Each year, millions of tons of xenobiotic compounds are applied globally as
herbicides in agricultural production area or in farmyards. As an outcome of
this extensive environment input, natural water in rivers, lakes and aquifers
has been contaminated with the trace amounts of herbicides compound. In Malaysia,
organochlorine herbicides are widely used especially to clear weeds or unwanted
crops. Environmental contamination of natural inland water have been a great
concern, since, most of these herbicide compounds are very persistent, bioaccumulative
and their toxicity can pose harmful effects to human and natural environment.
Most of Southeast Asian countries like Malaysia, Thailand, Indonesia and Vietnam
have banned the use of herbicide compounds since 1990s, but the residues are
still detected in water and soil or sediments at the significant levels (Ibrahim
et al., 2002).
Microbial catabolism of halogenated compound (Fig. 1) by
dehalogenase producing bacteria has been well studied by Hardman
(1991), Leisinger and Bader (1993),
Janssen et al. (1994), Olaniran et al.
(2001, 2004), Jing and Huyop (2007),
Jing et al. (2008) and Ismail
et al. (2008).
A variety of halogenated compounds such as haloacids, which are produced by
chemical industries, are degraded through dehalogenation by microbial dehalogenases
that involve carbon-halogen bond cleavage (Copley, 1998).
Previous investigation suggested that two different kinds of dehalogenases
were found in cells of Pseudomonas sp. strain S3 grown on D, L-2CP (Thasif
et al., 2009). Both enzymes have great potential in biotransformation
study. However, taxonomic identity and phylogeny of this unique halo-degrading
bacterium has not been determined so far. Thus, we are interested in identifying
the evolutionary relationship of this degradative bacteria among other Pseudomonas
sp. group and/or dehalogenase producing bacteria in order to verify which bacteria
was closely related and might be potentially degrade the same compound as Pseudomonas
sp. strain S3. Using phylogenetic analysis, a complete 16S rRNA gene sequence
of the Pseudomonas strain S3 was subjected to a specific computational
tool for the gene sequences analysis and phylogenetic tree construction. Computational
phylogenetics is the application of computational algorithms, methods and programs
to phylogenetic analyses. MEGA4 molecular software was used in reconstructing
a phylogram with the distance method approach to infer evolutionary distance
of genes sequence. This software functionality has evolved to include the creation
and exploration of sequence alignments, the estimation of sequence divergence,
the reconstruction and visualization of phylogenetic trees and testing of molecular
In this study, we have constructed two different phylograms which inferred the evolutionary relationship of Pseudomonas strain S3 to other Pseudomonas sp. and also their evolutionary distance among dehalogenase producing bacteria. The current goal is to assemble a phylogenetic tree representing a hypothesis about the evolutionary ancestry of a set of genes, species, or other taxa. The analysis will also able to predict the characteristics of an unknown microorganism based on their phylogenetic relationship.
MATERIALS AND METHODS
Characterization of Pseudomonas sp. S3: The 16S rRNA gene sequence
of Pseudomonas sp. S3 was obtained by sequencing analysis. Chromosomal
DNA of Pseudomonas sp. S3 was prepared and sent for sequencing (1st Base
Laboratory Malaysia) using initial primers as described by Fulton
and Cooper (2005). The 16S rRNA gene sequencing was subjected to MEGA4 computational
tool in order to find regions of local similarity between selected sequences
and also to generate a phylogenetic tree or phylogram which inferred evolutionary
relationship. The 16S rRNA gene sequence of Pseudomonas sp. S3 was used
to perform a BLAST search for homology study. From the selected of closely related
sequences, a phylogram was established in Mega4 software in order to investigate
the evolutionary pathway of Pseudomonas strain S3. In Mega4, homology
analysis of selected sequences are aligned together by aligned Explorer/Clustal
W. Phylogram was constructed using neighbor-joining method option (Tamura
et al., 2007). Phylograms were rebuilt based on pairwise distance
among sequence. Homology analysis of the 16S rRNA for S3 was also carried out
among dehalogenase producing bacteria 16S rRNA gene and its phylogenetic tree
was also constructed.
Collecting a set of homologous nucleotide sequences: The full 16S rRNA
gene sequence from Pseudomonas sp. S3 was analyzed at http://www.ncbi.nlm.nih.gov/BLAST/,
using BLASTn option. The BLASTn search will graphically displayed online on
the Distribution of Blast Hits on the Query Sequence.
Building the phylogram of Pseudomonas strain S3 using BLAST web page: Phylogenetic tree of Pseudomonas sp. S3 and others evolutionary related bacteria were constructed using distance tree of results icon that available in same page of the list of homologous sequences (BLAST web page). The phylogenetic tree of Pseudomonas sp. S3 was presented and neighbour joining method was used to show relatedness and distance matrix of Pseudomonas sp. S3 in evolutionary pathway.
Reconstruction of phylogram using Mega4 software: Initially, a selected 16S rRNA gene sequences were converted into FASTA format and analyzed using alignment explorer/Clustal W in Mega4 (Fig. 2).
After completing sequence alignment by Clustal W, all output data were used
to reconstruct phylogram. The evolutionary history was inferred using the neighbor
joining method (Saitou and Nei, 1987). The Neighbor Joining
(NJ) method constructs the tree by sequentially finding pairs of neighbors,
which are the pairs of Operational Taxonomic Units (OTUs) connected by a single
interior node. The clustering method used by this algorithm is quite different
and does not attempt to cluster the most closely related OTUs, but rather minimizes
the length of all internal branches and thus, the length of the entire tree.
sequence alignment analysis by Clustal W
The bootstrap consensus tree inferred from 500 replicates is taken to represent
the evolutionary history of the taxa analyzed. The percentage of replicate trees
in which the associated taxa clustered together in the bootstrap test (500 replicates)
are shown next to the branches (Felsenstein, 1985).
The tree is drawn to scale, with branch lengths (next to the branches) in the
same units as those of the evolutionary distances used to infer the phylogenetic
tree. The evolutionary distances were computed using the p-distance method (Tamura
et al., 2004) and are in the units of the number of base substitutions
per site. All positions containing gaps and missing data were eliminated from
the dataset (Complete deletion option).
Identification of Pseudomonas strain S3: A bacterial species
designated as S3 was isolated from paddy (rice) field agricultural area. The
bacteria were grown aerobically at 30°C in 100 mL of a liquid minimal medium
containing D, L-2-Chloropropionic acid as sole source of carbon. The 16S rRNA
gene analysis was sequenced using FD1 and rP1 primers (Fulton
and Cooper, 2005). The sequence comprises of 1469 nucleotides lacking the
very proximal 5 and terminal 3 regions corresponding to the universal
primers used and was submitted to the GenBank with accession number of FJ968758.
The S3 16S rRNA gene sequence was subjected to BLAST search (BLASTn). The sequence was compared with other sequences that contained in library database. This technique provides an algorithm for comparing primary biological sequence information. From BLASTn results, the most similar sequence was matched to the Pseudomonas sp. R1 (accession No. EU272817) with 98% sequence identity (Fig. 3). Hence, the bacteria was then designated as Pseudomonas sp. S3.
A phylogenetic tree of Pseudomonas sp. S3 and related species was also established using BLAST output homepage (Distance tree). From the phylogram, Pseudomonas sp. S3 located in the clade within others Pseudomonas sp. (Fig. 4). Strain S3 was diverged from a same node with Pseudomonas sp. strain R1. Strains S3 and R1 are sisters group which had minimum genetic distance.
Evolutionary relationship of Pseudomonas sp. S3 among Pseudomonas
sp.: In this study, ten different Pseudomonas sp. from each
major group of Pseudomonas were selected as operational taxonomic units
(OTUs) in order to investigate the evolutionary relationship of Pseudomonas
sp. S3 among other Pseudomonas sp. from various major groups. There
are 14, 678 base nucleotides from various 16S rRNA gene of Pseudomonas
sp. were analyzed and multiple alignment were constructed using Clustal W. All
results were based on the pairwise analysis of 10 sequences. The longest genetic
distance was between sequence of Pseudomonas sp. S3 and Pseudomonas
luteola (0.199) while the shortest pairwise distance was between Pseudomonas
sp. S3 and Pseudomonas chororaphis (0.170) as in Fig. 5.
The data was generated based on the proportion of different homologous sites
known as observed distance or p-distance and it is expressed as the number of
nucleotide differences site. All positions containing gaps and missing data
were eliminated from the dataset (Complete deletion option). There were a total
of 1309 positions in the final dataset.
16S rRNA gene identity using BLAST output obtained from NCBI database
tree of Pseudomonas sp. S3 among related species from reconstructed
of BLAST results
Data set illustrated in Fig. 5. was then converted into a
proper phylogenetic analysis (Fig. 6) suggesting Pseudomonas
sp. S3 was not located within the clade of other Pseudomonas species.
Operational Taxonomic Units (OTUs) was selected from different major groups
of Pseudomonas sp.
From all selected species, Pseudomonas chlororaphis from P.chlororaphis
group was closely related to the Pseudomonas sp. S3 (Fig.
6). Pseudomonas chlororaphis is known as a biocontrol agent against
certain fungal plant pathogens via production of phenazine type antibiotics
(Chin-A-Woeng et al., 2000). Other Pseudomonas
sp. that closely related to the S3 was Pseudomonas syringae strain BC2366.
Pseudomonas syringae is a rod shaped Gram-negative bacterium with polar
flagella. It is also a member of the Pseudomonas genus and placed in
the Pseudomonas syringae group (Anzai et al.,
number of base substitutions per site from analysis between sequences.
All results are based on the pairwise analysis of 10 sequences. Analyses
were conducted using the p-distance method in MEGA4. All positions containing
gaps and missing data were eliminated from the dataset (Complete deletion
option). There were a total of 1309 positions in the final dataset
Pseudomonas syringae is a plant pathogen which can infect a wide range
of plant species. Pseudomonas sp. S3 has distance relationship with Pseudomonas
luteola. Pseudomonas luteola is a Gram-negative, rod-shaped, motile
bacterium that can cause peritonitis, cellulitis and bacteremia (Kodama
et al., 1985). It has also been shown to reduce and hence decolorize
azo dyes (Hu, 2001). Based on 16S rRNA analysis, Pseudomonas
luteola has been placed in the Pseudomonas stutzeri group.
Evolutionary relationship of Pseudomonas sp. among dehalogenase producing bacteria: In this study, ten dehalogenase producing bacteria were selected. The dehalogenase producing bacteria were comprised of Pseudomonas strain R1 (EU272817), Pseudomonas strain S3 (FJ968758), Pseudomonas corrugata strain SB4 (AY050495), Methylobacterium extorquen strain DM4 (AF227128), Comamonas sp. strain CY01 (EU515237), Stenotrophomonas maltophilia strain SB5 (AY050496), Methylobacterium strain HN2006B (AM231910), Serratia marcescens strain HL1 (EU371058), Rhodococcus strain HN2006A (AM231909) and Dehalococcoide strain BAV1 (AY165308).
There was limited number of data of 16S rRNA gene sequence published in the
database for dehalogensase producing bacteria. All the gene sequences were obtained
from NCBI data base. There were 13, 318 base nucleotides of 16S rRNA gene from
ten different dehalogenase producing species were analyzed using multiple sequences
alignment constructed by Clustal W. The number of base substitutions per site
was shown in Fig. 7. One of the closest species was between
Methylobacterium HN2006b and Methylobacterium extorquens apart
from S3 and R1. All results were based on the pairwise analysis of 10 sequences
conducted using the p- distance method with 624 positions in the final dataset.
tree showing evolutionary relationships of Pseudomonas sp. S3 among
Pseudomonas species that was determined by the analysis of 16S
rRNA gene sequences. The scale bar represents 0.005 substitutions per
site. Bootstrap values above 40% are shown at the nodes (based on 500
Figure 8 showed all the dehalogenase producing bacteria in
the phylogenetic tree presented as Operational Taxonomic Units (OTUs) or external
nodes. There are eight Hypothetical Taxonomic Units (HTUs) or internal nodes
that illustrated in the bootstrap consensus phylogram. Pseudomonas sp.
S3 and Pseudomonas sp. R1 are sisters group because they sharing a common
node or ancestor. Pseudomonas sp. S3 was clustered with a good bootstrap
support (100%) within a clade consisting Pseudomonas sp. R1 and Pseudomonas
corrugata strains SB4. The results also suggested that Pseudomonas sp.
R1 was the most closely related to the Pseudomonas sp. S3 with a genetic
distance of 0.040 substitutions per site. Pseudomonas sp. R1 is a soil
pseudomonad that able to degrade monochloroacetic acid (MCA) (Ismail
et al., 2008). Other related species that located in the same clade
as Pseudomonas sp. S3 was Pseudomonas corrugata strain SB4. This
species was diverged from Pseudomonas sp. S3 at 0.082 base substitutions
per site. Pseudomonas corrugata strain SB4 was isolated from soil contain
a mixture of aniline and 4-chloroaniline (4CA) as principal carbon sources (Radianingtyas
et al., 2003).
number of base substitutions per site from analysis between sequences
is shown. All results are based on the pairwise analysis of 10 sequences
phylogenetic tree showing evolutionary relationships of Pseudomonas
sp. S3 among dehalogenase producing bacteria that was determined by
the analysis of 16S rRNA gene sequences. The scale bar represents 0.02
substitutions per site. Bootstrap values above 70% at the nodes (based
on 500 resamplings)
Pseudomonas sp. have ability to use many organic compounds as carbon and energy sources. On the other hand, Pseudomonas generally lacks the hydrolytic enzymes necessarily to break down polymer into their component monomers. These nutritionally versatile pseudomonads contain numerous inducible operons because the catabolism of unusual organic substrates often requires the activity of several different enzymes. Pseudomonads are ecologically importants organisms in soil and water and are probably responsible for degradation of many soluble compounds derived from breakdown of plant and animal materials. They are also capable of breaking down many xenobiotic compounds, such as pesticides and other toxic chemicals and thus, important agent of bioremediation in the environment.
S3 was originally isolated from a paddy rice field and capable of degrading
the D, L-2-Chloropropionic acid. The bacterial species was identified as gram
negative rods in chains (Thasif et al., 2009).
The identity of the bacteria was studied using phylogram and evolved from the
same ancestor with others Pseudomonas sp. Using molecular analysis of
phylogenetic software, strain S3 was closely related to the genus Pseudomonas
chlororaphis group. This is possibly due to this strain S3 have the same
characteristics to the species that belonged into this group which can degrade
Strain S3 was also closely related to the Pseudomonas sp. strain R1.
Both strains produced dehalogenases. Pseudomonas sp. S3 and R1 were classified
as gram negative bacteria, motile and also ubiquitous in soil and water. Both
bacteria produced dehalogenase enzyme and could degrade halogenated compound
as a carbon source. Even though both organisms were closed relationship, but
their enzyme characteristics were not identical. Pseudomonas sp. S3 for
example produced both types of dehalogenase specific to D- and L- isomer whereas,
Pseudomonas sp. R1 only produced dehalogenase that is non-stereospecific
(Ismail et al., 2008).
Pseudomonas sp. S3 has distant relationship to Dehalococcoide
sp. BAV1 even though they were classified as Dehalogenase producing bacteria.
Their features indicated that both bacteria were not similar in morphology as
expected. Dehalococcoides was related to the green non-sulfur bacteria
(Chloroflexus group). Dehalogenase from Dehalococcoides preferred
to de-chlorinated ethenes rather than chlorinated aliphatic acid compound (He
et al., 2003).
In conclusion, this study provides the identity of D, L-2-Chloropropionic acid
degrading bacteria using phylogenetic study. The evolutionary relationship of
Pseudomonas sp. S3 has been depicted from computational program based
on the molecular phylogenetic. The current research is very useful in identifying
the genus of the isolated bacterial species. From the results, it was also possible
to predict their general properties for further characterization.
This study was partly supported by Fundamental Research Grant Schemes 78307 from Ministry of Higher Education (MOHE) Malaysia.