Catabolic versatility of aromatic compound-degrading halophilic bacteria

FEMS Microbiology Ecology 54 (2005) 97–109 www.fems-microbiology.org

Catabolic versatility of aromatic compound-degrading halophilic bacteria Marı´a Teresa Garcı´a, Antonio Ventosa, Encarnacio´n Mellado

*

Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, C/ Profesor Garcia Gonzalez no. 2. 41012 Sevilla, Spain Received 14 December 2004; received in revised form 3 March 2005; accepted 8 March 2005 First published online 29 April 2005

Abstract There is growing interest in the development and optimization of bioremediation processes to deal with environments with high salinity that are contaminated with aromatic compounds. To estimate the diversity of moderately halophilic bacteria that could be used in such processes, enrichments were performed based on growth with a variety of aromatic compounds including phenol as a model pollutant. A group of bacteria that were able to grow over a wide range of salt concentrations were isolated, with the majority of these assigned to the genus Halomonas using phenotypic features and 16S rRNA sequences comparison. PCR amplification with degenerate primers revealed the presence in these isolates of genes encoding ring-cleaving enzymes in the b-ketoadipate pathway for aromatic catabolism: catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase. Furthermore, the activity of these two enzymes was detected in the newly described species Halomonas organivorans. Together, these studies indicate that moderately halophilic bacteria have the potential to catabolize aromatic compounds in environments with high salinity.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Extremophiles; Halophiles; Organic compounds

1. Introduction Moderately halophilic bacteria are extremophilic microorganisms adapted to live in saline environments. These halophiles grow optimally in media containing between 3% and 15% NaCl [1], although the main characteristic of this group of organisms is their ability to grow in a very wide range of salt concentrations. In recent years, moderate halophiles have been explored for their biotechnological potential in different fields, mainly as a source of extracellular enzymes or compatible solutes [2,3]. Saline and hypersaline environments are frequently contaminated with organic compounds as a result of industrial activities [4,5]. Contamination of these habi*

Corresponding author. Tel.: +34 95 455 38 06; fax: +34 95 4628162. E-mail address: [email protected] (E. Mellado).

tats constitutes a serious environmental problem mainly due to the high toxicity exhibited by some organic compounds. In most cases, biodegradation constitutes the primary mechanism for contaminant removal. However, biodegradation programs are difficult to perform under saline conditions [4,6]. In this case, compounds are not degraded or are degraded slowly and with low efficiency. One alternative for overcoming this problem is the use of halophilic bacteria adapted to live in such extreme conditions. Several studies have demonstrated bacterial degradation of aromatic compounds in saline conditions [3,5,7,8] and ecological studies concerning the ability of these microorganisms to degrade different aromatic compounds are still in their infancy. In fact, the extent and activity of halophilic aromatic compound-degrading microorganisms in nature is unknown. While moderate halophiles present not only an advantage with respect to their growth over a wide

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2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsec.2005.03.009

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M.T. Garcı´a et al. / FEMS Microbiology Ecology 54 (2005) 97–109

saline concentration, but also their ability to grown in simple media [2] little information is available on the use of these extremophiles for bioremediation. Maltseva et al. [9] described the first aerobic halophilic bacterium able to degrade a chloroaromatic compound. Moreover, a moderately halophilic bacterium, Arhodomonas aquaeoli that degraded oil aerobically has been isolated from a subterranean brine [10], and novel halophilic species of the genus Marinobacter have been reported to be able to degrade hydrocarbons and some crude oil components [11–13]. Phenol utilization by moderately halophilic bacteria under aerobic conditions has also been described [14–17]. More recently, Maskow and Kleinsteuber [18] reported the growth of a haloalkaliphilic Halomonas strain on phenol, describing the synthesis of compatible solutes to maintain the osmotic balance. Garcı´a et al. [19] described the characterization of a novel moderate halophile, Halomonas organivorans, able to degrade different aromatic compounds. In this work, we have analyzed a halophilic bacterial population that represents the dominant cultivable bacteria in enrichment cultures with different environmental aromatic compounds such as benzoic acid, p-hydroxybenzoic acid, cinnamic acid, phenylacetic acid, phenylpropionic acid, phenol, p-coumaric acid, ferulic acid, salicylic, or p-aminosalicylic acid as sole carbon and energy sources. These compounds have been selected as they represent low-molecular-weight aromatic compounds that persist in effluents from the industries located close to the saline areas studied, such as oil refineries and food-processing industries. Ring-cleavage dioxygenases constitute central enzymes in the bacterial recycling of aromatic compounds. In the b-ketoadipate pathway, catechol and protocatechuate are cleaved between their two hydroxyl groups by catechol 1,2-dioxygenase (1,2-CTD) or protocatechuate 3,4-dioxygenase (3,4-PCD). In the meta-cleavage pathway the ring fission occurs adjacent to one of the hydroxyls (extradiol cleavage) and the main enzymes involved in this fission are the catechol 2,3-dioxygenases (2,3-CTD) and protocatechuate 4,5-dioxygenases (4,5-PCD) [20]. These pathways have been identified in the main aromatic compounddegrader bacteria, including species of the genera Acinetobacter, Alcaligenes, Bacillus and Pseudomonas among others [20–22]. However, the prevalence of these catabolic pathways is unknown in moderately halophilic bacteria. In this sense, another objective of this work was to explore the degradative potential of the halophilic bacteria, by analysis of their catabolic mechanisms used for the degradation of different aromatic compounds. This work has given us the first analysis of a bacterial community able to degrade aromatic compounds under saline conditions and the isolation and characterization of new isolates constitutes a preliminary step toward understanding the ecology and fate of aromatic compounds in saline habitats.

2. Materials and methods 2.1. Sampling sites, isolation of bacteria and growth conditions Moderately halophilic strains were isolated from water and sediment of salterns and hypersaline soils in different areas of South Spain: Huelva estuary, Isla Bacuta, Isla Cristina (Huelva) and San Fernando (Cadiz). The sampling sites selected are subject to contamination with low-molecular weight aromatic compounds due to exposure to industrial wastes. Salinity on the sampling sites was measured with a salt analyzer (Extech Instruments, Waltham, USA). A standard enrichment method was used for isolation of organic compound-degraders. The organic substrates were provided as sole carbon and energy source in the minimal medium M63 [23] supplemented with 10% NaCl. A 250-ml flask containing 50 ml of the minimal medium was inoculated with a few grams of soil or 1 ml of saline water. The flask was incubated 48 h at 37 C while shaking (200 rpm). Subsequently, 2 ml of the culture was transferred twice to 50 ml of fresh M63 + 10% NaCl for a further 2-day incubation. Serial dilutions of the third culture were plated out on M63 + 10% NaCl plates supplemented with the substrate and incubated at 37 C. The fastest-growing colonies were selected for further characterization. A 250-ml flask with 50 ml of the mineral medium containing no substrate was used as negative control. Stock solutions (0.5 M) of benzoic acid, p-hydroxy-benzoic acid, cinnamic acid, salicylic acid, phenylacetic acid, phenylpropionic acid, p-coumaric acid, ferulic acid and p-aminosalicylic acid were prepared and when needed as substrates were added to the M63 saline medium at final concentrations of 1–5 mM. Phenol and p-cresol were added to the minimal medium at concentrations of 5 mM and 2 mM, respectively. To explore the ability of the isolates to grow on a variety of carbon sources, substrates were added to the minimal agar saline medium M63 + 10% NaCl at the different concentrations indicated above. The plates were inoculated with the selected strains and the utilization of the organic compound was tested by monitoring the growth of the isolates. 2.2. Phenotypic characterization of the isolates To characterize the isolates phenotypically, standard phenotypic tests were performed: Gram reaction, cell morphology, motility, growth at different salt concentrations, catalase and oxidase tests, hydrolytic activities [24], and the ability to use 95 different compounds. The capability to oxidize these compounds was performed with the Biolog automatic identification system (Biolog Inc., Hayward, USA). Strains were grown on

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isolate medium at 37 C for 24 h, and suspended in (warm) sterile saline medium (3% NaCl), within the density range specified by the manufacturer with a Biolog photometer model 21101. Immediately after suspending the cells in the saline solution, the suspensions were transferred into sterile pipetter reservoirs and the Biolog GP (for Gram-positive) and GN (for Gram-negative) MicroPlates inoculated with 125 ll of the cell suspension per well. The inoculated Biolog plates were incubated at 37 C for 24 h and the results were read with a MicroPlate Reader using the Microlog 3.59 computer software to perform automated reading and identification. 2.3. Determination and analysis of 16S ribosomal RNA gene DNA of isolated strains was extracted and precipitated using CTAB and following the standard protocol for bacterial genomic DNA preparations [25]. PCR amplification of the 16S rRNA gene with the forward primer 16F27 and the reverse primer 16R1488 was carried out using previously described methods [26]. Direct sequence determination of the PCR-amplified DNA was performed using an automated DNA sequencer model 3100 (Applied Biosystems). Data analyses were performed using the ARB software package [27]. Evolutionary distance matrices were calculated using the algorithm of Jukes and Cantor [28]. Different phylogenetic trees using different methods (distance matrix, maximum parsimony and maximum likelihood) were constructed and compared to elucidate the confidence of local topologies. 2.4. PCR detection of dioxygenase enzyme genes To detect the presence of three catabolic genes encoding key enzymes of the metabolic pathways, PCR amplification was performed using degenerate PCR primers for catechol 1,2-dioxygenase (1,2-CTD), catechol 2,3 dioxygenase (2,3-CTD) and protocatechuate 3,4dioxygenase (3,4-PCD) genes. The primers for the amplification were the following: cat1 (5 0 -ACCATCGARGGYCCSCTSTAY-3 0 ) and cat3 (5 0 -GTTRATCTGGGTGGTSAG-3 0 ) for 1,2-CTD genes, designed from two conserved regions of different catA proteins, spanning residues 100–106 and 232–237 in IsoB from Acinetobacter radioresistens (Accession No. AAG16896); cat2.3.1 (5 0 -GARCTSTAYGCSGAYAAGGAR-3 0 ) and cat2.3.2 (5 0 -RCCGCTSGGRTCGAAGAARTA-3 0 ) for 2,3-CTD genes, corresponding to positions 118–124 and 255–261 of NahH from Pseudomonas putida (Accession No. A27389); pro3.4.2 (5 0 -GCSCCSCTSGAGCCSAACTTC-3 0 ) and pro3.4.4 (5 0 -GCCGCGSAGSACGATRTCGAA-3 0 ), designed from conserved regions of PcaH, coding for the b-subunit of the enzyme (residues 118–124 and 224–230 of PcaH from Pseudomonas aeru-

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ginosa PA01, Accession No. NP_248843) for amplification of 3,4-PCD genes. These primers were designed by reference to the sequences of different dioxygenase genes. The most conserved regions of these genes were used to design primers. Each PCR mixture contained: 5 ll of 10· reaction buffer (Promega), 2.5 ll of 25 mM MgCl2, 8 ll of dNTPs (200 lM each), 50 pmol of the appropriate primers, 0.25 ll of Taq polymerase (Promega) and sterile distilled water to adjust the total volume to 50 ll. The PCR conditions for the amplification of 1,2-CTD and 3,4PCD encoding genes consisted of an initial cycle of 5 min at 95 C, followed by 35 cycles of: denaturation at 94 C for 1 min, annealing at 50 C for 1 min, and extension at 72 C for 1 min. Similar conditions were applied for amplification of the 2,3-CTD gene fragments, except that lower annealing temperatures were assayed (40–45 C). The amplified products were analyzed on 1% (w/v) agarose gels stained with ethidium bromide and photographed with UV illumination. 2.5. Cloning of Taq polymerase-amplified PCR products, sequence determination and analysis The amplified PCR products were cloned using the TOPO TA cloning kit (Invitrogen) following the instructions of the manufacturer. This kit provides an efficient strategy for the cloning of the PCR products into the plasmid vector pCR 2.1-TOPO, which possesses the specific priming sites for sequencing. DNA sequences were determined with double-stranded templates and primers that recognized the cloning vector. DNA sequencing was performed with an automated DNA sequencer model 3100 (Applied Biosystems). The DNA sequences were analysed using the BLAST program of the National Centre for Biotechnology Information (NCBI). 2.6. Ring-cleavage dioxygenase assays Isolate G-16.1T, designated H. organivorans was grown in the minimal saline medium M63 + 5% NaCl containing the different aromatic compounds. The cells were harvested by centrifugation and washed once with deionized water to remove any salts that might interfere with the enzymatic activity. The cell pellets were resuspended in a volume of breaking buffer (50 mM Tris HCl [pH 7.5], 1 M glycerol, 5 mM ammonium sulphate, 2.5 mM MgCl2, 1 mM EDTA, 1 mM DTT) and sonicated using a tip sonicator; the suspensions were centrifuged at 11,000 · g for 3 min at 4 C. The clear supernatants obtained were used for enzyme assays. Catechol 1,2-dioxygenase was assayed following the formation of cis,cismuconic acid at 260 nm [29]. Catechol 2,3-dioxygenase activity was determined spectrophotometrically by measuring the increase of A375 due to the formation of 2-hydroxy-muconic semialdehyde according to Fetzner et al.

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[30]. Protocatechuate 3,4-dioxygenase activity was measured by monitoring the decrease in A290 due to the oxidation of protocatechuate [31]. Protein concentrations were measured by the method of Bradford using bovine serum albumin as a standard [32]. Specific enzyme activities are reported as nmol product min
1mg protein
1. Activity assays were performed in triplicate and initial rates of the assays were determined and used for calculating mean and standard deviations.

and ferulic acid as substrates were obtained from the four sampling points. Enrichments were obtained from Huelva estuary when using all the compounds selected as experimental models. However, from San Fernando sampling site, enrichments were obtained only when using benzoic acid, p-hydroxy-benzoic acid, salicylic acid, phenylpropionic acid and ferulic acid as substrates. The highest number of degrading isolates was obtained using benzoic acid for enrichment. In this study, we were unable to enrich bacteria able to use p-cresol.

2.7. Nucleotide sequence accession numbers The nucleotide sequences reported in this work have been deposited in the EMBL Nucleotide Sequence Database under Accession Nos. AJ717686 to AJ717731. 3. Results 3.1. Isolation of moderately halophilic bacteria able to degrade different aromatic compounds Four different saline sites in South Spain were used for sampling. These sites were selected because of their proximity to industrial areas such as oil refineries (Huelva) and food-processing industries (Ca´diz). These were the sites where wastewaters containing lowmolecular weight aromatic compounds were discharged to the Atlantic Ocean. According to the earlier chemical analysis [33], phenolic compounds persist in effluents from the oil refineries in the area of Huelva. The salinity of the sampling sites ranged between 4% and 17% (Huelva estuary, 4%; Isla Bacuta, 17%; Isla Cristina, 12% and San Fernando, 10%). Enrichments were performed to screen the response of the halophilic bacterial communities to selected aromatic substrates representing compounds associated with environmental contamination. A collection of 322 isolates able to degrade these organic compounds (Table 1) was assembled following enrichment and selective plating (see Section 2). Controls without the carbon were established for comparison with the aromatic compounds enrichments. Enrichments using benzoic acid, p-hydroxy-benzoic acid Table 1 Numbers of isolates from saline and hypersaline environments in South Spain obtained from the different enrichment cultures using the organic compounds selected as experimental models Isolation site

A

B

C

D

E

F

G

H

I

J

Huelva estuary Isla Bacuta Isla Cristina San Fernando Total

15 11 10 12 48

11 10 8 9 38

6 8 12 – 26

8 – 10 11 29

16 – 14 – 30

8 10 – 8 26

15 9 8 – 32

8 16 10 – 34

8 9 7 11 35

14 10 – – 24

A, benzoic acid; B, p-hydroxy-benzoic acid; C, cinnamic acid; D, salicylic acid; E, phenylacetic acid; F, phenylpropionic acid; G, phenol; H, p-coumaric acid; I, ferulic acid; J, p-aminosalicylic acid.

3.2. Characterization of the isolates and comparative sequence analysis of 16S rRNA genes After performing a preliminary phenotypic characterization that included the colony morphology, Gram reaction, cell morphology, motility, catalase and oxidase tests in order to eliminate replicate strains, 110 of the 322 environmental isolates were selected. From these 110 isolates, 40 were isolated from Huelva estuary, 30 were isolated from Isla Bacuta, 24 were isolated from Isla Cristina and the other 16 were isolated from San Fernando (see supplementary material). The 16S rRNA genes of these selected strains were amplified. Restriction analysis of the amplified genes using the restriction endonucleases AluI and HhaI indicated the presence of different clusters. Forty seven unique isolates representing the major groups were further identified by partial sequencing of their 16S rRNA gene. Moreover, the ability to utilize 95 different compounds (Biolog system) was also tested for these 47 representative isolates of the different species. The Biolog MicroPlate assay assisted in the identification of the isolated bacterial species. In Table 2 and supplementary data, the site of sampling of these isolates is shown. Most studied strains have been isolated from the sampling site presenting the lowest salinity (4%), but located in close proximity to an oil refinery industry. Comparative sequence analyses of the 16S rRNA (approx. 700 bp corresponding to position 1–700 of 16S rRNA from Escherichia coli) of these 47 representative members of the different groups were performed. The sequences were aligned to the most similar 16S rRNA sequences in the data bases and used to construct the phylogenetic trees. Essentially, similar results were obtained with both neighbor-joining and maximumlikelihood method. Thus, only the maximum-likelihood trees are shown (Fig. 1). The majority of isolates were grouped within the family Halomonadaceae, and most of them were closely related to the genus Halomonas, exhibiting similarity values ranging from 91.8% to 100%. Four strains (strains C11.5, C12.3, F18.2 and H21.2) exhibited a high similarity (>99%) to 16S rRNA sequences of Halomonas salina and Halomonas halophila. On the other hand, an important group of 11 strains

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(a)

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(b) Salinicoccus alkaliphilus (AF275710) Salinicoccus hispanicus (AY028927) Salinicoccus roseus (X94559) D23.3 J8.8 Halobacillus karajensis (AJ486874) Halobacillus trueperi (AJ310149) Halobacillus litoralis (X94558) Halobacillus salinus (AF500003) Halobacillus locisalis (AY190534) Halobacillus halophilus (X62174) G19.1

D37.1 Halomonas alimentaria (AF211860) H21.2 F18.2 Halomonas salina (AJ295145) C12.3 C11.5 Halomonas halophila (M93353) B4.4 I14.3 C24.1 E3.1 E16.1 B20.4 C18.3 E34.3 A15.6 F16.2 B22.2 F26.3 F1.2 I21.2 I10.7 Halomonas elongata (X67023) A25.2 A17.6 Halomonas eurihalina (X87218) G17.1 H30.2 H17.3 G20.3 G18.1 G8.2 G2.5 C7.3 D27.5 G22.6 G21.5 Halomonas organivorans G16.1 (AJ616910) F12.7 F27.1 D25.3 E5.3 J21.8 Halomonas venusta (L42618) Halomonas glaciei (AJ431369) E25.7 J24.3 H38.1 H24.1 Chromohalobacter salexigens (X67023) Chromohalobacter israelensis (AJ295144) Chromohalobacter marismortui (X87219) Chromohalobacter canadensis (AJ295143) A27.8 Marinobacter lipolyticus (AY147906) Marinobacter litoralis (AF479689) Marinobacter squalenivorans (AJ439500)

Marinobacter hydrocarbonoclasticus (X67022) Marinobacter aquaeolei (AJ000726)

Fig. 1. Phylogenetic maximun-likelihood trees based on the analysis of 16S rRNA gene sequences, showing the diversity of the moderately halophilic isolates able to degrade different organic compounds. (a) The tree shows the relationship of the Gram-negative isolates to other members of the family Halomonadaceae and the genus Marinobacter (sequence accession numbers in parentheses), (b) tree showing the relationship of the Grampositive isolates to other member of the genera Salinicoccus and Halobacillus. Bar represents 2% sequence difference. In bold are shown the isolates described in the present study. 16S rRNA gene sequences from the isolates correspond to partial sequences of approximately 700 bp.

(strains C7.3, D27.5, G2.5, G8.2, G17.1, G18.1, G20.3, G21.5, G22.6, H17.3 and H30.2) formed a cluster also closely related to H. salina (98% sequence similarity), but the phenotypic differences and the low level of DNA–DNA hybridization suggest that these strains be placed as a new species within the genus Halomonas. The name H. organivorans was proposed for this new species [19]. Two other strains (strains F12.7 and F27.1) are also closely related to H. salina (97.3 and

97.1% sequence similarity, respectively), but when compared to the type species of their closest relatives, they showed important phenotypic differences, suggesting that these strains could constitute a new species within the genus Halomonas. The group with highest number of strains (Fig. 1(a)) shows high sequence similarity (>99.3%) to the type species of the genus, H. elongata. The remaining isolates were identified as organisms related to Halomonas

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Table 2 PCR amplifications of catabolic genes of selected isolates showing other characteristics of the individual strains Isolate

Isolation site

1,2-CTD primersa (414 bp)

3,4-PCD primersb (330 pb)

Enrichment medium

Closest relative

A15.6 A17.6 A25.2 A27.8 B4.4 B20.4 C7.3 C11.5 C12.3 C17.3 C18.3 C19.4 C22.1 C24.1 D27.5 D23.3 D25.3 D37.1 E3.1 E4.5 E5.3 E12.2 E16.1 E25.7 E34.3 F1.2 F12.7 F26.3 F27.1 G2.5 G8.2 G16.1 G17.1 G19.1 G20.3 H1.2 H17.3 H21.2 H24.1 H30.2 H38.1 I14.3 I10.7 I21.2 J4.2 J8.8 J11.3 J21.8 J24.3

Isla Bacuta San Fernando Isla Cristina San Fernando Isla Bacuta Isla Bacuta Huelva estuary Isla Cristina Isla Cristina Huelva estuary Isla Bacuta Huelva estuary Isla Cristina Isla Bacuta Huelva estuary San Fernando Isla Cristina Isla Cristina Huelva estuary Huelva estuary Huelva estuary Huelva estuary Isla Cristina Huelva estuary Huelva estuary Isla Bacuta Huelva estuary San Fernando Huelva estuary Huelva estuary Huelva estuary Huelva estuary Huelva estuary San Fernando Isla Cristina Isla Bacuta Isla Cristina Isla Cristina Isla Bacuta Huelva estuary Isla Cristina Isla Bacuta Isla Bacuta Isla Cristina Isla Bacuta Huelva estuary Huelva estuary Isla Bacuta Isla Bacuta

+ + + + + +






+ +
+
+ + +

+
+ +
+ + + + + + +
+ + + + +

+ +


+




+

+ + + + + + +
+
+ +






+ + + + + + + +
+ + + +
+

+ +

+
c


Benzoic acid Benzoic acid Benzoic acid Benzoic acid p-Hydroxy-benzoic acid p-Hydroxy-benzoic acid Cinnamic acid Cinnamic acid Cinnamic acid Cinnamic acid Cinnamic acid Cinnamic acid Cinnamic acid Cinnamic acid Salicylic acid Salicylic acid Salicylic acid Salicylic acid Phenylacetic acid Phenylacetic acid Phenylacetic acid Phenylacetic acid Phenylacetic acid Phenylacetic acid Phenylacetic acid Phenylpropionic acid Phenylpropionic acid Phenylpropionic acid Phenylpropionic acid Phenol Phenol Phenol Phenol Phenol Phenol p-Coumaric acid p-Coumaric acid p-Coumaric acid p-Coumaric acid p-Coumaric acid p-Coumaric acid Ferulic acid Ferulic acid Ferulic acid p-Aminosalicylic acid p-Aminosalicylic acid p-Aminosalicylic acid p-Aminosalicylic acid p-Aminosalicylic acid

H. elongata H. eurihalina H. eurihalina M. lipolyticus H. elongata H. elongata H. organivorans H. salina/H. halophila H. salina/H. halophila N.D. H. elongata N.D. N.D. H. elongata H. organivorans S. roseus H. venusta H. alimentaria H. elongata N.D. H. venusta N.D. H. elongata H. glaciei H. elongata H. elongata H. organivorans H. elongata H. organivorans H. organivorans H. organivorans H. organivorans H. organivorans H. trueperi H. organivorans N.D. H. organivorans H. salina C. israelensis H. organivorans C. israelensis H. elongata H. elongata H. elongata N.D. S. roseus N.D. H. venusta H. glaciei

N.D., not determined. a 1,2-CTD primers (cat1 and cat3 primers). b 3,4-PCD primers (pro3.4.2 and pro3.4.4 primers). c Larger band than the expected size (@500 bp) which sequence revealed a catA-like gene.

eurihalina (>99.6% sequence similarity), Halomonas alimentaria (>99.6% sequence similarity), Halomonas venusta (>99.1% sequence similarity), Halomonas glaciei (>98.7% sequence similarity), and Chromohalobacter israelensis (>99.7% sequence similarity) (Fig. 1(a)). Apart from these bacteria belonging to the family Halomonadaceae, among the Gram negative isolates,

only one strain not phylogenetically related to this family, strain A27.8, was isolated. This isolate exhibits high similarity (97.7% sequence similarity) to the moderately halophilic bacterium Marinobacter lipolyticus. Most strains assigned to the species H. elongata have been obtained from Isla Bacuta, the sampling site exhibiting the highest salinity (17%). In contrast, Huelva

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estuary, a sampling site with low salinity (4%) has been the source of many phenol-degrading strains assigned to the species H. organivorans (Table 2). Strains assigned to H. elongata have been isolated from the four sampling sites, indicating the wide presence of this species in the saline habitats studied. Only three Gram-positive strains (strains D23.3, G19.1 and J8.8) were isolated. Isolates D-23.3 and J8.8 were recovered from the salicylic and p-aminosalicylic acid enrichments, respectively. They had spherical morphology and were closely related (99.2% sequence similarity) to the moderately halophilic species Salinicoccus roseus (Fig. 1(b)). Isolate G-19.1 was recovered from the phenol enrichment and presented the highest phylogenetic relationship (96.3% sequence similarity) to Halobacillus trueperi (Fig. 1(b)). To determine the capacity of these isolates to grow in media with different salinities, a test using salt concentrations ranging from 0 to 30% was performed for the isolates (a Table showing these results is available as supplementary data). The isolates did not grow in the absence of NaCl and most of them were able to grow in a wide range of salinities (from 0.9% to 25% [w/v] salt). The results obtained showed that the isolates presenting the most extended range of NaCl concentrations (0.9% to 25% [w/v] salt) belonged to the genus Halomonas.

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3.4. Screening of dioxygenase enzyme genes We investigated the distribution of different pathways used by the isolated halophilic bacteria for mineralization of organic compounds. After comparing the amino acid sequences of different genes coding for catechol 1,2dioxygenases, catechol 2,3-dioxygenases and protocatechuate 3,4-dioxygenases, we identified some conserved sequence regions, which were used to design the degenerate PCR primers cat1, cat3; cat2.3.1; cat2.3.2, pro3.4.2 and pro3.4.4 (see Section 2). These primers allowed us to successfully amplify specific regions of the expected sizes from the DNA of different isolates (Fig. 2). Representative strains of the different phylogenetic groups were tested for the presence of the genes encoding these dioxygenase enzymes (Table 2). In general, isolates obtained from the enrichment cultures using benzoic and phydroxybenzoic acids as sole carbon and energy sources were positive using the primers designed for the amplification of catechol 1,2-dioxygenase encoding genes but negative for the protocatechuate 3,4-dioxygenase encoding genes. Among the cinnamic acid isolates, one strain (C24.1) was positive in PCR using the primers cat1 and cat3,

3.3. Substrate utilization by isolates All 110 selected isolates were tested for their capacity to use the different aromatic compounds utilized as experimental models in this work as the sole carbon and energy source (a Table showing these results is available as supplementary data). Benzoic acid, phydroxybenzoic acid, phenylpropionic acid, ferulic acid and p-aminosalicylic acid were the compounds that were used by many isolates (48%, 42%, 39%, 38% and 33%, respectively). In contrast, the other five substrates were used by a low proportion of isolates (ranging from 22% to 30%). None of the isolates utilized p-cresol. Most isolates were able to utilize at least three of the aromatic substrates provided as sole carbon source and the most versatile strains were obtained from the phenol and p-coumaric enrichments, exhibiting a broad range of degradation (6–7 aromatic compounds). Some strains isolated from Huelva estuary and obtained from the phenol enrichments (G8.2, G16.1 and G18.1) were able to grow at the expense of all compounds tested except p-cresol. Forty-one isolates presented a narrow range of degradation (one or two aromatic compounds). A color change in the medium from colorless to brown was observed in several cultures growing on phenol, p-coumaric acid, salicylic acid, cinnamic acid and p-aminosalicylic acid.

Fig. 2. PCR detection of the (a) catechol 1,2-dioxygenase (1,2-CTD) and (b) protocatechuate 3,4-dioxygenase (3,4-PCD) encoding genes. Numbers on the different lanes indicate the pollutant-degrading strain. DNA standard ladder (1 kb) was loaded in the first lane.

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while seven were negative. All other isolates able to degrade cinnamic acid were positive using the primers pro3.4.2 and pro3.4.4. As indicated in Table 2 all tested strains obtained from the phenylacetic acid enrichment were negative for the amplification using the primers pro3.4.2 and pro3.4.4 From the 23 strains able to degrade p-coumaric acid, phenol, ferulic acid and cinnamic acid, 18 were positive for amplification of a 330-bp internal fragment of the genes coding for protocatechuate 3,4-dioxygenases. The strain J21.8, that was related to H. venusta, (99.4% sequence homology) was negative in the amplification of the 414-bp fragment of the catechol 1,2dioxygenase encoding gene, however, a 500-bp band was obtained when the primers pro3.4.2 and pro3.4.4 were used. The sequence of this fragment showed the highest homology to the catA gene (Accession No. AF042281.1) of Ralstonia eutropha, the pheB gene (Accession No. M57500.1) codified in the plasmid pEST1226 of Pseudomonas sp. EST1001 and the gene catA2 of Burkholderia sp. TH2 (Accession No. 23491532). All these genes encode for catechol 1,2 dioxygenases. The degenerate primers designed for amplification of catechol 2,3 dioxygenase encoding genes yielded a PCR amplification product of the expected size (approx. 400 bp) when using the DNA of Pseudomonas putida harbouring the plasmid pTOL. However, no positive signals were detected when using the DNAs from our isolates, only slight signals of the correct sizes were obtained for the strains C7.3, F18.2 and J-24.3 obtained from the cinnamic acid, phenylpropionic acid and paminosalicylic enrichments, respectively. These strains are phylogenetically very closely related to the type strains of H. organivorans, H. salina and H. glaciei (see Fig. 1). The majority of the isolates yielded no PCR product or a PCR product smaller or higher than that of the positive control strain. These results are not indicated in Table 2.

3.5. Dioxygenase activity of H. organivorans Among the different isolates obtained in this study, the group formed by strains C7.3, D27.5, G2.5, G8.2, G17.1, G18.1, G20.3, G16.1T, G21.5, G22.6, H17.3 and H30.2, recently classified as H. organivorans [19], was selected for more detailed studies concerning the detection of enzymatic activities. The type strain of this species, G-16.1T grew on all the compounds tested except p-cresol. The activities of key enzymes, catechol 1,2-dioxygenase, catechol 2,3-dioxygenase and 3,4-protocatechuate dioxygenase were determined in cell-free extracts from cells grown in batch culture under aerobic conditions with the different organic compounds as inducers (Table 3). When H. organivorans was grown with benzoic acid (5 mM), cinnamic acid, (4 mM), salicylic acid (3 mM), phenylpropionic acid (4 mM), phenol (2.5 mM) and paminosalicylic acid (3 mM) the crude extracts showed catechol 1,2-dioxygenase activities. In contrast, no activity towards protocatechuate was obtained (Table 3). This would suggest that the catechol is possibly an intermediate of the degradation of these aromatic compounds. meta-Cleavage activity was not detected, also by varying several parameters in the assay, such as the substrate concentration, the buffer and pH values, the assay temperature or the concentration of crude extract. The crude extracts of the strain obtained after growing in minimal medium containing p-hydroxybenzoic acid (5 mM), p-coumaric acid (3 mM) and ferulic acid (4 mM) showed a strong protocatechuate 3,4-dioxygenase activities. However, only weak activities toward catechol were obtained indicating that protocatechuate is possibly an intermediate in the catabolism of these compounds in H. organivorans. Moreover, the described enzymatic assays revealed a higher level of activity of catechol 1,2-dioxygenase in the cells grown on benzoic acid than in the cells grown on compounds such as cinnamic acid, phenylpropionic acid and phenol. Higher

Table 3 Specific enzyme activities (nmol min
1 mg protein
1) in cell extracts of Halomonas organivorans G16.1 under different inducing conditions Inducer

Catechol 1,2-dioxygenase activity

Protocatechuate 3,4-dioxygenase activity

Benzoic acid p-Hydroxy-benzoic acid Cinnamic acid Salicylic acid Phenylacetic acid Phenylpropionic acid Phenol p-Coumaric acid Ferulic acid p-Aminosalicylic acid

360 ± 15 17.6 ± 6 103 ± 28 51 ± 8 <1 96 ± 11 61 ± 7 10 ± 2 8±3 35 ± 6

<1 423 ± 25 <1 <1 <1 <1 <1 900 ± 69 426 ± 36 <1

Each value is the mean of three determinations ± standard errors of the means. Catechol 2,3-dioxygenase activity was not detected.

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growth yield has been detected when H. organivorans was grown in the presence of benzoic acid than with the other substrates. This could explain the high induction of catechol 1,2-dioxygenase detected in cells grown with benzoic acid. However, further research has to be done in order to clarify this aspect, because the induction of other catabolic enzymes in H. organivorans grown with substrates as cinnamic acid, phenylpropionic acid and phenol could be evidence for an alternative pathway for the degradation of these compounds. The weak catechol 1,2 dioxygenase activity detected in these cultures could be explained by induction of this enzyme by intermediates of the metabolism presenting a similar structure. When H. organivorans was grown on phenylacetic acid no ring-cleaving dioxygenase activity was detected, suggesting that this substrate could be metabolized via another catabolic pathway. All ring-cleaving dioxygenases detected in this organism were of the ortho-type.

4. Discussion The diversity of indigenous microorganisms capable of degrading aromatic compounds in different environments has been of great interest in the last years [34] and different molecular techniques have been developed for the study of these communities [35]. However, research focused on halophilic bacterial communities that degrade aromatic compounds in saline habitats is very scarce, although the presence of aromatic compounds is abundant in these environments [5]. Among halophiles, moderately halophilic bacteria constitute a heterogeneous group exhibiting an eurihaline response. This group of microorganisms presents a great catabolic versatility [2]. However, the use of these catabolic capabilities in bioremediation processes under saline conditions has not been studied in a systematic way. This work describes the isolation and characterization of moderately halophilic bacteria able to degrade different aromatic compounds. The ecological study performed suggests that only a few genera could grow on the model compounds selected under the salinity conditions used. However, it is important to point out that the screening was performed in restrictive conditions of temperature and salinity. Interestingly, most of the aromatic compound-degraders belong to the genus Halomonas and despite much effort only three Gram-positive bacteria were enriched or isolated when the organic compounds tested were used as carbon sources. The genus Halomonas comprises a great number of species and constitutes the most common cultured inhabitant in saline habitats. Most of the species of this genus can grow in an extended range of salinities (1–30% NaCl), which is an advantage for biotechnological processes at different salinities [2].

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Based on the comparison of partial sequence of 16S rRNA genes, the majority of our isolates were closely related to species assigned to the phylogenetic group 1 of the family Halomonadaceae and contained the typical signature nucleotides characteristics of this family [36]. A total of 11 isolates presented high similarity to the recently described species H. organivorans [19], another important group of isolates was closely related to H. salina [37], H. elongata [38], H. halophila [39] and H. eurihalina [26], all these species belonging to the phylogenetic group 1. The rest of the isolates were assigned to H. alimentaria [40], H. venusta [36] and H. glaciei [41]. Two isolates resembling C. israelensis [42], were recovered only from the p-coumaric acid enrichments. This species is included in a genus forming a phylogenetic group closely related to Halomonas within the family Halomonadaceae [42]. Experimental studies of polycyclic aromatic hydrocarbon degradation in marine sediments have probed the presence of the genus Halomonas among the isolates able to grow on mono- and poly-aromatic compounds [15,43]. During the past three decades, much research has been done on elucidating the metabolism of different organic compounds in the non-halophilic bacterium Pseudomonas, a model genus in biodegradation studies. We propose the use of Halomonas as a model bacterium for the study of degradation of aromatic compounds at high salt concentrations. This genus includes species with few nutritional requirements, the ability to grow in a wide range of salinities and is very easy to culture. Moreover, the eurihaline response of these bacteria and the wide range for the mineralization of different aromatic compounds, render them as a useful model system for studying the catabolism of aromatic compounds. On the other hand, the isolate A27.8 from the benzoic acid enrichment exhibited a close relationship (97.7% sequence similarity) to a recently describe species, M. lipolyticus with lipolytic activity [44]. Some other species of the genus Marinobacter have been described as degraders of hydrocarbons and some crude oil components [11–13]. Up to now, representative members of the genus Salinicoccus and Halobacillus have not been described as strains able to degrade organic compounds in contaminated habitats. In this work, we described two strains very closely related to S. roseus [45] and one strain related to H. trueperi [46] thus presenting metabolic versatility and expanding the diversity of moderately halophilic bacteria with this ability. The substrate utilization of the different aromatic compounds revealed that most of the isolates were capable of growing on a moderately wide range of aromatic compounds. These results indicate that the isolated strains possessed the catabolic machinery necessary for the degradation of these compounds and also that

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functional abilities related to aromatic compounds degradation in the isolated strains generally did not correspond to phylogenetic grouping. The most versatile strains able to utilize a high number of substrates (supplementary data) have been obtained from Huelva estuary, the sampling site with the lowest salinity (4%). On the other hand, the results indicated that phenolenriched isolates are able to utilize a greater number of aromatic compounds than the rest of isolates enriched in other aromatic compounds (Table 1 and supplementary data). Most of these phenol-enriched isolates were positive for both, the catechol 1,2-dioxygenase and the protocatechuate 3,4-dioxygenase encoding genes, reflecting their wider substrate specificity. The design of specific primers allowed us to amplify conserved regions of the key dioxygenases of the catabolic pathways (see Table 2). Most of the sequences of these PCR-amplified fragments showed high similarity to dioxygenases of different species such as Pseudomonas aeruginosa (Accession No. AE004453), P. putida (Accession No. L14836), P. arvilla (Accession No. D37783), Agrobacterium radiobacter (Accession No. AF230649), A. tumefaciens (Accession No. U32867), Acinetobacter lwoffii (Accession No. U77659), Sagittula stellata (Accession No. AF253465), and Frateuria sp. (Accession No. AB009373). Most of the isolates thus seem to metabolize these aromatic compounds by the ortho-cleavage pathway. This pathway is chromosomally encoded and it is widely distributed in soil bacteria and fungi constituting the major pathway for aromatic compound catabolism in these organisms [22]. Both enzymes representing the two branches of this pathway, 3,4-PCD and 1,2-CTD, are nonheme iron-containing dioxygenases and show similar sequences [22,47]. b-Ketoadipate constitutes the metabolite in which the two branches of the pathway converge and two additional steps finally funnel this metabolite into the tricarboxylic acid cycle. Most studies performed on this pathway are related to soil bacteria, although there are some reports on the presence of the b-ketoadipate pathway in marine bacteria [48]. Most isolates able to degrade phenol, p-coumaric acid, ferulic acid, and cinnamic acid were positive for the amplification of the protochatechuate 3,4-dioxygenase encoding genes (Table 2). It has been reported that many soil and marine bacteria degrade these substrates converting them to protocatechuate and then continue the degradation via the b-ketoadipate pathway [22,48]. The wide presence of the protochatechuate 3,4-dioxygenase in the saline environments analyzed in this work indicates the importance of this key enzyme in the halophilic bacteria. This enzyme is composed of two nonidentical a and b subunits that are encoded by the pcaG and pcaH genes [49]. Among the isolates, strain G-16.1, described as H. organivorans has been selected for further studies be-

cause of the wide metabolic capabilities exhibited by this species. It is known that the ortho and meta fission of the intermediates catechol and protochatechuate are metabolic alternatives and the prevalence of one or the other depends not only on the bacterial species but also on the growth substrates. In this sense, we studied the induction of key enzymes by different organic compounds in H. organivorans to characterize the catabolic types. This halophilic bacterium possesses two different ring-cleaving enzymes, one active towards catechol and the other active towards protocatechuate thereby suggesting the presence of two catabolic pathways (Table 3). Phenolic compounds constitute environmental pollutants released by different industrial units, such as various chemical plants, wood preservation plants or oil refineries. In addition, they may also enter the environment as intermediates during the catabolism of other xenobiotic compounds [50]. Because of their toxicity, bioremediation of these compounds is necessary. Numerous studies on the microbial catabolism of phenol have been carried out and different catabolic routes have been described for this compound [34,51,52]. Phenol is usually degraded by hydroxylation of the ring and subsequent ring fission by meta or ortho cleavage. Our data suggest that the degradation of phenol by the halophilic species H. organivorans could occur through the intermediate catechol, via the b-ketoadipate metabolic pathway. However, the low level of induction of the catechol 1,2-dioxygenase when cells are grown on phenol could indicate the presence of an alternative pathway for phenol degradation. Further research concerning the characterization of other enzymes involved in the catabolism is planned to elucidate the phenol catabolic pathway in H. organivorans. Recently, the degradation of phenol by the haloalkaliphilic bacterium Halomonas campisalis through the b-ketoadipate metabolic pathway has been described [17]. Most microbial catabolic pathways for p-coumaric and ferulic acids yield either protocatechuate or gentisate [53,54]. According to our molecular and enzymatic data (Tables 2 and 3), in H. organivorans protocatechuate seems to be an intermediate in the metabolism of ferulic acid and p-coumaric acid, which is further degraded by the cleavage of the aromatic ring via the ortho pathway, however, cinnamic acid seems to be degraded through the catechol ortho pathway. Reports in the literature [53] revealed the transformation of cinnamic acid to benzoic acid in some microbial species, suggesting catechol as the most promising candidate for the ring fission. Four major pathways of ferulic acid degradation can be distinguished in a variety of microorganisms with respect to the initial reaction: (1) non-oxidative decarboxylation, (2) side-chain reduction, (3) coenzyme-A-independent deacetylation, and (4) coenzymeA-dependent deacetylation [55]. It is known that

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possible metabolic intermediates of ferulic acid are vanillin and vanillic acid [56]. Vanillic acid is further demethylated to protocatechuate and the genetics of this conversion has been described [57]. In H. organivorans vanillic acid might be metabolized via protocatechuate for its ring fission rather than via gentisate. This hypothesis should be further investigated concerning the detection of a vanillate demethylase activity in this strain. The catabolic routes of these phenylpropanoids described in other organisms, established that most microorganisms able to degrade cinnamic acid are also able to degrade their para-hydroxylated derivatives, however most of the ferulic acid degraders, fail to degrade cinnamic acid [53]. In this study, the enzymatic data obtained suggest that salicylic acid proceeds via a pathway similar to that described for the Gram-negative Acinetobacter sp. strain ADP1 through the b-ketoadipate pathway at the level of catechol [58]. However, several reports described the catabolic routes for salicylate and derivatives involving the conversion to the intermediate gentisate [59]. Moreover, the direct ring fission of salicylate by a salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans has also been described [60]. We have also shown that H. organivorans possesses a catechol 1,2-dioxygenase better induced with salicylic acid than with p-aminosalicylic acid. When phenylacetic acid was used as inducer, no activity of the three assayed dioxygenases was detected in the cell extracts. These results are not surprising because phenylacetic acid degradation in several bacteria has been described to follow unusual routes, which involves CoA derivatives, not similar to other known catabolic pathways for the degradation of aromatic compounds [61]. Catechol 2,3-dioxygenases constitute a group of enzymes that are considered crucial for degradation of a wide range of aromatic compounds in contaminated habitats [62]. However, enzymological assays revealed that catechol 2,3-dioxygenase activity was not induced in H. organivorans when using these compounds as the sole carbon and energy sources. The failure to detect this enzyme could be due to the absence of this activity or it could be that the method we used is not sensitive enough to detect low levels of enzyme activity. The presence of dioxygenases in the family Halomonadaceae has been shown in this work, but further work is needed to elucidate the early enzymes in the pathway and the mechanisms of action of these halophilic enzymes, which maintain their catalytic properties in saline environments. The comparison of primary and secondary sequences of these halophilic dioxygenases with their non-halophilic counterpart will help in the determination of the necessary conditions for the halophilic enzymes to be stable at high salt concentrations.

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The diversity of moderately halophilic bacteria able to catabolize aromatic compounds in saline conditions was until now unknown. Moreover, we have determined the specific catabolic abilities of distinct phylogenetic groups inhabiting saline environments and identified representative halophilic bacteria that might play an important ecological role.

Acknowledgements This work was supported by grants from the Spanish Ministerio de Ciencia y Tecnologı´a (REN2003-01650 and BMC2003-1344) and Junta de Andalucı´a.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version at doi:10.1016/ j.femsec.2005.03.009.

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