 2002. The reasons for the outbreaks of such severe
nosocomial infections have not been elucidated. The S.
marcescens in marine ecologies is also attracting attention.
White pox, a lethal disease of elkhorn coral, Acropora
palmate, which was first documented in 1996 on the East-
ern Dry Rocks Reef off Key West, Florida, is now prevail-
ing among reefs throughout the Caribbean. In 2002, S.
* Corresponding author; E-mail: tohey@med.niigata-u.ac.jp, Tel:
+81–25–227–2111, Fax: +81–25–222–7503
marcescens was identified as the etiological agent of this
reef-devastating disease9). Thus, for the first time, a bacterial
species associated with the human gut was shown to be a
marine invertebrate pathogen. The disease, white pox, is
highly contagious, and the rate of tissue loss by infection is
greatest during periods of seasonally elevated temperature.
Although the red pigment “prodigiosin” produced by S.
marcescens has made the bacteria famous in bacteriology13),
the temperature-dependent mode of pigment production by
S. marcescens is not well-known. At 30°C, pigmented S.
marcescens strains make red colonies via the production of
prodigiosin. At 37°C, the bacteria grow well, but make
white colonies lacking prodigiosin. The same temperature-
dependent mode has also been recognized in the production
of the biosurfactant “serrawettins” in S. marcescens6,8).
Thermo-regulated production mechanisms of these structur-
ally unrelated secondary metabolites15) have remained to be
elucidated11). When white colonies of S. marcescens cul-
tured at 37°C for one day were further cultivated at 30°C,
they became red. Whereas, when white colonies cultured at
Thermo-Responsive Bacteriostatic Activity
37°C for three days were further cultivated at 30°C, they
remained white. This finding14) seemed to suggest that an
unknown inhibitor of prodigiosin production had accumu-
lated in the agar media during longer incubation at 37°C. So,
analytical studies using various mutants (e.g., temperature-
insensitive mutants) were carried out to elucidate the
biochemical entity of this effect on the bacteria. Herein,
we report an unusual characteristic of S. marcescens, that
is, temperature-dependent bacteriostatic activity affecting
S. marcescens itself and other bacterial species.
Materials and Methods
Bacterial strains and growth conditions
S. marcescens 274, and the isogenic mutant strains
N0075 (thermo-insensitive mutant) and R074 (nonproducer
of prodigiosin and serrawettin W1 at 30°C), all of which are
laboratory stock, were used. Strains BB14 and ReTdrA
were constructed by marker rescue transformation of strain
N0075 with pUC19 containing genomic DNA of strain 274.
Other S. marcescens strains, Escherichia coli ATCC 25922,
Klebsiella pneumoniae Fu1-m21, Pseudomonas aeruginosa
ATCC 27853, Staphylococcus aureus ATCC 25923 and
Bacillus subtilis ATCC 21331 were described previously3,8).
The media used were peptone-glycerol (PG) agar (0.5%
Bacto-Peptone [Difco Laboratories, Detroit, USA], 1.0%
glycerol, 1.5% Bacto-Agar [Difco], pH, 7.0) and Luria
Bertani (LB) broth6). The examination for pH changes in
agar media was carried out using a flat surface electrode
of a pH BOY-P2 (SHINDENGEN, Tokyo) as described
previously7).
DNA manipulations
The isolation of chromosomal and plasmid DNA, restric-
tion enzyme digestions and ligations, and agarose gel elec-
trophoresis were performed using standard procedures1,10).
Detection of the bacteriostatic activity
A half part of PG agar medium was confluently inoculat-
ed with 50 ml of a bacterial suspension in sterile saline (0.15
M NaCl) which was prepared from an equal concentration
of LB broth-culture of bacteria (OD660, 1.00) by centrifuga-
tion and washing with saline. After cultivation at 30 or 37°C
for 3 days, growing bacteria and the agar medium just
underneath were removed with a sterile spatula. The left-
over semi-circular medium was inoculated with bacteria
and incubated at 30 or 37°C for 1 day to see the effects of
diffusible substances derived from the precedent bacteria.
237
Examination for butanediol fermentation
Acetoin, a characteristic product of butanediol fermen-
tation, was semi-quantitatively assayed using the Voges-
Proskauer (VP) test. An agar medium block (5´10´5 mm3)
dissected from the 3-day cultivated plate for examination of
accumulated activity (see above) was placed in a small test
tube, then, 400 ml of 6% a-naphthol in ethanol and 200 ml of
40% KOH with creatine were added successively. After aer-
ation by shaking, color changes were read. As standard
chemicals, acetoin (Wako Pure Chemical Industries, Osaka)
and 2,3-butanediol (Wako) were used.
Results and Discussion
Activities of the agar media on which S. marcescens
grew
Since S. marcescens colonies that grew on PG agar medi-
um at 37°C (non-permissive temperature for prodigiosin
production) for 3 days failed to became red on subsequent
cultivation at 30°C, the agar medium next to the white S.
marcescens lawn was examined for activity to inhibit the
production of pigment at 30°C (permissive temperature for
prodigiosin production). To that end, a semi-circular agar
medium was prepared (the right half of each plate shown in
Fig. 1) and inoculated with S. marcescens 274. After incu-
bation at 30°C for 1 day, inhibited bacterial growth was ob-
served near the confluent bacterial lawn (the empty area in
each plate, Fig. 1). It is worth noting that bacteria away
from the growth-inhibitory zone grew as red streaks. Thus,
S. marcescens 274 grown at 37°C was producing inhibitors
of bacterial growth rather than a specific inhibitor of prodi-
giosin production. The bacteria grown at 30°C did not show
such activity except the mutant R074 (non-producer of
prodigiosin even at the permissive temperature).
The observed growth inhibition by precedent bacterial
growth was temperature dependent. Bacterial species be-
longing to the same family (Enterobacteriaceae), E. coli
(mixed acid fermenter) and K. pneumoniae (butanediol
fermenter, as S. marcescens) were devoid of such activity
(data not shown). Among other strains of S. marcescens,
pigmented strain NS 38 and nonpigmented strains NS 25,
NS 45 and ATCC 8100 did not show such growth inhibitory
activity, but the type strain ATCC 13880 (pigment producer)
did show the same temperature-dependent growth inhibitory
activity (data not shown).
Examination of the growth inhibitory activity against
other species of bacteria revealed a broad spectrum simi-
lar to the effect of acetic acid (Fig. 2). In addition, the
238
Fig. 1. Bacteriostatic activity of S. marcescens In the left half of
each plate, S. marcescens 274 or its isogenic mutants (indicated
in each plate) were grown confluently at the indicated tempera-
ture for 3 days, then, the bacteria-grown parts were removed. On
the remaining semicircular agar medium, S. marcescens 274 was
inoculated as four parallel streaks and incubated at 30°C for 1
day. Growth inhibition of the streaked bacteria by the precedent
bacteria (274 [37°C] and R074 [30 and 37°C]) and no such inhi-
bition by the temperature-insensitive mutant (N0075, producer of
prodigiosin at 37°C) are shown. Bacterial growth is recognizable
as black streaks in this monochromatic photoprints. White bacte-
rial growth indicating inhibition restricted to prodigiosin produc-
tion was not observed. A color photograph is available at the
Online Journal Site (http://wwwsoc.nii.ac.jp/jsme2/).
streaked bacteria began to grow following growth inhibition
as shown in Fig. 2. Thus, this inhibitory activity of S.
marcescens is bacteriostatic rather than bacteriocidal, and
the substances responsible seemed to be volatile or unstable
during extended incubation.
TANAKA et al.
Fig. 2. Broad-spectrum bacteriostatic activity of Serratia marces-
cens and acetic acid. In the right half of each plate, Serratia
marcescens 274, P. aeruginosa ATCC 27853, E. coli ATCC
25922, Staphylococcus aureus ATCC 25923, and B. subtilis
ATCC 21331 were streaked in this order from the top and incu-
bated at 37°C for 1 day. The left half of each plate was grown
with S. marcescens 274 at 37°C for 3 days or soaked with 450 ml
of 0.5 M acetic acid (indicated in each plate), then removed
before inoculation of bacteria on the remaining right half of
the plate. Temporally inhibited bacterial growth (later, bacterial
growth began along the streaked line) was observed at 1-day post
inoculation. Since the plates were incubated at 37°C, the growth
of S. marcescens looks white.
Medium acidification by bacteriostatic S. marcescens
growth
The low specificity of the bacteriostatic activity of
S. marcescens cultivated at 37°C for 3 days suggested
enhanced pH changes caused by the growth. The S. marces-
cens PG-liquid culture at 37°C demonstrated a distinct
acidification of the medium and a tendency for the viable
count to decrease in the stationary phase. Such a tendency
was negligible in the 30°C-liquid culture (data not shown).
With respect to the solid agar surface, pH measurements in-
dicated a remarkable acidification of the medium on which
bacteriostasis was recognized (Figs. 1 and 3). In contrast,
the medium on which bacteriostasis was not recognized
showed minor pH changes (Figs. 1 and 3). Phosphate-buff-
ered PG medium which retained a neutral pH (Fig. 3) was
not bacteriostatic even after the growth of 274 at 37°C (data
not shown). Comparative studies with seven wild type
strains showing (e.g., S. marcescens ATCC 13880) or not
showing (e.g., E. coli) bacteriostatic activity, demonstrated
a clear correlation between bacteriostatic activity and acidi-
fication of the medium (data not shown).
In addition, S. marcescens 274 growing on phosphate-
buffered PG medium at 37°C developed white colonies.
Thus, it was shown that the inhibition of pigment produc-
tion was not a consequence of the medium’s acidification.
Thermo-Responsive Bacteriostatic Activity
Fig. 3. Changing pH of the PG agar medium on which S. marces-
cens is growing. The names of bacteria grown are given in each
graph, and special conditions are described in parentheses. Each
point is the average value with an error bar of standard deviation
(n³6). Cultivation was done at 30°C (closed circle) and 37°C
(open circle).
Pursuit of the bacteriostatic substance produced at 37°C
S. marcescens is known as a butanediol fermenter. In
contrast to those of mixed acid fermentation, products of
butanediol fermentation are not organic acids. So acidifica-
tion of the growth-supporting medium does not progress so
far. Referring to the well-known catabolism of S. marces-
cens, a possible metabolic shift from butanediol to mixed
acid fermentation in S. marcescens growing at 37°C was ex-
amined by conducting a comparative VP test with 30 and
37°C cultures. Unexpectedly, however, acetoin production
was remarkably higher at 37°C than 30°C. Then, 2,3-bu-
tanediol and acetoin were examined for their bacteriostatic
activity, and shown to be inactive (data not shown). Chemi-
cal extraction analysis of the 37°C culture medium has not
yet identified particular substances.
Marked acidification of the medium was shown to be a
critical factor for the bacteriostatic activity of S. marces-
cens. As the acidic substances responsible, the usual organic
acids derived from the bacterial metabolism seemed to be
involved. So, lactic acid, acetic acid, and formic acid were
examined for bacteriostatic activity. As shown in Fig. 2, the
effect of acetic acid was most similar to the bacteriostatic
effect by Serratia marcescens and relatively milder effect
239
on Staphylococcus aureus. These three organic acids were
all bacteriostatic and permitted bacterial growth later on.
Genetic basis of the bacteriostatic activity
As shown in Fig. 1, the bacteriostatic activity of S.
marcescens had an inverse relationship with prodigiosin
production which is, with serrawettin production, downreg-
ulated at 37°C. Since strain N0075 is a mutant defective in
such control systems, a marker rescue analysis was carried
out by transforming N0075 with pUC19 containing a
Sau3AI-digested DNA fragment of S. marcescens 274. The
transformant obtained, BB14, has the wild type phenotype,
that is, temperature-dependent production of prodigiosin
and serrawettin W1 and temperature-dependent bacterio-
static activity (data not shown). The sequencing of this
DNA fragment of 274, identified a putative regulator gene
tdrA (DDBJ accession number: AB077386, homologue of
E. coli yhcS encoding putative transcriptional regulator
belonging to LysR family). Then, a N0075 transformant
with pUC19 containing the DNA which covers tdrA and
its promoter region, was examined for its temperature-
dependent properties. This transformant, ReTdrA, produced
prodigiosin and serrawettin W1 in a temperature-dependent
manner, but was unable to acidify the medium to the level
achieved by strains 274 and BB14 (Fig. 4). With respect to
the bacteriostatic activity, ReTdrA was certainly devoid of
such activity at 37°C. So, a positive correlation between
marked acidification of the medium and bacteriostatic activ-
ity of S. marcescens was indicated again.
Biological role of the thermo-responsive bacteriostatic
activity
At the start of this study, we expected the presence of an
extracelluar inhibitor of prodigiosin production. Experi-
ments searching for such bacterial products, however, un-
covered a marked temperature-dependent acidification of
Fig. 4. Changing pH of the PG agar medium on which a marker-res-
cued S. marcescens transformant is growing. Each point is the
average value with an error bar of standard deviation (n³6). Culti-
vation was done at 30°C (closed circle) and 37°C (open circle).
240
the medium by S. marcescens. The acidification inhibited
the growth of S. marcescens itself and other unrelated bacte-
rial species. Is such bacteriostatic activity beneficial to the
long-term survival of bacteria in a stationary phase of
growth, or does it act to suppress subsequent microbes as
observed with Lactobacillus acidophilus?
pH changes in the environment will change the electric
charges of various molecules involved in multiple biologi-
cal activities. Herein, S. marcescens was shown to have a
specific ability to modify environmental pH in response
to environmental temperature. The genetic system involved
in such temperature-dependent activity seemed to have
evolved in S. marcescens. Why has temperature been so
critical for S. marcescens in nature? It is difficult to give
bacteriological and ecological explanations at the present
time.
Acknowledgements
This work was supported in part by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Cul-
ture, Sports, Science, and Technology of Japan (no.
12680628).
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