Characterization of blue pigmented bacteria isolated from Puerto Rico

V. Cardona-Cardona1, David Arroyo

1, J. Scellekens

2 and C. Rios-Velazquez

1

1 Microbial Biotechnology and Bioprospecting Laboratory, Department of Biology, University of Puerto Rico, Mayagüez

Campus, Puerto Rico 00681 2 Department of Physics, University of Puerto Rico, Mayagüez Campus, Puerto Rico 00681

In the microbial world, pigments are one of the most conspicuous traits. Blue Pigmented Bacteria (BPB) are very

uncommon; only four described genera share this characteristic. Scientific literature has also revealed the potential of this

bacterial group biotechnologically. The main objective of this study was to isolate and characterize this pigmented

microbes in Puerto Rico. Two BPB were isolated from different municipalities in the island and microscopic,

physiological, biochemical and molecular methods were used to its characterization. While the isolated gram-negative

bacterial rods optimal growth is 37oC and pH 7.0, the blue pigment is only produced at 25 °C. Physiologically, the isolates

can also grow in a media with a range of 0-1.5% of NaCl. The in silico analysis of the 16S rDNA gene suggest similarities

to the Vogesella genus, and the putative genes (igi) involved in pigment biosynthesis were also detected in the isolates by

PCR with differences at sequence levels only in igiE.

Keywords Blue Pigmented Bacteria; blue pigment; Vogesella sp.

1. Introduction

Pigments play an important role in physiological and molecular processes of microorganisms such as: photosynthesis,

survival to oxidative damage and resistance to UV-radiation. Through the years, pigments have been used as a

taxonomic tool for the identification and classification of algae, fungi, and bacteria [1,2]. Pigment diversity is due to

differences in their chemical structure compositions and the presence of specific chromatophores [3]. Pigmented

microorganisms have awakened the interest of the scientific community, because their role in taxonomic studies and

their biotechnological potential in processes like fermentation and bioprocess engineering [4]. Expression of the

pigments can be affected by a number of environmental factors, including oxygen availability, nutritional condition,

temperature, age of the colony, and strain variation [5]. For example, Arthrobacter atrocyaneus exhibits blue

pigmentation when grown at 24ºC, however no pigmentation is observed at 37ºC [6]. Other microbes such as

Rhodospirillum rubrum, Micrococcus luteus, and Sarcina aurantiaca exhibit pigmentation when grown at diverse

temperatures.

The most widely distributed pigments are the carotenoids. This type of pigment also plays an important role in

bacteria, for example, in photosynthetic processes, by preventing photo-damage, and conferring resistance to oxidative

damage due to the production of activated forms of oxygen. Purple and blue pigments in bacteria are less common.

Violacein (purple pigment) has been found in the genera Chromobacterium, Janthinobacter and Iodobacter [7].

Indigoidine (blue pigment) has been found in bacteria that belong to the genera Erwinia, Arthrobacter,

Corynebacterium and Vogesella.

Vogesella indigofera was first described in 1893 by Otto Voges [8] and he named it Bacillus indigofera. At the

beginning of the 20th century, the name of the bacterium was changed to Pseudomonas indigofera. After a systematic

study performed by Grimes et al. (1997) [9], P. indigofera was placed in the genus Vogesella. V. indigofera had been

isolated previously from pond sediments, natural aquatic systems, and soil. It is a member of the beta subdivision of the

Proteobacteria, closely related to the genus Chromobacterium. It is an organism which might easily be overlooked

during isolation of other oxidative bacteria, because the characteristic pigmentation occurs only in certain media and

then only when colonies are reasonably well separated [10].

The blue pigment produced by this bacterium was studied by Elzari-Volcani, who established that the formation of

the blue pigment was due to the production of indigoidine [8]. The specific function of this pigment is still unknown.

Very little is known about the pigment production in Blue Pigmented Bacteria (BPB). To our knowledge, the only

studies about the genes involved in the pigment biosynthesis are from the bacteria V. indigofera and E. chrysanthemi.

From these two, only for E. chrysanthemi exits a more complete study of the pigment biosynthesis and the genes that

are involved [11].

The only information of the V. indigofera genes involved in the pigment production includes two sequences

published in GenBank. The first sequence (accession number AF088856), provides the complete sequence for the

putative indigoidine biosynthesis locus. The second sequence (accession number is AF088857), describes the complete

sequence of the proposed biosynthesis regulatory locus. Based on sequence similarity, there are putative functions for

each gene According to the sequenced deposited and described in GenBank; the pecM gene (894bp) encodes for a

protein required for the production of indigoidine and is also a propose regulator for igiC. The PecS (513bp gene) is an

alleged regulative protein. The igiA gene (882bp) encodes for a putative 4'-phosphopantetheine transferase and also has

been proposed to be involved in the biosynthesis of indigoidine. The igiB gene (875bp) encodes for a supposed

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.)

©FORMATEX 2010 117

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glutamine dehydrogenase and probably involved in the biosynthesis of indigoidine. The igiC gene (830bp) encodes for

a putative N-carboxymaleamide decarboxylase, involve in the biosynthesis of indigoidine. The IgiD (encodes by a

3866bp gene) is a peptide synthetase homologous and is required for the biosynthesis of indigoidine. Finally, the igiE

gene (1226bp) encodes an ATPase component of an ABC transporter probably involved in indigoidine export.

Studies performed to Puerto Rican soils in a Geomicrobiological research project allowed the isolation of two blue

pigmented bacteria (BPB). To our knowledge these are the first BPB isolated and described on the island. Because of

the bacteria characteristic pigment, a full characterization of the organism was made. The main focus of this

investigation was to characterize biologically, molecularly, and physiologically the BPB’s. Molecular genetics was used

in order to identify and amplified the genes that were associated with the production of the blue pigment.

2. Materials and Methods

2.1 Soil analysis

A geological study was carried out for the sites where the BPB were isolated, to determine the mineral composition and

abundances in the collecting area.

2.1.1 Mineral analysis

The soil samples were analyzed to the Geology Department at the UPR-Mayagüez. The samples were analyzed using

Siemens D500 X-Ray diffraction instrument. The instrument produces a read out where the refractive ray produces a

characteristic peak at a specific angle of refraction (2Θ-) that corresponds to a plane in the crystal structure of the

mineral, distinguishing the mineral species.

2.2 Macroscopic and microscopic analysis

The isolates were analyzed at macroscopic level by general morphological description of the colony such as the

elevation, form, and margin, using the criteria described by Harley and Prescott (2007) (12). The size of the colonies

was also measured. The isolates were microscopically analyzed using the Olympus Optical Co., LTD CHT light

microscope and Scanning Electron Microscope (SEM). To determine the dimension of the bacteria, 100 of them were

selected and measured to calculate the average size. Gram stain was performed using heat-fixed smears. To observe the

morphology and dimension of the bacterium, simple stain was performed. Microscopic staining was done to determine

the presence of structure such as capsule and endospore (12). For the SEM the bacterium were observed using the JEOL

JSM-541 OL SEM microscope. For the analysis, the isolates were grown in Nutrient Broth for 24 hours at 25 ºC in a

orbital shaker at 120 RPM. Then, approximately, 1.5 mL of the culture was transferred to a microtube and centrifuged

for 1 min at 0.8 g (3,000 RPM) to obtain a cell pellet. Glutaraldehyde at 4% was added to fix the cell pellet and then

was left resting for 24 hours at 4ºC. The cell pellet was washed with 1X Phosphate Buffer (PBS) three times. To

dehydrate the pellet, different percentage of alcohol were used range from 10% to 100% in 10% intervals. Every ten

minutes the pellet was transferred from alcohol to another. To completely dry the sample, a critical point drier (Critical

Point Drying Apparatus Polaron E3000) was used. A sputter coater was used to give the samples a palladium-gold

cover in order to protect the sample and to add a better conductivity to the specimen. The parameters used above were

established by the Scanning Electron Microscopy Center at the University of Puerto Rico, Mayagüez Campus. Electron

micrographs were taken at an accelerating voltage of 15 kV.

2.3 Biochemical and physiological test

In order to determine the physiology of the isolated BPB, a series of biochemical tests were performed. As controls,

Vogesella indigofera (ATCC 19706) and Pseudomonas aeruginosa (ATCC 10145) were used. To determine the range

of NaCl that the bacteria can grow, different percentages of salt were used (0%, 0.5%, 1.0%, 1.5% and 2.0%) in

Nutrient Broth. Briefly, 5mL of the broth was transferred into tubes where the bacteria were inoculated. The samples

were incubated in an orbital shaker at 32°C for 48 hours. To determine the optimal temperature for the pigment

production, the isolates were streaked on Nutrient Agar plates. Then, the plates were incubated for four days at different

temperatures ranging from 4 ºC to 42 ºC. To determine the optimal pH for the bacteria, tubes with Nutrient Broth at

different pH were prepared. The pH range used was from 3.0 to 10.0 at 0.5 units intervals. HCl and NaOH were used to

adjust the corresponding pH. Then the medium was inoculated with the isolates bacteria at logarithmic stage. The

samples were incubated at 32 ºC for four days. To determine the different enzymes that the bacteria may have, different

tests were done. Urea broth was used to see if the bacteria can degrade urea. SIM medium were used to see if the

isolates could degrade tryptophan to indole, using the enzyme tryptophanase, and to determine the production of H2S

and to see if the bacterium in motile. Starch Agar Media was used to see if the bacterium produced the enzyme amylase.

The Nitrate Broth was used to see if the bacterium has the ability to reduce the nitrate to nitrite. The Nutrient Gelatin

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Agar was used to see if the isolate produced the exo-enzyme gelatinase. To determine the carbohydrate utilization,

assays were performed with sucrose, dextrose, maltose, and mannitol broths with phenol red as indicator. The ability to

use glucose, lactose, and sucrose was measured using Triple Sugar Iron Agar (TSIA) slant, the production of H2S and

gas were also observed. The utilization of citrate as only carbon source was measured using Simmons Citrate Agar

slants. To detect the conversion of pyruvic acid in acetoin, the Methyl Red (MR) test was used and to see the

fermentation of glucose and its transformation to pyruvic acid, we used Voges Proskauer (VP) test. All samples were

incubated at 32 ºC for 48 hours. Then a post incubation treatment was added to those tests that required it. To the SIM

medium, 2-3 drops of the Kovac reagent were added. To the MR medium 7 drops of Methyl Red reagent were added.

To the VP medium 10 drops of α–naphthol and 5 drops of KOH 40% were added, and then results were observed after

30 minutes. To the nutrient gelatin medium the post incubation treatment was 30 minutes in the refrigerator. To the

starch agar medium 2-3 drops of iodine were added. To the nitrate medium 1 drop of sulfanilic acid and 1 drop of α-

naphtilamine were added; if the result was negative, a small portion of zinc powder was added. These tests are

described by Harley and Prescott (2007).

2.4 DNA extraction

The genomic DNA of the BPB isolated was extracted using the method described by Chen and Kuo (1993) (13).

Briefly, the samples were inoculated into Nutrient Broth for 24 hours and 1.5 µl of the sample were centrifuged at

15.7xg (13,000 RPM) to form a cell pellet. The cell lysis was performed using lysis buffer (40mM Tris-acetate pH 7.8,

20mM sodium-acetate pH 8.0, 1.0mM EDTA pH 8.0 and 1% SDS) and sodium chloride 5M, then treated with RNAse

(20 µg/µl) for 30 minutes at 37 ºC. An organic extraction was made using one volume of chloroform and then

centrifuged at 15.7xg (13,000 RPM); this step was repeated twice. Then the sample was precipitated with absolute

ethanol. The isolated genomic DNA was resuspended in 50 µl of distillated sterile water. The final concentration of the

DNA was measured using an Eppendorff biophotometer.

2.5 Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) was performed with the genomic DNA of the isolate as template in order to amplify

part of the 16S rDNA gene. The amplification reaction was made using the Green Taq Master Mix by Promega and

Oligo 14-F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492-R (5’-GGTTACCTTGTTAGGACTT-3’) as universal

bacterial primers (Willmotte and Wachter 1993) (14). The following parameters were used for the reaction: initial

denaturalization at 94ºC for 3 minutes; followed by 30 cycles which included denaturalization at 94ºC for 30 seconds,

annealing at 52.7ºC for 30 seconds, extension at 72ºC for 1 minute. Then, a final extension at 72ºC was done for 10

minutes. The amplicons were purified using a PCR purification kit (Quagene Inc). The PCR product was sequenced in

the facilities Macrogene USA (www.macrogenusa.com). In silico analysis was done by using available online databases

such as GenBank in NCBI (http://www.ncbi.nil.nih.gov) by the program BLAST. The complete 16S rDNA sequences

were obtained by sending the genomic DNA to the facilities of Macrogene USA. The in-silico analysis was done as

mentioned above.

2.6 Phylogenetic Analysis

The BPB isolated 16S rDNA sequence were edited using the program Chromas Lite 2.0.0 and then aligned and edited

with BioEdit 7.0.0 The phylogenetic analysis was performed using the MEGA 3.1 program. The distance model used

was p-distance and the bootstrap test of phylogeny was calculated for 1000 replicates. The creation of the consensus

tree was performed by the Neighboor Joining method and the final tree was drawn with the use of MEGA 3 Tree

Explorer.

2.7 Primer design to amplify the genes involved in pigment production

To amplify the genes proposed to be involved in the indigoidine biosynthesis, the generation of specific primers was

necessary. Due to the lack of available primers to amplify igi genes, we used the gene igi sequences described in

GeneBank to design the forward and reverse primers.

2.8 Amplification of the indigoidine pigment genes

Polymerase Chain Reaction (PCR) was used to amplify each gene with the genomic DNA of the isolate as template.

The amplification reaction was made using the Green Taq Master Mix by Promega and primers designed in the

Microbial Biotechnology and Bioprospecting laboratory at UPR-Mayaguez. The following parameters were used for the

reaction: initial denaturalization at 94 ºC for 3 minutes; 30 cycles of denaturalization at 94 ºC for 30 seconds, annealing

at 46.9 ºC for the igiA gene, 45.0 ºC for the igiB gene, 54.9 ºC for the igiC gene and 51.5 ºC for the igiE gene for 30

seconds, extension at 72 ºC for 1 minute and final extension at 72ºC for 10 minutes. The igiD gene measures 3,866 bp.

To amplify the gene completely, two reactions were made. For the first reaction the specific primers were igiD1 P1 and

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igiD1 P2. The following parameters were used for the reaction: initial denaturalization at 94 ºC for 3 minutes; 30 cycles

of denaturalization at 94 ºC for 30 seconds, annealing at 47.0 ºC for 30 seconds, extension at 72 ºC for 1 minute and

final extension at 72 ºC for 10 minutes. For the second reaction the specific primers were IgiD2 P1 and igiD2 P2. The

following parameters were used for the reaction: initial denaturalization at 94 ºC for 3 minutes; 30 cycles of

denaturalization at 94 ºC for 30 seconds, annealing at 50.0 ºC for 30 seconds, extension at 72 ºC for 1 minute and final

extension at 72 ºC for 10 minutes.

The PCR products were purified with a PCR purification kit (Quagene Inc) following the manufacturer

especifications. DNA sequencing of the PCR product was done in the facilities Macrogene USA

(www.macrogenusa.com). In silico analysis was done by using available online databases such as Gen Bank in NCBI

using the program BLAST.

3. Results

3.1 Soil analysis, isolation and characterization of two BPB

Two blue pigmented bacteria were characterized in this study. One isolated from the south region of Puerto Rico (BPB-

W) (accession number HM236169) and the other one from the south-west region of Puerto Rico (BPB-SW) (accession

number HM236170). The mineral composition test performed to the soil sample from where the BPB-S was isolated

showed strong signals for quartz as a common mineral, follow by vermiculate. In the soils where the BPB-SW was

isolated besides quartz, the other weak peaks that represent minerals like anorthite, calcite and vermiculite were

detected (data not shown).

Macroscopically, and in both blue pigmented isolates, the colonies showed a circular form, convex elevation, entire

margin, and metallic deep blue color. Both isolated bacteria were gram-negative. The BPB from the south region forms

single rods or pairs of approximately 1.0- 2.5 µm of length by 0.4 µm of width, and the isolated from the south-west

region form curved rods that measured approximately 1.0-1.9 µm of length by 0.4 µm of width. Endospores or capsules

were not observed.

1mm

1mm

Figure 1. Macroscopic and microscopic analysis of the Blue Pigmented Bacteria (BPB) isolated in Puerto Rico. The colonies

from the BPB-W (A) and the colonies from the BPB-SW (B) were round, entire, and elevated and the colonies from both measured

approximately 2mm. Gram staining of the isolated from the south region (C) and south-west region (D) demonstrates that both

bacteria are gram- rods (bars measure 5µm). Scanning Electron Microscopy of the BPB-W (E) and BPB-SW (F), were performed at

the UPR-Mayagüez (Biology Department Microscopy Center).

Physiologically, the optimal grow parameters was determined. The results indicate that the bacteria grow better at

32 ºC, but without pigmentation. The optimal temperature for the pigment production was 25 ºC. Both bacteria were not

able to grow in the absence of oxygen. The bacteria were also able to grow in a pH that range from 5.5 to 8.5.

Biochemically, the tests performed to the BPB isolates and compared with the results obtained with V. indigofera

(ATCC 19706), showed no differences.

C

B

A

D

E

F

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3.2 Molecular analysis

Amplification of the 16S rDNA was done by PCR with positive amplification of approximately 1500bp . Sequencing

of the whole 16S rDNA gene was performed by Macrogene USA. In-silico analysis of both BPB suggested that they are

99% similar with each other, but 97% similar with V. indigofera. The phylogenetic analysis demonstrates the two

isolates bacteria are very similar to V. indigofera.

Figure 2. Phylogenetic analysis of the BPB. The generation of the consensus tree was made by the Neighbor-Joining method and

the final tree was drawn with the use of MEGA 3 Tree Explorer. Numbers in the nodes are the bootstrap values. For the first figure

(A) the bar represents 0.02 substitutions per nucleotide position. The second figure (B) is a magnification of the fragment showing

the BPB isolates with Vogesella indigofera. The bar represents 0.002 substitutions per nucleotide position.

3.3 Primer design and amplification of the genes involved in the production of the pigment

The igiA, igiB, igiC, igiD and igiE genes were successfully amplified in both BPB from Puerto Rico using the primers

designed in the laboratory (Fig. 3).

Gulbenkiania mobilis 16S strain E4FC31T

Paludimonas yongneupensis strain 5YN8-15

Chromobacterium violaceum ATCC 12472

Vogesella indigofera strain ATCC 19706T

BPB-W

BPB-SW

Laribacter hongkongensis

Burkholderia cepacia

Janthinobacterium lividum ATCC 12473

Pseudomonas aeruginosa strain ATCC 10145

Pseudomonas putida strain ATCC 12633

Pseudomonas fluorescens strain ATCC 1352

Pseudomonas syringae85

100

100

100

100

100

83

78

52

99

0.02

Vogesella indigofera strain ATCC 19706T

BPB-W

BPB-SW100

0.002

A

B

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Figure 3. Amplification of the indigoidine genes of the Blue Pigmented Bacteria (BPB) from Puerto Rico. The putative

indigoidine biosynthetic genes (igi) were amplified for both BPB-S (A), and BPB-SW (B). The samples were analyzed by

electrophoresis in a 1% agarose gel, using 1Kb as molecular marker. The sample from lane 1-6 represents the amplicons from the

genes igiA, igiB, igiC igiD (lane 4 and 5) and igiE, respectively.

The in silico analysis of the putative indigoidine biosynthesis genes are shown in Table 1. The identity of the genes

when compare to the sequence publish in the GenBank data base range from 87 to 95% in BPB-S and a range from 92

to 95% in BPB-SW of max identity. In both cases the gene with the lowest identity was igiE and the most similar was

igiB.

Table 1. In-silico analysis of the indigoidine genes from Blue Pigmented Bacteria-South and Blue Pigmented Bacteria-South West.

BPB-W= Blue pigmented bacterium from the west; BPB-SW= Blue Pigmented bacterium from the south west.

For each one of the amplifications the E value was 0.

4. Discussion

The main purpose of this research was to identify and characterized blue pigmented bacteria (BPB) isolated from Puerto

Rico. BPB are very rare so XRD analysis were carried out to see if some unusual mineral or metals were present. The

minerals present in both soils are common in these types of soils, so no unusual minerals were present to explain the

occurrence of BPB. The common feature of the soils was that they occur near water bodies, suggesting that this

bacterium prefers environments with high water activity (Aw).

The data obtained from both BPB were compared with the published data by Grimes (1997) [9], it can be concluded

that there is no macroscopic, morphological, or physiologic (based on the tests performed) differences between them.

Both bacteria are gram-negative rods. But they are different when compared in size. It appeared that the isolated from

Gene length in V. indigofera

igiA

igiB

igiC

igiD

igiE

BPB-W

igiA

igiB

igiC

igiD

igiE

BPB-SW

Query length Query coverage Max Identity

882bp 816bp 98% 93% HM488354

875bp 798bp 98% 95% HM488355

830bp 743bp 98% 92% HM488356

3866bp 2502bp 83% 93% HM488357

1226bp 1150bp 79% 92% HM488358

882bp 814bp 100% 93% HM488359

875bp 797bp 98% 95% HM488360

830bp 720bp 98% 92% HM488361

3866bp 2375bp 99% 94% HM488362

1226bp 917bp 92% 87% HM488363

Accession number

A B

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the south range from 1.0- 2.5 µm and the isolated from the south-west range from 1.0 -1.9 µm. So both bacteria differ in

size. Physiologically both are aerobic, the optimal growth pH is at 7 and they can not grow in excess of 1.5% of NaCl.

With the tests that were performed in this work, no physiological differences were found with the two isolated bacteria

and Vogesella indigofera., but further analysis is recommended to determine if there are any physiological differences

between them.

The optimal temperature for growth for both bacteria was 32 °C, but at this temperature neither of the BPB produced

the blue pigmentation. The optimal temperature to produce the pigment was measured at 25 °C. Pigments have been a

very important taxonomic tool to identify bacteria species, but identification it’s complicated because there are many

physical conditions that can make a bacterium change its pigmentation. Some bacteria may lose the pigments when they

are in stress (6). In our case it seems that temperature is the stress point that can make the bacteria change its color. The

experiment performed suggests that the bacteria may be sensitive to thermal shock, causing the bacteria to lose the

pigment at temperatures higher or lower than 25 °C. This phenomenon can be observed in Serratia marcescens which

produce the red pigmentation only when grown at 25°C [15].

Molecularly, all of the genes that have been proposed to be involved in the pigment production (indigoidine) were

amplify in both BPB. This suggests that both isolates have genetic potential to produce the blue pigment. All of the

genes were very similar between them. This is confirmed phonotypically by the presence of the blue pigment in the

colonies growing on solid media. The igi gene that shows to be the most diverse id the igiE gene. Because it has been

reported that this gene encodes a component of a ABC transporter probably involved in indigoidine export, the DNA

difference could be related to changes that can affect the structure or function of igiE.

When the results are compared to the portion of the 16S rDNA gene, both bacteria have a 99% homology to each

other and a 97% homology with Vogesella indigofera. Also the molecular analysis of the whole 16S rDNA gene and the

phylogenetic tree suggest that the bacteria may be of the Vogesella genus, but they may be different species. The

phylogenetic tree place the isolates in the same branch together and near V. indigofera. This suggests that both BPB are

very similar and are closely evolutionary related to each other, but not identical to V. indigofera, suggesting two

possible new Vogesella-BPB species. In order to confirm this, DNA-DNA hybridization using the Vogesella indigofera

type specie needs to be done. Also, the BPB isolates from Puerto Rico have some physiological difference with the

Vogesella sp., such as the inability to produce the pigment in liquid medium. This also reinforces the possibility of

having a new species or sub-species.

Acknowledgements. To the UPR Mayagues Marc and Sloan Programs and PRLSAMP.

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