― 18 ―Characterization of Massilia sp. BS-1, a Novel
Violacein-producing Bacterium Isolated from Soil
Hitosi AGEMATU（平成28年12月19日受理）
Massilia sp. BS-1, a novel bacterium, was characterized from a view point of violacein
production. During cultivation, strain BS-1 began to produce violacein in the early stage oflog phase growth at an incubation time of 16 h, and the amount of violacein rapidly reacheda maximum value of 62 mg/L for 5 h. This observation was suggested that the violacein
production was regulated by quorum sensing because it was not growth-associated and occurred
over a short period. Strain BS-1 did not produce violacein in a synthetic MM2 medium without
L-histidine. Neither N-hexanoyl-DL-homoserine lactone nor N-（3-oxooctanoyl）-DL-homoserine
lactone, known as autoinducers of quorum sensing, induced violacein production of strain BS-1.
Any acetyl homoserine lactones were not detected in the culture broth by LC-MS analysis.
However, the supernatant of the culture broth of strain BS-1 induced violacein production.
Key word: Massilia, Violacein, Quorum sensing, Autoinducer

1.

Introduction

Violacein is a bluish-purple pigment that hasvarious important biological activities including[1]
antibacterial（against gram-positive bacteria）,
antifungal [2], anti-protozoan [3], anti-malarial [4], antitumor [5], anti-viral [6], anti-oxidant [7], and anti-diarrheal [8]activities. The violacein carbon skeleton is derived
from 2 molecules of L-tryptophan（Fig. 1）.
Although further investigation to elucidate thepharmaceutical potential of violacein is needed,
mass production of violacein is diﬃcult due to its
low productivity. The role of violacein productionin its producers has not been understood, but it issuggested that it provides a survival advantage
to species over other microorganisms in theenvironment.
It has been reported that various bacteria,such as Chromobacterium violaceum [9], C. fluviatile [9],Janthinobacterium lividum [10], Alteromonas luteoviolacea [11],Pseudoalteromonas luteoviolacea [12], Duganella sp. [13],and Collimonas sp. [14], produce violacein. We havealready reported that a novel bacterium, Massilia
sp. BS-1, was isolated from a soil sample andproduced violacein and deoxyviolacein [15].
The production of violacein is known to be
regulated via quorum-sensing molecules, forexample, N-hexanoyl-L-homoserine lactone（C6HSL）in C. violaceum [16] and N-（3-oxooctanoyl）L-homoserine lactone in Pseudoalteromonas sp.
520P1 [17]. Among the Gram-negative bacteria,
N-acylhomoserine lactones（AHLs）are common
quorum sensing molecules（autoinducers）.
Quorum sensing allows a species to measure its
population density and control gene expression ina population density-dependent manner. It
is now clear that quorum sensing is the norm

Fig. 1. Chemical structures of violacein(R＝OH)anddeoxyviolacein(R＝H).
平成29年２月

― 19 ―
Characterization of Massilia sp. BS-1, a Novel Violacein-producing Bacterium Isolated from Soilin the bacterial world and that this process isfundamental to microbiology. Therefore, it is very
important to discover new producers of violaceinto effectively produce it and to elucidate themechanism of quorum sensing.
In this study, we characterize Massilia sp.
BS-1 from a view point of violacein productionand examine whether the violacein production isregulated by quorum sensing.
2. Materials and methods
Bacterial strain and growth conditions  
Massilia sp. BS-1 was isolated in our laboratory [15].
Strain BS-1 was cultivated on a nutrient agar platecontaining 0.1% yeast extract, 0.1% polypeptone,
0.1% KH2PO4, and 1.5% agar（pH 6.8）. The agar
plate was incubated at 28℃ for 2-3 days. The
strain was also cultivated in nutrient broth consistingof the same components described above without
agar at 28℃ for 1-2 days with shaking. A
synthetic MM2 medium containing 0.2% glucose,0 . 1 % （ N H 4 ）2 S O 4 , 0 . 4 % N a 2 H P O 4 · 7 H 2 O , 0 . 2 %KH 2PO 4, and 0.01% MgSO 4·7H 2O was used forthe characterization of strain BS-1. N-HexanoylDL-homoserine lacton and N-tetradecanoyl-DLhomoserine lactone were purchased and used toinduce violacein production as AHLs.
Production of violacein To produce violacein,strain BS-1 was cultivated in 30 mL of nutrient
broth on a rotary shaker at 28℃ for 18 h. Thirty
milliliters of the seed culture was transferred to a
3-L jar fermentor containing 2.0 L of nutrient brothfollowed by cultivation with aeration（0.5 v/v/m)and agitation（300 rpm）at 25℃. Samples（3.0mL）withdrawn from the culture broth at regularintervals were used for measuring pH, bacterial
growth, and violacein production. Violacein was
harvested from the sample by centrifugation,
extracted by methanol（3.0 mL）, and quantified
using a spectrophotometer at 577 nm. Bacterial
growth was monitored by measuring the turbidity
at 660 nm. After 67 h of cultivation, the cells
were centrifuged at 8,000× g for 10 min, andthe supernatant was discarded. The cell pellet
was then mixed with 200 mL of methanol. The
mixture of the cells and methanol was treated
秋田高専研究紀要第52号

by ultrasonication until the cells were completely
bleached. The methanol extract was thenseparated from the cells by centrifugation at 8,000
× g for 10 min. The extract was concentrated toremove methanol in a rotary vacuum evaporator
and was extracted twice with an equal volume ofethylacetate. The extract was evaporated undervacuum to obtain a crude mixture of violacein anddeoxyviolacein.
Quantitative and qualitative analysis The
amount of violet pigment, consisting of violaceinand deoxyviolacein, in the culture broth wasestimated using their molecular absorption[19]
coefficient（ε＝1.7×104 M-1 cm-1, λ＝577 nm）.
Quantitative analysis of the pigment was performed
using high performance liquid chromatography（HPLC）with a ZORBAX SB-Aq column（150×4.6 mm i.d.）（Agilent Technologies, Inc., USA）
at 577 nm and 40℃ with MeOH / water（70:30［V/V］）as the mobile phase at a flow rate of
0.5 mL/min. Qualitative analysis of the culture
broth was carried out by liquid chromatographymass spectrometry（LC-MS）. The analysis
was performed using a time of flight mass
spectrometer（Xevo QTof MS, Waters Corp.,
USA）. HPLC separation was performed using
an ACQUITY UPLC BEH C18 1.7µm column（50×2.1mm i.d.）at 40℃ with a mixture of waterand acetonitrile contained 0.1% formic acid as
the mobile phase at a flow rate of 0.2 mL/min.
To detect trace amounts of AHLs in the culture
broth, we used the selected reaction monitoring
technique. The characteristic fragment ion of m/z 102, derived from the homoserine lactone moietyof the AHLs, was monitored selectively to detect
all kinds of AHLs.
Antibacterial activities of violacein The
growth inhibitory and lethal effect of the violet
pigment, a mixture of violacein and deoxyviolacein,
isolated from strain BS-1 were examined forseveral bacteria such as Bacillus subtilis,
Staphylococcus aureus and Escherihia coli using thestandard methods [20].
Assay for autoinducers of violacein To
determine whether autoinducers are produced
by strain BS-1, we attempted the following
assay（Fig. 2）: Strain BS-1 was cultivated in― 20 ―
Hitosi AGEMATU

nutrient broth with shaking as described above.
Two ml of the culture broth was sampled atvarious times for the cultivation time from 6 h to
19 h, and was centrifuged to get a supernatant.
The supernatant was assayed for autoinducer
activity. The autoinducer activity showed theviolacein production induced by autoinducers inthe supetnatant.

Fig. 3. Cultures on an agar plate(A)and formation of
biofilm(B).

Fig. 4. Non-production of violacein in a synthetic MM2
Fig. 2. Assay for autoinducers of violacein production.

medium(B).
Strain BS-1 was cultivated in nutrient medium(A)and MM2 medium(B)supplemented with 1.0 mM

3.

Results and Discussion

Cultivation of strain BS-1 Massilia sp. BS-1
is a rod-shaped Gram-negative bacterium thatexhibits motility and ﬂocculation. On a nutrient
agar plate, it formed a violet colony whose surface
was wavelike and hard. Although it is an obligate
aerobe, it grew under sessile conditions, forming
extended biofilms on the surface of the culture
broth（Fig. 3）.
However, strain BS-1 did not produce violacein
in a synthetic MM2 medium, even if L-tryptophan
was added, with under aerobic conditions,although the strain grew well and produced
bioﬁlms（Fig. 4）.
Time courses for growth and production
When strain BS-1 was cultivated in the nutrient
broth with shaking, it grew to stationary phaseand ended violacein production by an incubation
time of 24 h. It began to produce a bluish-purple
pigment in the early period of log phase growth at
an incubation time of 16 h, and production rapidly
reached a maximum value of 62 mg/L pigment at
5 h（Fig. 5）. The color of the culture changed
to a deep violet due to the pigment. It appeared

L-tryptophan under the same conditions.

Fig. 5. Time course profile of Massilia sp. BS-1
cultivated in nutrient broth.
S y m b o l s : (♦) O D 6 6 0 ; (●) c r u d e v i o l a c e i nconcentration;(▲)pH.

that pigment production was regulated, because
it was not growth-associated and occurred over ashort period.
Characterization of the pigment The pigment
平成29年２月

― 21 ―
Characterization of Massilia sp. BS-1, a Novel Violacein-producing Bacterium Isolated from Soilis water-insoluble and is present in the extracellularpolymers produced by strain BS-1. The pigment was
separated into two components with maximum
absorption wavelengths of 577 nm and 555 nm
by preparative HPLC. Under analytical HPLC
conditions, the former（violacein）and thelatter（deoxyviolacein）components were eluted
at 5.51 min and 7.82 min respectively（Fig. 6）.

Fig. 6. HPLC analysis of the extract of the culture
broth. HPLC conditions are described in Materials andMethods.

The violacein concentration was about 10fold higher than that of deoxyviolacein. The
molecular masses（m/z）of these two components
were determined to be 344.24（positive）and
328.23（positive）respectively by LC-MS analysis.
The entirety of the carbon, hydrogen, and nitrogenskeleton of violacein is derived from 2 moleculesof L-tryptophan, and the 3 oxygen atoms originate
from molecular oxygen [2].
Antibacterial activities of violacein The
antibacterial eﬀect of the violet pigment containing
violacein and deoxyviolacein was confirmed for
putrefactive bacteria such as B. subtilis, S. aureus
and E. coli. The minimum inhibitory concentration（MIC）of the violet pigment for B. subtilis and S.
aureus was 2.5 mg/L. MIC is deﬁned as the lowest
concentration of antibacterial material that caninhibit cell growth completely. The concentrationof the violet pigment more than 20 mg/L caused
lethal effects on B. subtilis and S. aureus. The
lethal effect means the death of logarithmicallygrowing bacterium. However, the violet pigment
could not inhibit the growth of E. coli even if morethan 30 mg/L.
Effect of L-histidine on violacein production
In C. violaceum, the production of violacein is under
the control of signal molecules called autoinducers [16].
The signal molecules are detected by cognate
cytoplasmic receptors, and then the activatedreceptors bind DNA and induce transcription of
秋田高専研究紀要第52号

target quorum-sensing genes. [20] Therefore, we
considered that strain BS-1 could not produce
autoinducers for violacein production and tried todetermine which amino acids were necessary for
violacein production in a synthetic MM2 medium.
We found that L-histidine was necessary for
violacein production（Fig. 7A）. When strain
BS-1 was cultivated in MM2 medium containing
0.04 mM L-histidine and various amounts of
L-tryptophan（0-5 mM）, violacein production
increased with the increase in L-tryptophan till it
reached a value of 0.38 mM violacein（Fig. 7B）.
These results suggest that L-histidine is necessary
for the production of the autoinducer because
L-histidine is not the source of violacein.
Putative quorum sensing in Massilia sp. BS-1
In Gram-negative bacteria, autoinducers are
generally acylhomoserine lactones（AHLs）that
diﬀer in the structure of their N-acyl side chains. [21]Fig. 7. Effect of L-histidine(A)and L-tryptophan(B)on violacein production by strain BS-1 grown in MM2
medium in the presence of 1.0 mM L- tryptophan(A)or
0.04 mM L-histidine(B).

― 22 ―
Hitosi AGEMATU

However, strain BS-1 did not produce violaceinin MM2 medium containing 0.1 mM N-hexanoylDL-homoserine lacton or 0.1 mM N-tetradecanoylDL-homoserine lactone. Moreover, to determine
whether AHLs are produced by strain BS-1, we
attempted to extract autoinducers by ethylacetatefrom a culture broth sampled just before violacein
production. Strain BS-1 was subsequently
cultivated in MM2 medium containing bothL-tryptophan and the concentrated extract for 2days. However strain BS-1 did not produce
violacein. These results showed that autoinducersof strain BS-1 were not hydrophobic compounds
like AHLs. We also used LC-MS to detect AHLs
in the culture broth of strain BS-1. To identify
the selected ions as AHLs, the characteristic
fragment ion of m/z 102, derived from thehomoserine lactone moiety of the AHLs, wasmonitored selectively. A fragment ion of m/z 102 was not detected in the sample from theculture extract. Gram-positive quorum-sensing
bacteria, such as Streptococcus and Bacillus,
predominantly communicate with short peptides
that often contain chemical modiﬁcations [20]. The
autoinducer of strain BS-1 might be a hydrophiliccompound like peptide. So we assayed theautoinducer activity in the culture broth of strain
BS-1 sampled at various times for the cultivation
time from 6 h to 19 h. We detected the autoinducer
activity in the early stages of violacein production（Fig. 8）. The peak of the autoinducer activity
was after 10 hours of the cultivation time, and theFig. 8. Autoinducer activity in the early stages ofviolacein production.
S y m b o l s : ( ●) A u t o i n d u c e r a c t i v i t y ( A 5 7 0 ) ; ( ○)Violacein(A570).

activity was decreased rapidly as the violacein
production progressed.
The role of L-histidine and the elucidationof chemical structure of autoinducer in violacein
synthesis in Massilia sp. BS-1 are currently underinvestigation.
4. Conclusion
1）Massilia sp. BS-1 produced a bluish-purple
pigment and biofilms in nutrient medium. The
pigment consists of violacein and deoxyviolacein.
2）Massilia sp. BS-1 grew well in a synthetic
MM2 medium, while both L-tryptophan and
40µM L-histidine were neede to produce violacein.
L-Tryptophan is a building block of violacein.
However, the role of L-histidine is unknown.
3）Neither N-hexanoyl-DL-homoserine lacton nor
N-tetradecanoyl-DL-homoserine lactone induced
violacein production. Any acetyl homoserine
lactones were not detected in the culture brothby LC-MS analysisi. However, the supernatant of
the culture broth of strain BS-1 induced violacein
production.
Acknowledgement
I thank Hiroaki Tsuya, Kazuya Suzuki, Kévin
Bounoua, Aleksi Saariranta, and Mamoru Washiya
for their help with the experiments.
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秋田高専研究紀要第52号