Identification and Characterization of Rhodopseudomonas Spp., a Purple

Biomolecular Engineering 18 (2001) 49–56 www.elsevier.com/locate/geneanabioeng

Identiﬁcation and characterization of Rhodopseudomonas spp., a purple, non-sulfur bacterium from microbial mats

Sharifeh Mehrabi a,*, Udoudo M. Ekanemesang a, Felix O. Aikhionbare b, K. Sean Kimbro a, Judith Bender a

a Department of Biological Sciences, Clark Atlanta Uni6ersity, Atlanta, GA 30314, USA b Department of Microbiology/Biochemistry/Immunology, Morehouse School of Medicine, Atlanta, GA 30310, USA Received 2 March 2001; received in revised form 10 June 2001; accepted 10 June 2001

Abstract

A species of facultative photo-organotrophic, purple, non-sulfur bacterium was isolated from mixed-species microbial mats, characterized and examined for metal tolerance and bioremediation potential. Contributing mats were natural consortia of microbes, dominated by cyanobacteria and containing several species of bacteria arranged in a laminar structure, stabilized within a gel matrix. Constructed microbial mats were used for bioremediation of heavy metals and organic chemical pollutants. Purple, non-sulfur bacteria are characteristically found in lower strata of intact mats, but their contributing function in mats survival and function by mediating the chemical environment has not been explored. The gram-negative rod-shaped bacterium, reported here, produced a dark red culture under phototrophic conditions, reproduced by budding and formed a lamellar intracytoplasmic membrane (ICM) system parallel to cytoplasmic membrane, which contained bacteriochlorophyll a and carotenoids. This strain was found to have multiple metal resistances and to be effective in the reductive removal of Cr(VI) and the degradation of 2,4,6-trichlorophenol. Based on the results obtained from morphology, nutrient requirements, major bacteriochlorophyll content, GC content, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) profile and 16S-rDNA phylogenetic analysis, this member of the microbial mats may be identified as a new strain of the genus Rhodopseudomonas. © 2001 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Microbial mat; Rhodopseudomonas, non-sulfur purple bacteria; Intracytoplasmic membrane; RAPD-PCR; 16S-rDNA; TCP; Cr(VI)

1. Introduction types (maximum absorption at 450–550 nm) and bacteriochlorophylls [3]. Absorption spectra of cell suspen- Species of the genus Rhodopseudomonas constitute sions yield primary information on predominant the majority of phototrophic purple non-sulfur bacte- bacteriochlorophylls and carotenoids. This color trait is useful in initial characterization of these species [2]. The ria, and are characterized as rod-shaped, motile cells major carotenoids are lycopene, rhodopin (463, 490, that show polar growth and asymmetrical division or 524 nm), spirilloxanthin (486, 515, 552 nm), okenone budding as a mode of reproduction [1]. They form (521 nm) and spheroidene (450, 482, 514 nm). The lamellar intracytoplasmic membranes that are adjacent spirilloxanthin series (lycopene, rhodopin, spirilloxan- and parallel to cytoplasmic membrane and contain thin) as the major component gives a pink or red color, photosynthetic pigments under phototrophic conditions increasing amounts of rhodopin turn the color to red– [2]. The colors of cell suspensions of many species brown, okenone (521 nm) results in purple–red. The grown under phototrophic conditions vary from species spheroidenone series gives a green or brownish red and to species, indicating the presence of major carotenoid greenish brown under reducing conditions [2,3]. Rhodopseudomonas species have shown a high degree * Corresponding author. Tel.: +1-404-8806297; fax: +1-404- of morphological and structural similarities, however, 8806756. they are phylogenetically quite diverse. Comparisons of E-mail address: [email protected] (S. Mehrabi). rRNA sequences, DNA–DNA and DNA–RNA hy-

1050-3862/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S1389-0344(01)00086-7 50 S. Mehrabi et al. / Biomolecular Engineering 18 (2001) 49–56 bridization have demonstrated that these bacteria be- application of microbial mats have been reported pre- long to several different lines of descent within the viously [17,18,20–22]. Samples of microbial mat were alpha-2 subclass of Proteobacteria [3]. Morphological, blended in sterile saline solution (NaCl 8.5 g/l) and cytological and physiological properties of the genus serially diluted. Colony count pour plates were pre- Rhodopseudomonas have been summarized by Imhoff pared from each dilution in nutrient agar medium et al. [4]. Recent phylogenetic studies of Rhodopseu- (Dickerson Microbiology Systems, Cokeysville, MD), domonas species demonstrated that Rhodopseudomonas incubated at room temperature in the dark and anaer- palustris is more closely related to some chemotrophic obically under 60 W incandescent light in an anaero- taxa, such as Afipia felis, Bradyrhizobium japonicum, bic chamber (Forma Scientific, Marietta, OH). Several Blastobacter denitrificans and Nitrobacter winograd- red or red–brown colonies of different sizes and tex- skyi, than to any of the other Rhodopseudomonas spe- tures appeared after five to seven days incubation un- cies for which rDNA sequences are available [5,6]. der anaerobic/light conditions. The most dominant Phototrophic non-sulfur bacteria have been used in red colony was selected for purification. Several repre- sewage treatment processes [7,8], for biomass and vi- sentatives of single red colonies were individually tamin production [9] and for production of molecular transferred into screw cap tubes filled with nutrient hydrogen [10]. In bioenergetic research, they have broth medium and incubated under the light at room been used as a cell-free system for performing photo- temperature for enrichment. To ensure the purity of synthesis [11,12]. Biodegradation of aromatic com- cell culture, the streak dilution plates were prepared pounds by Rhodopseudomonas species such as from each tube containing the originally isolated benzoate [13], dihydroxylates, methyoxylate [14] and colony on nutrient agar medium, and checked for pu- 4-hydrobenzoate [15] have been reported. Non-sulfur rity. bacteria display a high resistance to heavy metals [16], giving these cells another potential application in 2.2. Determination of nutritional and physiological bioreclamation of rare-earth metals. characteristics In attempts to isolate the bacterial members of mats and elucidate their roles in bioremediation, we The stock solutions of different carbon sources isolated and partially characterized a phototrophic, were filter-sterilized and added to the AT salt medium purple, non-sulfur bacterium reported in this study. [2] to a final concentration of 1–2g/l.ThepHofthe The mats were developed for bioremediation and con- media was adjusted to 7 with HCl or NaOH for structed by stimulating the formation of a microbial routine analysis or to 5, 6, 7 or 8 for pH profile consortium with specific substrates (ensilaged grass) analysis. Heterotrophic cultures were grown in 100 ml and inocula composed of Oscillatoria spp. and allow- of medium in 250 ml flasks incubated in a rotary ing volunteer heterotrophic groups from the soil to shaker at 150 rpm in the dark. Phototrophic cultures colonize the preparation [17]. When the mat was used were grown in completely filled 250 ml screw cap cul- for treatment of organic [18,19] and/or inorganic [20– 23] chemicals, the population of purple bacteria ture bottles and incubated under 60 W incandescent within the mat significantly increased and the mat bulbs at a distance of 60 cm in an anaerobic chamber. changed from dark green to reddish purple in color. The growth was monitored turbidimetrically, using a This strain was selected for identification and charac- Beckman DU 650 UV/VIS scanning spectrophotome- terization because of its dominance during the sewage ter (Beckman, Fullerton, CA) at 600 nm. The number treatment process. The isolate is characterized accord- of colony forming units (CFU) was determined by the ing to its phenotypic, genotypic and phylogenetic spread plate count method using serial dilutions. The properties and compared with other members of growth rate constant (K), the number of generations Rhodopseudomonas spp. The contribution of this (n) and the mean generation time (g) were calculated strain in microbial mats bioremediation properties will using standard mathematical growth equations [24]. be reported in future. The measurement of optimum growth temperature was carried out in nutrient broth medium, in a water bath set to the desired temperature. Assay for formation of intracellular sulfur globules was performed ac- 2. Materials and methods cording to the method described by Balows et al. [2]. Briefly, the cells were incubated on a microscopic

2.1. Bacterium source and isolation technique slide for 20 min with neutralized Na2S solution and monitored for intracellular sulfur globules formation. The bacterium was isolated from a mature micro- Chromatium 6inosum (ATCC 17899), a sulfur purple bial mat used for metal sequestration and organic pol- bacterium was used as a positive control. All experi- lutant degradation experiments. The development and ments were performed in triplicate. S. Mehrabi et al. / Biomolecular Engineering 18 (2001) 49–56 51

2.3. Absorption spectra and electron microscopy Peaks were quantiﬁed by external standards using system Gold. Determination of the predominant bacteriochlorophyll and the carotenoid composition of the isolate 2.6. Sample source and GC content analysis were performed as described by Balows et al. [2]. The absorption spectra of cell suspensions in 4 M sucrose Seven species of Rhodopseudomonas that share phe- were recorded against a blank of 4 M sucrose solution notypic characteristics with the isolate were purchased using a Beckman DU 650 UV/VIS scanning spec- from ATCC (American Type Culture Collection) trophotometer. The approximate maximum absorption (Table 1). These strains were cultured in media recom- of major peaks was determined. A transmission elec- mended by ATCC under phototrophic conditions. Cells tron microscope (TEM, Hitachi H-6000), set at 0.3 nm were harvested at their exponential growth phase. The point-to-point resolution and 50 000× magniﬁcation, G+C base content of the isolate was determined by was used to examine the isolate. Standard microscopic the thermal denaturarion method as described by Mar- methods were used for slide preparation and Gram mur and Doty [25]. staining. 2.7. DNA preparation and 16S-rDNA sequence 2.4. Reducti6e remo6al of Cr(VI) analysis

Cells were grown to the late log phase in nutrient DNA extraction was performed using the methods broth harvested, washed and resuspended in 100 ml of described by Maniatis et al. [26]. DNA was then precip- minimal salt medium at cell density equivalent to opti- itated with chilled ethanol, washed with 70% ethanol, cal density reading of 1.5 at 600 nm. Varying concen- dried and finally dissolved in TE (10:1) and stored at trations of dichromate solution were added to the cells. −20 °C until diluted to about 100 ng/ml prior to use. Aliquots were removed at time intervals, filtered and Amplification of 16S ribosomal DNA (rDNA) was analyzed for hexavalent chromium. The concentration performed on the isolated DNA using primers (5%- of Cr(VI) was determined colorimetrically by reaction CAGCCAGCCGCGGTAATAC-3%)and(5%-CCGT- with diphenylcarbazide in acid solution as described in CAATTCCTTTGAGTT-3%). The PCR reaction mix section 3500 of the Standard Method for Examination was performed as previously described [27,28]. Am- of Water and Wastewater (American Public Health plified products were resolved on 1.5% agarose gel, then Association, 1995). An aliquot of 2 ml of sample was excised from the gel and purified. The purified products added to 9.8 ml of 0.2 N H2SO4, followed by the addi- were cloned into pGEM-T (Promega) according to tion of 0.5 ml of diphenylcarbazide reagent (0.5% in manufacturer’s instructions and subsequently se- acetone, Labchem Inc., Pittsburgh, PA). The ab- quenced using Big Dye Terminator chemistry (ABI, sorbance of the solution was measured at 540 nm using Foster, CA). The sequences were assembled using Au- a Beckman DU 650 UV/VIS scanning toassembler and sequence navigator (ABI, Foster, CA). spectrophotometer. A 407 bp 16S-rDNA sequence was obtained from this study. 2.5. Degradation of trichlorophenols 2.8. RAPD-PCR analysis Cells were grown in nutrient broth to the late log phase, harvested, washed and resuspended in 100 ml of A total of 20 random and previous 16S-rRNA se- minimal salt medium at cell density equivalent to opti- quences of Rhodopseudomonas species were used in the cal density reading of 1.5 at 600 nm. 2,4.6- selection of RAPD primers, then obtained from Trichlorophenol (100 mg/ml) was added to the culture Genosys Biotechnologies Inc. (Woodlands, TX). All flasks and incubated in an anaerobic chamber at 30 °C on a rotary shaker at 150 rpm. At time intervals Table 1 aliquots of the culture (2.0 ml) were removed; cells were Rhodopseudomonas species used in RAPD-PCR removed by centrifugation. Equal volume of methanol SampleSpecies ATCC Reference was added to the supernatant and filtered through a 0.45 mm pore-size filter (Millipor, Bedford, MA) into a 1 Rps. palustris (RPS p) 17001 [31] HPLC sample vial. Samples were analyzed using a 2 Rps. acidophila (RPS a) 25092 [31] Beckman System Gold HPLC equipped with a model 3Rps. rutila (RPS r) 33872 [32] 502 autosampler, 126 solvent module, diode array de- 4 Rps. rosea (PRS ro)49724 [33] 5 Rps. 6iridis (RPS v)19567 [31] tector and c18 column (15 cm×4.6 mm; particle size 6 Rps. marina (RPS m) 35675 [34] 5 mm). Injection volume was 20 ml. The mobile phase 7 Isolate This study was methanol/water (60:40) at a flow rate of 1 ml/min. 52 S. Mehrabi et al. / Biomolecular Engineering 18 (2001) 49–56

Fig. 1. Scanning electron micrograph of the isolate; circle shows a budding cell; bar, 1 mm.

RAPD primers used in this study were 10-mers, with a ORS278, AF239255; Bradyrhizobium elkanii PRY52, 60% or 70% G+C content. RAPD-PCR reactions were AF239846; Bradyrhizobium japonicum, AF293382; m obtained in 25 l volume similar to previously described Bradyrhizobium spp. strain Cj3-3, AF321215; Bradyrhi- [29]. Ampliﬁed PCR products and controls were resolved zobium liaoningense strain 2281, AF363132; Bradyrhizo- in 1.5% agarose gels. All the resultant bands were scored bium japonicum strain USDA 123, AF363151; Aﬁpia in reference to the 100 bp molecular weight ladder genosp. U87771; Rhizospere soil bacterium isolate RSI-33, (GibcoBRL, Gaitherburg, MD) as either present or AJ252600; Bradyrhizobium spp. MSDJ5726, AF363149; absent across the lanes. Bradyrhizobium elkanii strain USDA 94, AF363152; Bradyrhizobium genospp. J, Z94820; Rhodopseudomonas 2.9. Phylogenetic analysis palustris, D89811; Rhodopseudomonas spp. g–c 16S AF123086; R. palustris, X87279. The program Clustal W 407 bp of the16S-rDNA sequence of the isolate was [30] was used to create a multiple-sequence alignment of matched to other Rhodopseudomonas spp. obtained from the sequences and to construct the tree from the align- GenBank databases. Names and accession numbers are ment. Bootstrapped distance matrix analysis was per- as follows: Rhodopseudomonas spp. B29, AB027692; formed using Clustal W with 1000 bootstrapping trials. Uncultured alpha proteobacterium A, AB035489; Uncul- We used the PHYLIP program DRAWTREE to draw tured alpha proteobacterium, AB035490; Bradyrhizobium the tree from the output of the Clustal W program. spp. Mml-3, AF159438; Bradyrhizobium spp. Pel-3; AF159436; Bradyrhizobium spp. Pe4, AF159437; Bradyrhizobium spp. DesB1, AF178436; Bradyrhizobium 3. Results spp. jwc91-2, AF178437; Bradyrhizobium spp. th-b2, AF178438; Bradyrhizobium spp. ORS2012, AF230721; 3.1. Morphology and phototrophic properties Bradyrhizobium spp. ORS285, AF230722; Bradyrhizo- bium elkanii strain SEMIA 5019, AF237422; Bradyrhizo- The isolate was a gram-negative rod-shaped motile bium spp. STM461, AF239254; Bradyrhizobium spp. bacterium with a length of 2.0–2.4 mm. The cells divided S. Mehrabi et al. / Biomolecular Engineering 18 (2001) 49–56 53

Table 2 Physiological properties of the isolate

Substrate, reaction or enzyme Phototrophic growth

Succinate + Ethanol + Pyruvate + Malate − Acetate + Valerate + Benzoate − Tween 80 hydrolysis − Nitrate reduction + Catalase − 3% NaCl − Arginine − Lysine − Ornithine − Fig. 2. Thin section electron micrograph of the isolate showing Sodium citrate − intracytoplasmic membrane (ICM); bar, 0.3 mm. Sodium thiosulfate − Urea − asymmetrically by budding. Bud formation at the end Tryptophan − of the mother cell gives a dumbbell-shaped appearance Gelatinase 9 before separation of the daughter cell [1] (Fig. 1). The Galactosidase − Glucose + electron micrographs of thin sections of phototrophi- Mannitol − cally grown cells showed a lamellar intracytoplasmic Starch hydrolysis − membrane (ICM) system (Fig. 2). The bacteria form Sucrose + smooth, round and dark red colonies with a diameter Inositol + of 2–3 mm on agar media and red cell suspensions in Rhamnose + Melibiose − liquid media under phototrophic conditions. The strain Amygdalin − formed photosynthetic pigments when grown under Arabinose − anoxygenic phototrophic conditions. Absorption spectra of cells suspended in sucrose had three major peaks +, Positive growth or reaction; −, negative growth or reaction; 9, at 593, 805 and 871 nm, indicating the presence of partially positive. bacteriochlorophyll a (590, 800–900 nm), and three peaks at 463, 490 and 530 nm, indicating the presence 1025–1035 nm) and c (660–668 nm), which are charac- of lycopene and rhodopin. There were no absorption teristic of purple non-sulfur bacteria (Fig. 3) [2,3]. peaks for bacteriochlorophylls b (400, 605, 840 and 3.2. Physiological and metabolic properties

Optimal growth occurred at 30–35 °C and at pH 6–8 under both heterotrophic and phototrophic conditions. Phototrophic growth was observed on several carbon and electron donor sources (Table 2). The growth of the bacteria in nutrient broth was signiﬁcantly higher than in minimal salt media with a single carbon source. The K values (mean growth rate constants) of the bacterial growth in nutrient broth and other carbon substrates under phototrophic and heterotrophic conditions are presented in Table 3.

3.3. Strain identiﬁcation

The isolate was sent to two commercial organiza- Fig. 3. Absorption spectrum of cell suspension of isolate grown under tions, ATCC (Bethesda, MD) and Analytical Services phototrophic conditions showing major absorption peaks at 590, 805 and 871 nm, indicating the presence of bacteriochlorophyll a and 463, Inc. (Essex Junction, VT) for their independent confir- 490 and 530 nm, indicating the presence of carotenoid (lycopene and mation of our identification. Analytical Services was rhodopin). unable to obtain a match on its microbial identification 54 S. Mehrabi et al. / Biomolecular Engineering 18 (2001) 49–56 system (MIS) database using the fatty acid profile. Based on microscopic examination, cultural requirements and metabolic properties, ATCC identified the bacterium as an unknown species of Rhodopseudomonas genus.

3.4. Bioremediation property

The bacterium reduced Cr(VI) to Cr(III). The reduction was rapid at lower concentrations of Cr(IV) and decreased at higher concentrations as toxicity of Cr(IV) increased. The reduction rates were as follows: 10 mg/l per 2 h and 80 mg/l per 2 days. TCP degradation rate was about 10 mg/l per day. The metabolic products of 2,4,6-TCP and 2,4-DCP, 4-CP also appeared in approx- imately 3 h and 2 days and disappeared in 4 and 8 days, respectively. These products were identiﬁed using known standards. 4,6-DCP, 2,6-DCP, 2-CP and 6-CP were not detected, suggesting that groups at positions 6 and 2 are most labile.

3.5. RAPD

While 20 RAPD primers produced multiple amplification products for the seven bacterial species (Fig. 4), three primers: SMO1 (5%-ACGGTGCCTG-3%), SMO2 (5%-GAGATCCGCG-3%) and SMO3 (5%-CGGGTC- GATC-3%) produced the most variable banding profile. RAPD-PCR produced distinguishable bands with the studied species (lane 7) from all other six species (lanes 1, 2, 3, 4, 5, 6). Fig. 4. RAPD-PCR profiles obtained with primers: SMO1 (a); SMO2 (b); SMO3 (c) show variations among the DNA of the seven Rhodopseudomonas spp. (lane 1, Rps. palustris; lane 2, Rps. 3.6. Phylogeny acidophila; lane 3, Rps. rutila; lane 4, Rps. rosea; lane 5, Rps 6iridis; lane 6, Rps marina; lane 7, isolate). Lanes M, Kb ladder. To classify the isolate, we used Clustal W [29] to align the 407 bp sequence obtained from the isolate to Bradyrhizobium spp. and Rhodopseudomonas with several non-sulfur alpha-2 Proteobacteria groups. palustris. From this alignment, we construct a rooted phylogenetic tree (Fig. 5). Phylogenetic analysis of this isolate using the 16S-rDNA sequence data suggests that the 4. Discussion isolate was an unidentified species that is closely related One of the objectives of this study was to identify Table 3 and characterize some of the dominant species in a Growth parameters of the isolate in different nutrients synthetic microbial mat designed for bioremediation applications. Results from this investigation provided Media Ta (h) g (h) information on a heretofore-unidentified strain of Rhodopseudomonas spp., a dominant bacterium in the Nutrient broth95 4:45 Acetate 164 16 mats. In order to assess the bioremediation potential of Succinate168 21 the isolate for metal removal from contaminated water, Ethanol234 18 an initial scan of tolerance to various metals was per- Glucose 234 23 formed (data not shown). Based on these results, a Pyruvate 270 19 selection of Cr(VI) was made for further studies in metal removal rates from contaminated water. Al- Value Ta is end of exponential phase in hours determined directly from growth curves; g is mean generation time in hours calculated though Cr(VI) was the only metal included in these from growth equations [24]. studies, metal tolerances indicate that this microbe may S. Mehrabi et al. / Biomolecular Engineering 18 (2001) 49–56 55 be important in the bioremediation of other metals as that was clearly distinguishable from other known well. The identification of this bacterium as a putative Rhodopseudomonas species. The physiological and mor- new member of non-sulfur purple bacteria was based phological properties of the isolate are also different on its morphological, physiological characteristics, and from those of the other known Rhodopseudomonas spe- DNA analyses. These results suggest that this isolate is cies [2], which was consistent with the RAPD-PCR a mesothermophilic gram-negative rod-shaped bac- results. terium that shares general characteristics with the Comparison of the partial 16S-rDNA sequence Rhodopseudomonas a genus of Rhodospirillaceae family. (407 bp) of this isolate with the sequences found in the Cellular and morphological similarities of the isolate to GenBank database yielded 97% homology to Nitrobac- the previously described species in the genus ter and several Bradyrhizobia and Rhodopseudomonas Rhodopseudomonas are: the presence of a lamellar intra- palustris strains. Using SEQUENCE-MATCH from the cytoplasmic membrane system, the bactochlorophyll a Ribosomal Database Project II we determined that this and reproduction by budding [1–3]. Electron micro- isolate belongs to the purple bacteria, alpha subdivision graphs of the isolate also exhibited these characteristics of proteobacteria based on the fact that sequence ho- (Figs. 1 and 2). mology corresponded with the rhizobium-agrobacterium group, bradyrhizobium subgroup. This study and other The isolate did not grow on sulfide-containing phylogenetic studies using 16S-rRNA sequence ho- medium and failed to form intracellular sulfur globules mologies [5,6] have demonstrated that species that distinguished this strain from the sulfur purple Rhodopseudomonas palustris is more closely related to bacteria. The general identification of the isolate as a some chemotrophic taxa, such as Bradyrhizobium spe- Rhodopseudomonas spp. is consistent with results from cies, Nitrobacter winogradskyi and Afipia felis than to ATCC. any of the other Rhodopseudomonas species. Although The RAPD-PCR profile of the isolate had a pattern data obtained from this study clearly provide reliable evidence for close relationships between this isolate and Rhodopseudomonas palustris, it would be unduly opti- mistic to hope that analysis of 407 nucleotide positions of 16S-rDNA, many of which are invariant, could allow an unambiguous reconstruction of all deeper branches in the phylogeny of this isolate to already known R. species. However, our results show that the isolate, Bradyrhizobium spp. and Rhodopseudomonas palustris belong to the same lineage, and are distinct from all other Rhodopseudomonas species. Based on the results of this study, we suggest that this isolate is an unidentified strain of Rhodopseudomonas species. The bioremediation properties of this isolate will be presented in another report.

Acknowledgements

This project was partially supported by the US Envi- ronmental Protection Agency, Ofﬁce of Exploratory Research, Washington, DC 20460; Assistance ID NO R 821301-01-0 and a grant of the Department of Energy, No. DE-Fco4-90AL66158.

References

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