BIOTROPIA NO BIOTROPIA NO. 15, 2000 : 26 - 35 PRESENCE OF hemA-LIKE AND hemT-LlKE GENES IN A JSUMBER OF ANOXYGENIC PHOTOSYNTHETIC BACTERIAL ISOLATES FROM INDONESIA AND SOIL SAMPLES FROM BOGOR AREA NURUL AINI'AND ANTONIUS SUWANTO' 2 -1 'Laboratory of Microbiology and Biochemistry, Inter University Centre for Biotechnology, Bogor Agricultural University, Bogor-16680 Indonesia. Department of Biology, Faculty of Science and Mathematics, Bogor Agricultural University, Bogor 16144, Indonesia. 'South-East Asian Regional Center for Tropical Biology, Bogor-16001, Indonesia. ABSTRACT The Rhodobacter sphaeroides hemA and hemT are known to encode a distinct 5-aminolevulinic acid (ALA)-synthase isozyme. This enzyme catalyzes the first and rate limiting step in ALA biosynthesis through the C4 pathway. This study was carried out to detect hemA-\\ke and hemT-\\ke genes in twenty Anoxygenic Photosynthetic Bacterial (APB) isolates from several wetland areas in Indonesia, and four DNA samples that were isolated from four soil samples obtained from Bogor area. Hybridization techniques of Southern and dot blot were used, using hemA and hemT fragment as probes. Southern hybridization analyses indicated the presence of hemA-\\ke gene in five of APB isolates, i.e., MB15, MB16, MB21.2, MB55 and MB6, whereas hemT-\\ke gene was detected only in MB15. Dot blot hybridization analyses suggested that the soil samples from waterlogged paddy-field, dry paddy-field as well as a mud pond were predominantly occupied by prokaryotic organisms which harboured hemA-]\ke gene. However, /iem7"-like sequences were also found in soil sample from dry paddy-field. Key words: hemA-\\ks gene / hemT-\\ke gene / Southern hybridization analysis / dot blot hybridization analysis. INTRODUCTION The Rhodobacter sphaeroides hemA and hemT genes encode a distinct 5- aminolevulinic acid (ALA) synthase isozyme (Neidle & Kaplan 1993). ALA- synthase catalyzes the first and rate-limiting step in ALA biosynthesis through the C4 pathway. ALA is the first committed precursor in the common tetrapyrrole pathway (Goodwin & Mercer 1986; Beale & Weinstein 1991; Beale 1995). Recently, ALA has received attention as a new biodegradable herbicide (Sasaki et al. 1987) and insecticide (Sasaki et al. 1990). The DNA sequences of hemA and hemT genes and their location on R. sphaeroides physical map have been determined. The hemA gene is located on the large chromosome whereas hemT gene is found on the small chromosome (Neidle & Kaplan 1993a). The hemA and hemT genes encode peptides that are 53% similar to each other, and these peptides are also significantly similar to ALA-synthase from several bacteria and eucaryotic species (Neidle & Kaplan 1993). The hemA frag- ment has been cloned in R. sphaeroides (Tai et al. 1988) as well as in Escherichia 26 BIOTROPIA NO. 15, 2000 coli (Werf & Zeikus 1996). The cloned hemA fragment is expressed well in E. coli and able to enhance ALA production (Werf & Zeikus 1996). This evidence shows that hemA can be used as a genetic material for enhancing ALA production. Since hemA and hemT genes have high homology to gene encoding ALA- synthase from other organisms, hemA-V\ke and hemT-V\ke genes might be found in other bacteria that form ALA through the C4 pathway. In this study, we detected the presence of hemA-\\ke and hemT-\ike genes in Anoxygenic Photosynthetic Bacterial (APB) isolates, because most member of APB use the C4 pathway to produce ALA. Soil, especially paddy-field soil, is known as a common habitat of APB (Habte & Alexander 1980: Gest et al. 1985). Therefore, we also carried out an experiment to detect these genes in four soil samples. The results of this study would be expected to generate some insights on the distribution and population density of APB as well as R, sphaeroides strains. In addition, the specificity of hemT would be assessed to be used as a specific molecular marker for R. sphaeroides isolates. MATERIALS AND METHODS Bacterial strains, plasmids, growth conditions and soil samples The bacterial strains and plasmids used are listed in Table 1. APB isolates were grown photoheterotrophically in Sistrom's minimal medium (Lueking et al. 1978) in full filled screw-cap tubes, pH 7.2. E. coli were grown at 37°C in Luria Bertani (LB) Table 1. Bacterial strains and plasmids 27 Presence of hemA-like and hemT-like genes – Nurul Aini & Antonius Suwanto Table 1. Continued (Sambrook et al. 1989) supplemented as needed with antibiotics. Antibiotics were added at the following concentrations: 100|ag Ampicillin/ml (for maintaining pUC- 19, pUI1004, pUI1014, and pUI612), 25|ig Chloramphenicol/ml (for maintaining pHFl.l). Soil samples were taken from waterlogged paddy-field, dry paddy-field, LSI pond and Grawida yard, Bogor Agricultural University, Darmaga Campus. All sampling areas were located at Darmaga, Bogor. The description of the soil samples are listed in Table 2. Table 2. Soil samples DNA isolation Plasmids DNA were isolated by using Wizard Miniprep DNA Purification System (Promega, Wise.) according to the manufacturer's instruction. Genomic DNA was extracted from each APB isolate using the phenol extraction method with slight modification as follows. The cell pellet was suspended in EDTA solution containing 15 mg lysozyme/ml, and incubated at 37°C for 1 hour. The lysis was accomplished by adding 300 ul SDS buffer (0.1 M NaCl; 4% SDS; 0.5M Tris-HCl, pH 8). The extract was freeze-thawed. The DNA was phenol-extracted and ethanol- 28 BIOTROPIA NO. 15, 2000 precipitated as in standard protocol (Sambrook et al. 1989). The E. coli genomic DNA was isolated as described previously (Leach et al. 1994). The DNA was extracted from soil by using modified Tiedje Method (Keller 1997, unpublished). The soil sample (10 g) was finely grinded. The 5 g grinded soil was mixed with 13.5 ml Tiedje buffer (100 mM Tris-HCl, pH 8; 100 mM Na- EDTA, pH 8; 100 mM Na2PO4, pH 8; 1.5 M NaCl; 1% CTAB) in 100 ml centrifuge tube, and freeze-thawed 3x. After freeze-thawing, 100 ul proteinase-K (20 mg/ml) (Sigma, Singapore) was added and incubated at 37°C for 30 minutes, then 10 ml 10 % SDS was added and incubated at 65°C for 2 hours. The mixture was centrifuged at 6000 g for 10 minutes. The supernatant was extracted with 1 volume of chloroform (Merck, Jakarta) and centrifuged at 6000 g for 1 minute. The aqueous phase was transferred to a new 100 ml centrifuge tube and 0.6 volume of isopropanol (Merck, Jakarta) was added at room temperature. The DNA was recovered by centrifugation at 16,000 g for 20 minutes at 4°C. The supernatant was discarded and the pellet was washed with 70 % ethanol. The DNA was dried at room temperature and dissolved in 100 ul TE buffer (OmM Tris-HCl, pH 8; I m M EDTA). Southern hybridization analysis The 1.2 kb Bamttl (NEB, Singapore) fragment from p U I 1 0 1 4 and 1.8 kb Bam\\\ (NEB, Singapore) fragment from pUI1004 were isolated for preparing hemA and hemT probes. The fragments were purified from agarose gel using the Gene Clean Kit (Bio 101 Inc, La Jolla, Calif.), and labeled with biotin-14-ATP using Nick Translation System (GIBCO/BRL, Grand Island, NY) according to the manufac- turer's instruction. The unincorporated nucleotides were removed from the probes with nuctrap push columns (Stratagene, La Jolla, Calif.). The genomic DNAs extracted from APB isolates were digested with EcoRl (NEB, Singapore), except the DNA from MB 15 isolate, which was digested with Bam\\\ (NEB, Singapore). Digestions were performed in appropriate buffer at 37°C for 12 hours. The digested DNA was electrophoretically separated. The DNA fragments were transferred to a nylon membrane (Photogene, GIBCO/BRL, Grand Island, NY) by capillary action with standard method (Sambrook et al. 1989). Hybridization was carried out as described previously at 42°C for 12 hours (Sambrook et al. 1989), followed by washing at 37°C for 2x 15 minutes each, and detection using a chemiluminescent method (Photogene Detection System, GIBCO/ BRL, Grand Island, NY). Dot blot hybridization analysis Four biotinilated DNA probes, i.e. hemA, hemT, pucBA, and I6S rRNA genes were used. The probes were prepared as described above. To avoid bias in calculation, the population density of prokaryotes was based on the same amount of soil samples, and not on the same amount or standardization of DNA concentration. DNA isolated from soil samples were denatured as described previously (Keller and 29 Presence of AemA-like and hemT-\ike genes - Nurul Aini & Antonius Suwanto Manak 1992), and applied to nylon membrane (Photogene, GIBCO/BRL, Grand Island, NY) by spotting directly onto the membrane. High stringency hybridization and washing conditions were used. Hybridization was carried out at 42°C for 12 hours, with washing temperature of 55°C (Sambrook et al. 1989). Detection was done using the Photogene Detection System (GIBCO/BRL, Grand Island, NY). RESULTS AND DISCUSSION Southern hybridization analyses Southern hybridization analyses were performed to determine the presence of hemA-like and hemT-\\ke genes in twenty APB isolates from Indonesia (Table 1). R. sphaeroides 2.4.1 (Rsp 2.4.1) hemA and hemT genes were used as probe. The analyses identified some homologous regions the homology to hemA in the genomic DNA of MB6, MB 15, MB 16, MB21.2 and MBS 5. The region of homology to hemA probe in the genomic DNA of each APB isolate is shown in Figure la, lanes 3-7. The hemTprobe hybridized only to 4.8 kb BamHl fragment in MB15 genomic DNA (Fig. Ib, lane 3). A B Figure 1. A. Southern hybridization analyses using hemA probe. Lanes (1) Rhodobacter sphaeroides 2.4.1, (2) KBslEll, (3) MB15, (4)MB16, (5) MB21.2, (6) MB55, and (7) MB6 B. Southern Hybridization analyses using hemTprobe Lanes (1) Rhodobacter sphaeroides 2.4.1, (2) KBslEll, and (3) MB 15 30 BIOTROPIA NO. 15, 2000 The results implied the presence of hetnA-like gene in MB6, MB15, MB16, MB21.2 and MB55, whereas hemT-like gene was only implied in MB 15. The presence of hemA-like and/or hemT-like genes in the five APB isolates indicates the ALA biosynthesis in these APB isolates is employed through the C4 pathway, because the C4 pathway is mediated by ALA synthase which encoded by hemA and/or hemTgenes. Moreover, the presence of hemA-like and/or hemT-\ike genes in the five APB isolates indicates that these APB isolates could be classified under sub group cc-proteobacteria. In photosynthetic bacteria, C4 pathway is utilized by purple non-sulfur bacteria, especially sub group a-proteobacteria (Avissar et al. 1989; Beale 1995). The sub group a-proteobacteria contains species of genera Rhodos- pirillum, Rhodopila, Rhodopseudomonas, Rhodomicrobium and Rhodobacter (Imhoff 1995). Neither hemA-like gene nor hemT-like gene was identified in the other 15 APB isolates. The data suggested that the C4 pathway is not utilized by these APB isolates to produce ALA. The 15 APB isolates might produce ALA through C5 pathway, which does not require ALA synthase. These APB isolates used are not the member of sub group a-proteobacteria, although all APB isolates studied here belong to purple non-sulfur bacteria. The hemT-like gene was only identified in MB 15. This APB isolate also carries hemA-like gene. Interestingly, the hemA-like gene in MB 15 was detected on the same locations with Rsp. 2.4.1 hemA, i.e., at 1.2 kb and 2.4 kb BamHl fragments (Fig. la, lane 1 and 3). The strong intensity of the hybridization signals revealed that the similarity between MB 15 hemA-like gene and Rsp.2.4.1 hemA was very high. Moreover, the color comparison of MB 15 culture with Rsp. 2.4.1 culture also showed a high similarity. Based on these findings, it is very likely that MB 15 was Rhodobacter sphaeroides. We tentatively conclude that hemT-like gene harbors specifically in R. sphaeroides. Neidle and Kaplan (1993) reported that the R. sphaeroides is the only bacterial species that produces two ALA synthase isozymes. However, previously ALA synthase isozymes are found in some vertebrates (Dierks 1990), while no previous information on bacterial ALA synthase isozymes have been reported. Dot blot hybridization analysis Dot blot hybridization analysis was employed to detect the presence of hemA- like and hemT-like genes in DNA extracted from four soil samples (Table 2). Four DNA probes, i.e., hemA, hemT, pucBA, and 16S rRNA gene were used. This analysis also revealed the relationship of the activity of prokaryote and APB in the soil samples with the presence of hemA-\ike and hemT-like genes. Figure 2 shows the results of dot blot hybridization using the four probes. Interpretation of these results is described in Table 3. Based on the hybridization using hemA probe (Fig. 2a), the presence of hemA-like gene was detected in soils taken from waterlogged paddy field, dry paddy field and LSI pond. The presence of hemA-like gene in these soil samples indicates the activity of organisms producing 31 Presence of AemA-like and hemT-\\ke genes - Nurul Aini & Antonius Suwanto ALA through the C4 pathway. The C4 pathway is utilized by animals, fungi, protozoa and sub group cc-proteobacteria (Avissar et al. 1989; Beale & Weinstein 1991; Beale 1995). Figure 2. Dot blot hybridization analyses using HemA (A), hemT(B), 16S rRNA (C), and pucBA (D) as probes. Dots (1) Rhodobacter sphaeroides 2.4.1., (2) soil sample from, waterlogged paddy field, (2) soil sample from dry paddy field, (3) soil sample from LSI pond, and (5) soil sample from Grawida yard Table 3. Interpretation of the result of dot blot hybridization + : Detected - : Nodetected The number of (+) represent the degree of intensity of hybridization signal 32 BIOTROPIA NO. 15, 2000 Using 16S rRNA probe, we identified the presence of prokaryotic micro- organisms in all four soil samples (Fig. 2c). The results of hybridization to pucBA probe indicated that the APB could be detected in all soil samples (Fig. 2d). Thus, it is possible to find the ct-proteobacteria in the soil samples, because some members of oc-proteobacteria are classified as APB group. The possibility for the ct-proteo- bacteria to exist in the four soil samples correlates with the presence of hemA-l\ke gene in these soil samples. However, in spite of the detection of the APB activities in soil from Grawida yard, hemA-\ike gene was not found in this soil. The intensity of hybridization signal to both 16S rRNA (Fig. 2c) and pucBA (Fig. 2d) probes shows that the population densities of both prokaryote and APB in soil from Grawida yard are much lower than those found in the other three soil samples. The a-proteobacteria might not exist in the soil from Grawida yard or the cc- proteobacteria were actually present in this soil, but the population density is very low. Therefore, presence of hemA-Yike gene could not be detected. Soils obtained from waterlogged paddy field, dry paddy field and LSI pond were found to have high population density of prokaryotes and APB. The high population density of APB enables the a-proteobacteria to proliferate. The presence of hemA-V\ke gene in these soil samples is related to the population density of APB. The population density of APB as well as other microorganisms in the soil is dependent on the availability of growth nutrient, O2 and water (Brock & Madigan, 1991). The fertility and the soil texture are responsible for the availability of these factors. Therefore, the presence of HemA-\ike gene in soil is indirectly affected by the fertility and the soil texture Based on the results of hybridization to hemA probe, the hemA-\ike gene might be present in the soil taken from APB habitat. In nature, APB occur in moist soil, paddy field, sewage water, fresh water, brackish water, waste water, marine habitat and in extreme condition of the Antartic (Sasikala et al. 1985). The presence of hemA-\ike gene in the soil is related to the fertility of the soil, because the availability of growth nutrient affects APB growth. The dot blot hybridization using hemT probe identified the presence of hemT- like gene only in soil taken from dry paddy field (Fig. 2b, dot 3). By comparing the intensity of hybridization signal to hemT probe with the hybridization signal to hemA probe, it is clear that the homologous sequence to hemT probe is present in relatively fewer amount than the homologous sequence to hemA probe. The results indicate that not all organisms carrying hemA-\\ke gene also carry the hemT-\ike gene. These findings confirm the data obtained from Southern hybridization analy- ses that the presence of hemT-\ike gene is more specific than that of hemA-\ike gene. The hemT or hemT-\\ke gene might be specific to R. sphaeroides. 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