Antonie van Leeuwenhoek (2014) 105:207–219 DOI 10.1007/s10482-013-0067-2
ORIGINAL PAPER
Salinispora arenicola from temperate marine sediments: new intra-species variations and atypical distribution of secondary metabolic genes Kian-Sim Goo • Masashi Tsuda • Dana Ulanova
Received: 26 September 2013 / Accepted: 24 October 2013 / Published online: 1 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract The obligate marine actinobacterium Salinispora arenicola was successfully cultured from temperate sediments of the Pacific Ocean (Tosa Bay, offshore Kochi Prefecture, Japan) with the highest latitude of 33°N ever reported for this genus. Based on 16S rRNA gene sequence analysis, the Tosa Bay strains are of the same phylotype as the type strain S. arenicola NBRC105043. However, sequence analysis of their 16S-23S rRNA intergenic spacer (ITS) revealed novel sequence variations. In total, five new ITS sequences were discovered and further phylogenetic analyses using gyrase B and rifamycin ketosynthase (KS) domain sequences supported the phylogenetic diversity of the novel Salinispora
isolates. Screening of secondary metabolite genes in these strains revealed the presence of KS1 domain sequences previously reported in S. arenicola strains isolated from the Sea of Cortez, the Bahamas and the Red Sea. Moreover, salinosporamide biosynthetic genes, which are highly homologous to those of Bahamas-endemic S. tropica, were detected in several Tosa Bay isolates, making this report the first detection of salinosporamide genes in S. arenicola. The results of this study provide evidence of a much wider geographical distribution and secondary metabolism diversity in this genus than previously projected. Keywords Marine actinomycetes Salinispora arenicola Secondary metabolism Salinosporamide
Electronic supplementary material The online version of this article (doi:10.1007/s10482-013-0067-2) contains supplementary material, which is available to authorized users.
Introduction K.-S. Goo Evaluation and Support Organization for Young Researchers, Kochi University, Kohasu, Oko-cho, Nankoku, Kochi 783-8505, Japan M. Tsuda Center for Advanced Marine Core Research, Kochi University, Monobe-otsu 200, Nankoku, Kochi 783-8502, Japan D. Ulanova (&) Oceanography Section, Science Research Center, Kochi University, IMT-MEXT, Kohasu, Oko-cho, Nankoku, Kochi 783-8505, Japan e-mail: [email protected]
Actinobacteria have been isolated from a large variety of samples collected worldwide (Okazaki 2006; Ward and Bora 2006; Valverde et al. 2012), with some genera showing preferential occurrence in specific environments (Mincer et al. 2002; Zhi et al. 2007). However, their global distribution patterns, as well as the environmental factors in different locations that affect biodiversity, are poorly understood. For example, some terrestrial species were found in marine environments, but it is believed that they were washed out from the shoreline. Further studies on these marine
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species revealed higher tolerance of salinity and some differences in their metabolite production, suggesting possible adaptation of these species to the marine environment (Okazaki 2006). Nevertheless, such ambiguities over terrestrial/marine origins often complicate environmental studies. In recent years, marine actinobacteria have been extensively isolated and studied for the presence of bioactive natural compounds that are useful for clinical purposes (Ward and Bora 2006; Bull and Stach 2007; Blunt et al. 2013). Compared to their terrestrial counterparts, marine actinobacteria are relatively underexplored, and thus there exists the potential of discovering novel metabolites with as yet unknown activities (Zotchev 2012). The genus Salinispora contains the first reported obligate marine actinomycetes, which have a strict requirement for seawater in order to survive and proliferate (Jensen et al. 1991; Mincer et al. 2002). To date, only three Salinispora species have been described, namely Salinispora arenicola, Salinispora tropica and Salinispora pacifica (Maldonado et al. 2005; Ahmed et al. 2013). Although these three species share more than 99 % identity in their 16S rRNA gene sequences, they are distinguished into different species due to them having less than 60 % of DNA–DNA relatedness (Maldonado et al. 2005; Ahmed et al. 2013). Nonetheless, they can be differentiated by their 16S rRNA gene sequences (Freel et al. 2012). So far, five phylotypes based on signature nucleotide positions of 16S rRNA gene sequences have been described for S. arenicola, 15 for S. pacifica and only one for S. tropica (Freel et al. 2012; Vidgen et al. 2012). Salinispora species are widely found in tropical and subtropical marine sediments, showing species-specific biogeographical distributions (Jensen and Mafnas 2006). At present, S. arenicola has been reported to have a more diverse distribution, where strains of this species have been isolated from the Bahamas, several locations in the tropical and subtropical Pacific Ocean, and the Red Sea (Jensen and Mafnas 2006; Freel et al. 2012). On the other hand, S. pacifica was only reported in seven out of the 12 locations studied, mainly in the Pacific regions and the Red Sea (Freel et al. 2012). As for S. tropica, it appears to be endemic to the Bahamas (Mincer et al. 2002). Interestingly, no Salinispora species has been cultivated from temperate regions, leading to the hypothesis that low temperature affects
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Salinispora viability (Jensen and Mafnas 2006). However, Salinispora strains have been successfully isolated from deep-sea sediment samples obtained from the Bahamas at a depth of 1,100 m with temperatures near 4 °C (Mincer et al. 2005). Therefore, it is very likely that there are other factors affecting Salinispora viability and distribution in temperate regions. This hypothesis is supported by culture-independent methods, where the DNA of Salinispora species has been detected in temperate marine environments (San Diego, 32°N) and deep-sea sediments of 5,699 m, although attempts to cultivate them from these samples remain unsuccessful (PrietoDavo et al. 2013). The most significant aspect of studying this actinobacterial genus is the diverse range of secondary metabolites it produces. Extensive screening studies of Salinispora species have revealed species-specific production of secondary metabolites and geographical production patterns. The profiling of over 30 S. arenicola strains from six geographically distinct locations has revealed that all of them produce rifamycin, staurosporine and saliniketal. Similar profiling studies of several Bahamas-derived S. tropica strains have revealed that they produce salinosporamide and sporolide. In addition to the core chemotype, some S. arenicola strains were found to specifically produce compounds unique to certain locations (Jensen et al. 2007). These results, combined with the analysis of the Salinispora genome, led to the conclusion that secondary metabolism is a major speciation factor for this genus and that secondary metabolite profiles could be location-specific (Udwary et al. 2007; Penn et al. 2009). However, it remains unclear how these location-specific metabolites influence Salinispora species diversity, geographical distribution and their adaption to the local environment. Nevertheless, there lies the possibility of strains isolated from new locations that produce yet unknown secondary metabolites with new bioactivities. In this study, we report the successful isolation of Salinispora strains from offshore sediments of Tosa Bay (Kochi Prefecture, Japan) at the highest latitude of 33°N recorded so far. These strains were identified as S. arenicola based on physiological properties and 16S rRNA gene sequence analysis. However, detailed sequence analysis of the 16S-23S rRNA intergenic region, housekeeping and secondary metabolic genes revealed variations between the isolates, despite their
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conserved 16S rRNA sequence. Interestingly, phylogenetic analysis with deposited genome sequences of S. arenicola showed the relatedness of some Tosa Bay strains with isolates from other reported locations, especially regarding their secondary metabolism.
209
filamentous hyphae were detected by eye and/or using a microscope (SZX7, Olympus). Thereafter, colonies were transferred to fresh culture plates of the same medium until pure cultures were obtained. Manipulation of type strains
Materials and methods Sampling of marine sediment and selective actinobacterial isolation Sediment samples were collected in January 2012 from Tosa Bay, off Kochi Prefecture, Japan, at depths of 10, 30, 50, 80 and 100 m below seawater surface (33°27.110 N, 133°28.500 E; 33°26.060 N, 133°29.240 E; 33°24.260 N, 133°30.090 E; 33°21.350 N, 133°31.230 E; 33°18.530 N, 133°32.320 E, respectively), using a Smith-McIntyre sediment sampler (Smith and McIntyre 1954). Sediment samples were stored at 10–20 °C and processed within one week using methods described by Jensen et al. (2005). Briefly, three pretreatment methods were used: 1. Dry method: About nine grams of each sample were dried in a biosafety cabinet for four to five hours. After drying, sediment samples were ground and inoculated by a plate stamping technique (Mincer et al. 2002); 2. Dry and heat method: Dried sediment samples were added to sterile deep seawater (DSW, Kochi Prefectural Deep Seawater Laboratory) in 1:4 dilution and heated for 6 min at 55 °C. After heating, samples were mixed by vortexing and 50 lL of each sample were inoculated; 3. Wet method: One gram of wet sediment was added to 9 mL of sterile DSW and one gram of glass beads, following by shaking at room temperature at 180 rpm for 1 h. Ten and 100 times dilution of samples were inoculated onto plates using sterile spreaders. Seven high-nutrient cultivation media (AMM, Bennet’s, ISP1, starch-casein agar, non-sporulation agar, Mueller–Hinton agar, Middlebrook agar), as well as six low-nutrient media (SMC, SMP, SNC, SPC, STC, SMY) were used (Jensen et al. 2005; Maldonado et al. 2009). All media were prepared using DSW. Cycloheximide (100 mg L-1) or nystatin (50 mg L-1) was added to suppress fungal growth and some media were also supplemented with rifampicin (5 mg L-1) to reduce the growth of fast growing bacteria. All culture plates were incubated at 28 °C for up to 4 months. Actinobacterial-like colonies forming
Type strains of S. arenicola (CNH643/ATCC BAA917) and S. tropica (CNB440/ATCC BAA916) were obtained from NBRC (Biological Resource Center, National Institute of Technology and Evaluation, Japan) and are referred as NBRC105043 and NBRC105044 in this study, respectively. They were cultivated according to supplier’s recommendations and used as a positive control to validate gene amplification conditions. Low-temperature sensitivity, seawater requirement and rifampicin resistance For testing of seawater requirement and rifampicin resistance, isolates were cultivated on AMM media prepared using DSW or distilled water with or without addition of rifampicin (20 mg L-1) for 2–3 weeks at 28 °C. For testing of viability after prolonged lowtemperature incubation, selected strains were first cultivated at 28 °C for 3 weeks on two sets of AMM agar plates. Thereafter, one set of plates was transferred to 4 °C. Every week, isolates from the 28 and 4 °C plate sets were transferred to fresh AMM agar and cultivated at 28 °C. Viability of strains was visually estimated after 2–3 weeks of cultivation. BOX-PCR Genomic DNA of marine isolates was extracted by lysozyme treatment, followed by phenol:chloroform extraction and ethanol precipitation (Kieser et al. 2000). The final BOX-PCR reaction mixture contained 19 Ex TaqÒ buffer (TaKaRa), 0.5 mM dNTPs, 10 % (v/v) DMSO, 0.5 lM BOX-A1R primer (Lanoot et al. 2004), 0.2 mg mL-1 BSA, 2.5 U Ex TaqÒ DNA polymerase (TaKaRa) and 100 lg mL-1 genomic DNA. The cycling condition used was initial denaturation at 95 °C for 5 min, followed by 35 cycles of 92 °C for 30 s, 53 °C for 30 s and 68 °C for 6 min, and final extension at 68 °C for 16 min. The PCR products were subjected to agarose gel electrophoresis using 2 % (w/v) agarose gels prepared in 19 TAE (Tris–Acetate–
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EDTA) buffer and electrophoresed at 50 volts for 60 min. Subsequently, the agarose gels were stained using SYBRÒ Safe DNA stain (Invitrogen). Amplification of 16S rRNA and 16S-23S rRNA intergenic spacer sequences 16S rRNA gene sequencing was used to determine the identity of the marine isolates that morphologically resembled members of the family Micromonosporaceae. The amplification of nearly complete 16S rRNA gene sequences was performed using the primers 28F and 1492R (Lane 1991; Jensen and Mafnas 2006). The amplification reaction mixture contained 19 Ex TaqÒ buffer (TaKaRa), 0.5 mM dNTPs, 5 % (v/v) DMSO, 3 lM of each primer, 2.5 U Ex TaqÒ DNA polymerase (TaKaRa) and *100 lg mL-1 genomic DNA. The cycling condition used was initial denaturation at 94 °C for 1 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 90 s, and final extension at 72 °C for 5 min. Amplification of PCR products was detected using agarose gel electrophoresis and the amplimers subjected to DNA sequencing using the ABI PRISMTM dye terminator cycle sequencing kit and ABI PRISMTM 3100 Genetic Analyser, according to manufacturer’s recommendations. The same primers for gene amplification were used for sequencing, including additional primers 530R and 936R (Jensen and Mafnas 2006). For the amplification of the 16S-23S rRNA intergenic spacer (ITS), the reaction mixture contained 19 Ex TaqÒ buffer (TaKaRa), 0.5 mM dNTPs, 5 % (v/v) DMSO, 3 lM of each primer, ITS_F and ITS_R (Table S1), 2.5 U Ex TaqÒ DNA polymerase (TaKaRa) and *100 lg mL-1 genomic DNA. The cycling condition used was initial denaturation at 95 °C for 1 min, followed by 30 cycles of 95 °C for 30 s, 65 °C for 30 s and 72 °C for 30 s. Amplification of PCR products was detected using agarose gel electrophoresis and subjected to DNA sequencing as described above, using the same amplification primers. Amplification of gyrB sequence Gene amplification of gyrase B subunit (gyrB) was based on the primers gyr33F, gyr611F, gyr662R and gyr1300R reported by Jensen and Mafnas (2006), which included M13 primer sequences on 50 end. The reaction mixture contained 19 Ex TaqÒ buffer
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(TaKaRa), 0.5 mM dNTPs, 5 % (v/v) DMSO, 0.6 lM of each primer, 2.5 U Ex TaqÒ DNA polymerase (TaKaRa) and 100 lg mL-1 genomic DNA. The cycling condition used was initial denaturation at 95 °C for 2 min, followed by 30 cycles of 95 °C for 30 s, 65 °C for 30 s and 72 °C for 30 s. Amplification of gene products was detected using agarose gel electrophoresis and subjected to DNA sequencing as described above, using M13 primers. Detection of ketosynthase sequences Gene amplification of ketosynthase (KS) sequences, i.e., KS1-4 and the rifamycin polyketide synthase KS domain, was based on the specific primers reported by Edlund et al. (2011). The reaction mixture contained 19 Ex TaqÒ buffer (TaKaRa), 0.5 mM dNTPs, 5 % (v/v) DMSO, 0.6 lM of each primer, 2.5 U Ex TaqÒ DNA polymerase (TaKaRa) and 100 lg mL-1 genomic DNA. The gene amplification cycling condition reported by Edlund et al. (2011) was also adopted. Amplification of gene products was detected using agarose gel electrophoresis and subjected to DNA sequencing as described above. Detection of S. tropica-specific secondary metabolite gene clusters Representative genes from S. tropica-specific secondary metabolite gene clusters were amplified using the primers listed in Table S1. The reaction mixture contained 19 Ex TaqÒ buffer (TaKaRa), 0.5 mM dNTPs, 5 % (v/v) DMSO, 0.6 lM of each primer, 2.5 U Ex TaqÒ DNA polymerase (TaKaRa) and 100 lg mL-1 genomic DNA. The gene amplification cycling condition was initial denaturation at 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 64–72 °C for 30 s, depending on the primer pairs (Table S1), and 72 °C for 30 s. Amplification of gene products was detected using agarose gel electrophoresis and subjected to DNA sequencing as described above. Sequence manipulation and phylogenetic analysis DNA sequences were manipulated using Geneious R6 sequence analysis software (Biomatters Ltd). DNA sequences were aligned and sequence similarities were
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calculated using the Geneious alignment method. Phylogenetic trees were constructed using MEGA5 software (Tamura et al. 2011). All sequences were verified by at least two independent DNA amplification and sequencing experiments. New sequence data have been deposited in the GenBank database and their accession numbers are provided in the phylogenetic trees and Supplementary Table S2. Sequence comparison was also performed using the BLAST program (Basic Local Alignment Search Tool from National Center for Biotechnology Information) in both nucleotide collection and whole-genome shotgun contigs databases to search for homologous sequences, up to 11th June 2013.
Results Isolation and identification of Salinispora species In this study, three methods were used to pretreat the Tosa Bay sediments collected at five different depths. A total of 13 culture media, comprising seven highnutrient and six low-nutrient media, were used to isolate actinobacteria from these sediments. 101 actinobacterial-like colonies were isolated and the majority of these colonies (80 %) were obtained using the dry-stamping method. The most effective media for isolation were high-nutrient Bennet’s, AMM and ISP1 culture media. Interestingly, a higher yield of actinobacterial-like colonies per gram of processed sediment was obtained from deeper sediments (30–100 m) (Table 1). Among the 101 actinobacterial-like colonies isolated, 38 colonies possessed the typical characteristics of the family Micromonosporaceae with orange Table 1 Yield of Salinispora isolates from marine sediments of Tosa Bay
Depth (m)
pigmentation and no aerial mycelium formation. Partial 16S rRNA analysis revealed that six of them belong to the genus Micromonospora, three to the genus Nocardia and the majority (29 colonies) to the genus Salinispora. Subsequently, the 16S rRNA gene sequences of the Salinispora isolates were fully sequenced to reveal their species identities. All the sequences obtained were identical to the 16S rRNA gene sequence of the S. arenicola type strain NBRC105043 (Jensen and Mafnas 2006). Neither other phylotypes of S. arenicola, nor those of strains of S. pacifica and S. tropica were detected. Subsequently, the Salinispora isolates were tested for seawater requirement, low-temperature sensitivity and rifampicin resistance, in order to further validate their identities. As five of the 29 isolates lost their viability after several rounds of sub-culturing, only 24 isolates were used for further analysis. All 24 isolates failed to grow on culture media without seawater, indicating an obligatory requirement of seawater for growth, which is a typical characteristic of the genus Salinispora. Furthermore, selected isolates showed reduced viability after prolonged storage at 4 °C (up to 9–23 weeks), similar to previously reported Salinispora strains (Jensen and Mafnas 2006). All strains grew well on culture media supplemented with a high concentration of rifampicin, thus confirming the identity of these isolates to be S. arenicola since only this species, out of the three known Salinispora species, is rifampicin-resistant. Phylogenetic analyses of Salinispora isolates In order to reduce clonal replicates of Salinispora isolates, BOX-PCR using BOX-A1R primer, which has proved to be useful method for the whole-genome
No. of Salinispora colonies
Mean no. of Salinispora colonies per gram of sediment
No. of actinobacterial colonies
Mean no. of actinobacterial colonies per gram of sediment
10
0
0
11
0.55
30
7
0.35
18
0.9
50
7
0.35
31
1.55
80
9
0.45
27
1.35
100
6
0.6
14
1.4
Total number of isolates
29
–
101
–
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212
level analysis and reduction of Salinispora clonal isolates (Vidgen et al. 2012), was performed to compare the Salinispora isolates on a whole-genomic level. From the 24 isolates analysed, ten different BOX profiles were obtained (Fig. S1). For a more comprehensive analysis of the interspecies diversity observed in the BOX-PCR results, the 16S-23S rRNA ITS sequence of the isolates were amplified, sequenced and compared with those deposited in GenBank database. To date, only five ITS sequences of S. arenicola have been reported (Freel et al. 2012) and they are highly homologous to each other, differing only at two positions—G9 and T218 (nucleotide numbering based on the S. arenicola CNS205 ITS sequence) are deleted in the type strain CNH643, and T218 is deleted in the strain CNH964. The rest are identical to that of strain CNH964. Strain NBRC105043, which is clonal to the type strain CNH643, was used as a control in our analysis. Contrary to the sequence deposited in GenBank (AY371897), the ITS sequence of strain NBRC105043 does not have the G9 deletion, suggesting there are only two S. arenicola ITS phylotypes instead of the initial three sequence variations. This sequence was verified by triplicate sequencing experiments from independent DNA isolations and PCR amplifications. More importantly, the S. arenicola isolates from Tosa Bay possessed several differences in the ITS sequence that have not been reported previously (Table 2). Only two isolates had the same ITS sequence as the type strain NBRC105043 and CNH964, where they are referred to as ITS group 0 in this study. The remaining 22 isolates were divided into five different ITS groups. Phylogenetic analysis of all Salinispora species ITS sequences showed a close relationship of these new sequences with those of other S. arenicola strains reported earlier (Fig. S2). Additionally, analysis of yet-to-be-annotated sequences deposited in the whole-genome shotgun contigs GenBank database revealed that some previously reported Salinispora strains possess ITS sequences identical to the Tosa Bay isolates. For example, strains CNS991 and CNH962 have the same ITS sequences as ITS groups 3 and 4, respectively. Nevertheless, the ITS sequences of ITS groups 1, 2 and 5 were found to be unique to the Tosa Bay Salinispora isolates. In order to further validate the ITS sequence diversity, another common phylogenetic marker, gyrase subunit B gene (gyrB), was used (Jensen and
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Mafnas 2006; Peeters and Willems 2011). Nearly completed gyrB sequences were amplified, sequenced and compared. The isolates of the same ITS group were found to possess the same gyrB sequences, which differed from other groups (Table 3). Although ITS group 0 has the same ITS sequence as the type strain NBRC105043, there was some nucleotide sequence variations observed in their gyrB sequences (Table 3). Similarly, sequence comparison of gyrB sequences of ITS groups 3 and 4 with those of strains CNS991 and CNH962 deposited in the whole-genome shotgun contigs GenBank database revealed distinct sequence variations, respectively (Fig. S3). However, all these nucleotide differences do not translate into amino acid differences from the type strain NBRC105043. Interestingly, gyrB sequences of ITS groups 0, 1 and 2 clustered, with high bootstrap values, with sequences of strains from the Sea of Cortez that belong to 16S rRNA phylotype B (Fig. S3) (Jensen and Mafnas 2006). To reflect the 16S rRNA and ITS diversity of the Tosa Bay isolates, as well as to provide an improved support for the separation of ITS groups 1 and 2, a phylogenetic tree of concatenated 16S rRNAITS-gyrB sequence was constructed (Fig. 1). This concatenated tree supports the notion that the Tosa Bay Salinispora isolates are phylogenetically different from other reported strains, despite small variations in the three sequences analysed. Ketosynthase gene analysis Several location-specific sequences coding for newtype ketosynthase domains (designated as KS1–KS4) have been reported in S. arenicola, where these domains are presumably involved in the biosynthesis of as yet unknown polyketide compounds (Edlund et al. 2011). Since secondary metabolism is an important speciation factor in the genus Salinispora, homologues of these sequences were also probed in this study to determine the relatedness of Tosa Bay isolates to reported strains. For this and further analyses, we used one representative strain from each ITS group, designated as JP00–JP05. Each representative strain was screened using the specific primers and gene amplification procedures reported by Edlund et al. (2011). Interestingly, S. arenicola strains JP00, JP01, JP02 and JP03 were found to possess KS1 sequences, which were mainly detected previously in S. arenicola strains isolated from the Sea of Cortez, the Bahamas and the Red Sea. These KS1
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Table 2 Sequence variations in 16S-23S rRNA ITS of S. arenicola isolates ITS group
Representative strain (GenBank accession number)
Number of isolates
218a
231
312–314
338–339
349
–
CNS205 (HQ642901)b
–
T
T
CTG
GG
T
–
CNH964 (AY371900)
–
D
–
CNH962 (ARKS01000023)
–
D
–
CNS991 (ARBB01000003)
–
D
–
NBRC105043
–
D
0
JP00
2
D
1
JP01
10
D
C
2
JP02
4
D
C
3
JP03
2
D
4
JP04
2
D
5
JP05
4
D
a
Nucleotide numbering based on S. arenicola strain CNS205 16S-23S rRNA ITS
b
Data were obtained from whole-genome shotgun GenBank database using BLAST
sequences (575 bp) of JP strains were identical to that of the type strain NBRC105043, which was isolated from the Bahamas. However, KS2, KS3 and KS4, reported from Guam, the Sea of Cortez and the Bahamas, were not detected in any of the JP strains using the specific primers and gene amplification conditions reported by Edlund et al. (2011) (Table S3). In this study, we also analysed a ketosynthase sequence associated with rifamycin biosynthesis (Wilson et al. 2010). Rifamycin has been reported to be produced by all S. arenicola isolated so far and so it is considered a core metabolite specific for this species. The sequences for comparison were based on the S. arenicola CNS205 Sare1247 gene (FJ844623). The Sare1247 partial sequence of Salinispora JP strains and the type strain NBRC105043 were amplified and compared with S. arenicola strain CNS205 (Table S4). Similarly to the ITS and gyrB sequences, all partial Sare1247 sequences of JP strains varied from that of strain CNS205 and the type strain NBRC105043. Although strain JP00 has the same ITS sequence as the type strain NBRC105043, its rifamycin ketosynthase domain sequence differed and thus, further supported the phylogenetic difference between ITS group 0 isolates and the type strain. Detection of S. tropica secondary metabolite biosynthetic gene clusters in S. arenicola isolates Currently, two genomic sequences of Salinispora species are available, i.e., S. tropica CNB440 and S.
D GGTGG
GGTGG GGTGG D C
arenicola CNS205 (Udwary et al. 2007; Penn et al. 2009). Out of the 30 secondary metabolite gene clusters in S. arenicola, 12 clusters were homologous with S. tropica (Penn et al. 2009). We were curious as to whether the new phylogenetically distinct JP strains could possess secondary metabolite gene clusters that have been previously reported as unique for S. tropica. Seven S. tropica gene clusters reported to be absent in S. arenicola CNS205, i.e., pks1, sal (salinosporamide gene cluster), lom (lomaiviticin gene cluster), spo (sporolide gene cluster), slm (salinilactam gene cluster), sid3 and sid4 (Penn et al. 2009; Kersten et al. 2013), were selected. The presence of representative genes from each cluster was analysed by PCR detection. No representative genes of the pks1, lom, spo, slm, sid3 and sid4 gene clusters were detected. Interestingly, salRIII sequence was detected in four of the six representative JP strains with identities of 94 and 90 % with the S. tropica CNB440 salRIII gene and protein sequences, respectively (Table 4). To further validate these findings, specific primers targeting salA, salI, salJ, salL and salZ, which spanned across the sal cluster in S. tropica CNB440, were designed and screened against these strains. Interestingly, nearly all these sequences were present in the four strains that tested positive for salRIII, except for salJ (Table 4). Next, salA sequences of JP strains were used to search for homologues in the GenBank whole-genome shotgun contigs database in order to screen yet-to-beannotated sequences of various S. arenicola strains. Interestingly, three S. arenicola strains (CNP105,
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T A
A
C
C
T C
C G
C C T
T
JP02 JP03
JP04
A
T JP01
T JP05
A NBRC105043/CNH643
Only nucleotide variation at nucleotide position 916 leads to amino acid difference between strain CNS205 (Ala306) and other strains (Thr306). The nucleotide and amino acid numberings are based on S. arenicola strain CNS205 gyrB (Sare0007) sequence numbering
T C G
T
T
T A A A
A
A A
A C
C T JP00
G C G CNS205
123
A T
C
T
T
C
C C
G
C
G C T C G G
A C
T C C
C
T
C
1,377 1,338 1,332 1,296 1,224 1,221 1,197 966 924 916 813 696 636 561 555 528 426 396 Strain\nucleotide no.
Table 3 Sequence variations in gyrB gene of S. arenicola isolates
C
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CNP193 and CNY280) were found to harbour salA homologues. Phylogenetic analysis of salA sequences showed distinct branching of S. arenicola salA sequences from those of S. tropica and S. pacifica, with higher homology to S. tropica sequence (Fig. 2). Similar to the 16S rRNA-ITS-gyrB concatenated tree (Fig. 1), salA sequences from strains JP00, JP01 and JP02 clustered with sequences of strains CNP105 and CNP193 from the Sea of Cortez, whereas strains JP03 and CNY280 showed distinct branching that was well supported by bootstrap analysis.
Discussion The distribution patterns of Salinispora species have been studied extensively during the last decade, which makes this genus a model for studying marine bacterial biogeography and specialisation (Freel et al. 2012). Various methods and cultivation media were adopted in this study to isolate Salinispora species from Tosa Bay marine sediments. Similar to the observation reported by Jensen et al. (2005), the majority of the Salinispora isolates were obtained using the dry-stamping method. One possible explanation could be that the bacterial cells were tightly attached to sediment particles, such that common separation methods based on sediment washing with water were less effective for their isolation. Another observation was that a higher yield of actinobacterial colonies, including Salinispora isolates, per gram of processed sediment was obtained from deeper sediments. This result could be due to the size of sediment particles; shallower sediments consisted of larger sand particles, which could be more difficult to homogenise by grinding, as compared to the fine particles in deeper sediments. Another explanation could be due to the overgrowth of fast-growing bacteria, which are more abundant in shallow sediments close to the shore, as previously reported by Becerril-Espinosa et al. (2013). Among the 29 Salinispora isolates, 24 of them were isolated from AMM, Bennet’s and Mueller–Hinton culture media, which indicated the need for enriched cultivation media to isolate Salinispora species from this region, in contrast to those reported by Jensen et al. (2005), which were mostly isolated using lownutrient media. In this study, viable Salinispora strains were isolated from offshore marine sediments of Japan at
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215
Fig. 1 Neighbour-joining phylogenetic tree generated from concatenated 16S rRNA, ITS and gyrB sequences (2,620 bp total). Bootstrap values above 60 %, calculated from 1,000 resamplings, are shown for neighbour-joining (NJ) and
maximum likelihood (ML) methods on the respective nodes. The two-letter code after the strain numbers denotes the location of strains reported previously by other groups. BA the Bahamas, FJ Fiji, PA Palau, RS the Red Sea, SC Sea of Cortez
Table 4 Screening of selected S. tropica sal genes in S. arenicola JP strains ST salA (Strop1024)a
ST salI (Strop1015)
ST salJ (Strop1013)
ST salL (Strop1026)
ST salRIII (Strop1032)
ST salZ (Strop1043)
S. tropica NBRC105044
?
?
?
?
?
?
S. arenicola NBRC105043
-
-
-
-
-
-
JP00
?
?
-
?
?
?
JP01 JP02
? ?
? ?
-
? ?
? ?
? ?
JP03
?
?
-
?
?
?
JP04
-
-
-
-
-
-
JP05
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CNP105 (ARGZ01000046)
b
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CNP193 (ARGY01000047)
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CNY280 (ARHH01000035 and ARHH01000052)
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a
Gene identifier of S. tropica CNB440 showed in parentheses was selected for screening of sal gene cluster
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Data were obtained from whole-genome shotgun GenBank database using BLAST
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Fig. 2 Neighbour-joining phylogenetic tree generated from salA KS domain sequences (623 bp). Bootstrap values above 50 %, calculated from 1,000 resamplings, are shown for neighbour-joining (NJ) and maximum likelihood (ML) methods
on the respective nodes. The two-letter code after the strain numbers denotes the location of strains reported previously by other groups. BA the Bahamas, FJ Fiji, GU Guam, PA Palau, SC Sea of Cortez
the highest latitude (33°N) recorded so far for members of this genus, compared to the last reported highest latitude of 28°N at Los Angeles Bay, Gulf of California (Fig. S4) (Becerril-Espinosa et al. 2013). Contrary to the unsuccessful attempt to isolate Salinispora species from offshore temperate sediments of San Diego (32°N) (Prieto-Davo et al. 2013), it is surprising that Salinispora was successfully isolated from Tosa Bay, even though it is one degree higher in latitude than San Diego. One possible reason for the successful isolation of Salinispora in Tosa Bay could be due to the flow of the Kuroshio Current, which is considered a warm current with an annual mean sea surface temperature of 22 °C. This is as opposed to the
California Current, which is colder with an annual mean sea surface temperature of 17 °C (Fig. S4) (Yashayaev and Zveryaev 2001). Since Salinispora strains have been reported to be cultivable at 10 °C (Maldonado et al. 2005), sediment temperature is likely not the sole factor that affects the survival of Salinispora. It could be an interplay of various biological factors, such as microbial community composition, or physical factors, such as nutrient, pressure, salinity, etc., which determine the presence and survival of any organism. Hence, other environmental and culturing studies have to be carried out in order to elucidate and determine the impact of each of these factors.
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Based on 16S rRNA gene sequence analysis, the Tosa Bay isolates were classified as S. arenicola of the type strain phylotype. This classification was further supported by phenotypic characteristics, such as rifampicin resistance and the positive detection of a rifamycin biosynthetic gene. We did not detect any S. tropica colony, which was expected since this species appears to be endemic to the Bahamas region (Maldonado et al. 2005; Jensen and Mafnas 2006). However, it was surprising that no S. pacifica strains were isolated, given that many have been found in different regions of the Pacific Ocean (Freel et al. 2012). This is probably due to a number of reasons, such as the overgrowth of other microorganisms in culture media, a low abundance of S. pacifica strains in this region or the small sample size. One of the challenges in identifying Salinispora species is the low diversity of its 16S rRNA gene sequence. This is particularly true as horizontal gene transfer is very common in this genus (Penn et al. 2009) and thus identification based on the conserved 16S rRNA gene sequence does not reflect the true diversity of the various strains isolated. The diversity of 16S rRNA gene sequences of S. arenicola strains isolated hitherto is relatively conserved as compared to that of S. pacifica. The majority of S. arenicola strains isolated, including the Tosa Bay strains, belong to the type strain phylotype, while the remaining are divided into four phylotypes A–D (Freel et al. 2012). However, we have observed diversity within the Tosa Bay strains using BOX-PCR. Using 16S-23S rRNA ITS sequencing and analysis of the housekeeping gene gyrB, five new ITS variants were identified in this study and the concatenated 16S rRNA-ITS-gyrB sequence analysis clearly demonstrated the Tosa Bay isolates to be phylogenetically distinct from other known S. arenicola strains. Secondary metabolism is an important aspect in the study of Salinispora. In fact, it has been proposed as a speciation marker because despite having highly similar 16S rRNA gene sequences, the genomes of the three Salinispora species are very diverse due to the presence of different secondary metabolite biosynthetic gene clusters. Since rifamycin production is a core chemotype of S. arenicola, the ketosynthase sequence associated with rifamycin biosynthesis was compared and sequence variations were observed between each representative strain, as well as the type strain. This further supports the suggestion that the
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Tosa Bay Salinispora isolates are phylogenetically distinct from each other and the type strain. Interestingly, a location-specific KS1 sequence reported by Edlund et al. (2011) was detected in four strains of the Tosa Bay isolates. This KS1 sequence was only detected previously in strains isolated from the Sea of Cortez, the Red Sea and the Bahamas, but not in Guam and Palau, both of which are geographically closer to Tosa Bay (Edlund et al. 2011). Therefore, this result has important implications with respect to the biogeographical distribution and secondary metabolite evolution of S. arenicola. Although the other three ketosynthase sequences were not detected in the Tosa Bay isolates, the possibility of sequence variations resulting in unsuccessful gene amplification could not be ruled out. An earlier study by Jensen and co-workers (2007) suggested that salinosporamide is uniquely produced by S. tropica. However, recent studies on S. pacifica revealed the production of a salinosporamide analogue in some strains (Eustaquio et al. 2011; Freel et al. 2011). This is hardly surprising since phylogenetic studies have indicated a closer relationship between S. tropica and S. pacifica, rather than with S. arenicola. In this study, the salinosporamide gene cluster was detected for the first time in S. arenicola. The selected genes used in this study span across the whole sal cluster in S. tropica. Thus, these results suggest that an almost complete S. tropica sal cluster is present in S. arenicola strains, including the salL gene, which is absent in S. pacifica. Although salJ was not detected in JP strains, it is possible that the salJ gene was displaced by a transposition event in the Tosa Bay strains. This is suggested by the presence of an annotated transposase sequence upstream of salJ in the sal cluster of S. tropica (Eustaquio et al. 2009). It is also possible that the detection primers were not specific enough to amplify the salJ sequences. Preliminary RT-PCR analysis detected the presence of salL transcript in S. arenicola strain JP00, indicating the expression of this cluster during broth cultivation in Bennet’s medium (data not shown). Therefore, it is very likely that the sal gene cluster is functional in Tosa Bay strains. Complete sequence analysis, coupled with further studies on the production and structural analysis of salinosporamide in these S. arenicola strains will provide more insights into the structural relationship of salinosporamide produced by S. tropica.
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Importantly, the discovery of sal genes in S. arenicola refutes the earlier hypothesis that salinosporamide is produced only by the closely related S. tropica and S. pacifica (Freel et al. 2011). Perhaps, salinosporamide biosynthesis is a common characteristic of all Salinispora species and is not unique to S. tropica and S. pacifica, as previously thought. Yet, the phylogenetic analysis of salA from all three Salinispora species in this study suggested a separate evolutionary trajectory between S. tropica and S. pacifica, much earlier than with S. arenicola, which contradicts the description of S. tropica and S. pacifica as sister taxa (Freel et al. 2012). Since S. arenicola and S. tropica have been reported to co-occur in the Bahamas (Freel et al. 2012; Maldonado et al. 2005), it seems to be more likely that a horizontal gene transfer had occurred between both species. This hypothesis is supported by a sequence comparison study of staD genes of staurosporine biosynthesis from S. arenicola and S. pacifica strains, which also suggested interspecies recombination (Freel et al. 2011). Furthermore, sequence searches in the whole-genome shotgun contigs database using BLAST revealed the presence of sal genes in three other S. arenicola strains (CNP105, CNP193 and CNY280), indicating a higher occurrence of sal gene clusters in S. arenicola, and not just in the Tosa Bay strains. Although an earlier secondary metabolite profiling study of Salinispora species did not reveal that strains CNP105 and CNP193 produce salinosporamide (Jensen et al. 2007), this observation could have been due to silencing of the sal genes under the specified culturing conditions. Therefore, with more genome sequences of S. arenicola made available, perhaps more strains may be revealed to possess the sal gene cluster, which in turn allows a more thorough investigation of their evolution. In summary, this study has provided novel insights into the evolution and biodiversity of the unique marine genus Salinispora. The distribution of secondary metabolic genes in this genus may be more diverse or random than previously thought. Hence, continuing efforts in isolating them from new regions may unravel new clues to further strengthen the current understanding of their biogeographical distribution and secondary metabolic potential. Acknowledgments The authors thank C. Ihara for technical assistance, T. Nishisaka for assistance during sediment collection,
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Antonie van Leeuwenhoek (2014) 105:207–219 Z. Imoto and T. Hashimura of Usa Marine Biological Institute, Kochi University, for helping with sediment collection using research vessel Ms. Neptune, and Kochi Prefectural Deep Seawater Laboratory for providing deep seawater. This work was performed through Program to Disseminate Tenure Tracking System of the Ministry of Education, Culture, Sports, Science and Technology, The Japanese Government.
References Ahmed L, Jensen PR, Freel KC, Brown R, Jones AL, Kim BY, Goodfellow M (2013) Salinispora pacifica sp. nov., an actinomycete from marine sediments. Antonie Van Leeuwenhoek 103:1069–1078 Becerril-Espinosa A, Freel KC, Jensen PR, Soria-Mercado IE (2013) Marine actinobacteria from the Gulf of California: diversity, abundance and secondary metabolite biosynthetic potential. Antonie Van Leeuwenhoek 103:809–819 Blunt JW, Copp BR, Keyzers RA, Munro MH, Prinsep MR (2013) Marine natural products. Nat Prod Rep 30:237–323 Bull AT, Stach JE (2007) Marine actinobacteria: new opportunities for natural product search and discovery. Trends Microbiol 15:491–499 Edlund A, Loesgen S, Fenical W, Jensen PR (2011) Geographic distribution of secondary metabolite genes in the marine actinomycete Salinispora arenicola. Appl Environ Microbiol 77:5916–5925 Eustaquio AS, McGlinchey RP, Liu Y, Hazzard C, Beer LL, Florova G, Alhamadsheh MM, Lechner A, Kale AJ, Kobayashi Y, Reynolds KA, Moore BS (2009) Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S-adenosyl-Lmethionine. Proc Natl Acad Sci USA 106:12295–12300 Eustaquio AS, Nam SJ, Penn K, Lechner A, Wilson MC, Fenical W, Jensen PR, Moore BS (2011) The discovery of salinosporamide K from the marine bacterium ‘‘Salinispora pacifica’’ by genome mining gives insight into pathway evolution. ChemBioChem 12:61–64 Freel KC, Nam SJ, Fenical W, Jensen PR (2011) Evolution of secondary metabolite genes in three closely related marine actinomycete species. Appl Environ Microbiol 77:7261–7270 Freel KC, Edlund A, Jensen PR (2012) Microdiversity and evidence for high dispersal rates in the marine actinomycete ‘Salinispora pacifica’. Environ Microbiol 14:480–493 Jensen PR, Mafnas C (2006) Biogeography of the marine actinomycete Salinispora. Environ Microbiol 8:1881–1888 Jensen PR, Dwight R, Fenical W (1991) Distribution of actinomycetes in near-shore tropical marine sediments. Appl Environ Microbiol 57:1102–1108 Jensen PR, Gontang E, Mafnas C, Mincer TJ, Fenical W (2005) Culturable marine actinomycete diversity from tropical Pacific Ocean sediments. Environ Microbiol 7:1039–1048 Jensen PR, Williams PG, Oh DC, Zeigler L, Fenical W (2007) Species-specific secondary metabolite production in marine actinomycetes of the genus Salinispora. Appl Environ Microbiol 73:1146–1152 Kersten RD, Lane AL, Nett M, Richter TK, Duggan BM, Dorrestein PC, Moore BS (2013) Bioactivity-guided genome
Antonie van Leeuwenhoek (2014) 105:207–219 mining reveals the lomaiviticin biosynthetic gene cluster in Salinispora tropica. ChemBioChem 14:955–962 Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics. John Innes Foundation, Norwich Lane DJ (1991) 16S/23S rRNA sequencing. Nucleic acid techniques in bacterial systematics. Wiley, New York Lanoot B, Vancanneyt M, Dawyndt P, Cnockaert M, Zhang J, Huang Y, Liu Z, Swings J (2004) BOX-pCR fingerprinting as a powerful tool to reveal synonymous names in the genus Streptomyces. Emended descriptions are proposed for the species Streptomyces cinereorectus, S. fradiae, S. tricolor, S. colombiensis, S. filamentosus, S. vinaceus and S. phaeopurpureus. Syst Appl Microbiol 27:84–92 Maldonado LA, Fenical W, Jensen PR, Kauffman CA, Mincer TJ, Ward AC, Bull AT, Goodfellow M (2005) Salinispora arenicola gen. nov., sp. nov. and Salinispora tropica sp. nov., obligate marine actinomycetes belonging to the family Micromonosporaceae. Int J Syst Evol Microbiol 55:1759–1766 Maldonado LA, Fragoso-Yanez D, Perez-Garcia A, RosellonDruker J, Quintana ET (2009) Actinobacterial diversity from marine sediments collected in Mexico. Antonie Van Leeuwenhoek 95:111–120 Mincer TJ, Jensen PR, Kauffman CA, Fenical W (2002) Widespread and persistent populations of a major new marine actinomycete taxon in ocean sediments. Appl Environ Microbiol 68:5005–5011 Mincer TJ, Fenical W, Jensen PR (2005) Culture-dependent and culture-independent diversity within the obligate marine actinomycete genus Salinispora. Appl Environ Microbiol 71:7019–7028 Okazaki T (2006) Intrigued by actinomycete diversity. Actinomycetologica 20:15–22 Peeters K, Willems A (2011) The gyrB gene is a useful phylogenetic marker for exploring the diversity of Flavobacterium strains isolated from terrestrial and aquatic habitats in Antarctica. FEMS Microbiol Lett 321:130–140 Penn K, Jenkins C, Nett M, Udwary DW, Gontang EA, McGlinchey RP, Foster B, Lapidus A, Podell S, Allen EE, Moore BS, Jensen PR (2009) Genomic islands link
219 secondary metabolism to functional adaptation in marine Actinobacteria. ISME J 3:1193–1203 Prieto-Davo A, Villarreal-Gomez LJ, Forschner-Dancause S, Bull AT, Stach JE, Smith DC, Rowley DC, Jensen PR (2013) Targeted search for actinomycetes from nearshore and deep-sea marine sediments. FEMS Microbiol Ecol 84:510–518 Smith W, McIntyre AD (1954) A spring-loaded bottom-sampler. J Mar Biol Assoc UK 33:257–264 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Udwary DW, Zeigler L, Asolkar RN, Singan V, Lapidus A, Fenical W, Jensen PR, Moore BS (2007) Genome sequencing reveals complex secondary metabolome in the marine actinomycete Salinispora tropica. Proc Natl Acad Sci USA 104:10376–10381 Valverde A, Tuffin M, Cowan DA (2012) Biogeography of bacterial communities in hot springs: a focus on the actinobacteria. Extremophiles 16:669–679 Vidgen ME, Hooper JN, Fuerst JA (2012) Diversity and distribution of the bioactive actinobacterial genus Salinispora from sponges along the Great Barrier Reef. Antonie Van Leeuwenhoek 101:603–618 Ward AC, Bora N (2006) Diversity and biogeography of marine actinobacteria. Curr Opin Microbiol 9:279–286 Wilson MC, Gulder TA, Mahmud T, Moore BS (2010) Shared biosynthesis of the saliniketals and rifamycins in Salinispora arenicola is controlled by the sare1259-encoded cytochrome P450. J Am Chem Soc 132:12757–12765 Yashayaev IM, Zveryaev II (2001) Climate of the seasonal cycle in the North Pacific and the North Atlantic oceans. Int J Climatol 21:401–417 Zhi XY, Yang LL, Wu JY, Tang SK, Li WJ (2007) Multiplex specific PCR for identification of the genera Actinopolyspora and Streptomonospora, two groups of strictly halophilic filamentous actinomycetes. Extremophiles 11:543–548 Zotchev SB (2012) Marine actinomycetes as an emerging resource for the drug development pipelines. J Biotechnol 158:168–175
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ORIGINAL PAPER
Salinispora arenicola from temperate marine sediments: new intra-species variations and atypical distribution of secondary metabolic genes Kian-Sim Goo • Masashi Tsuda • Dana Ulanova
Received: 26 September 2013 / Accepted: 24 October 2013 / Published online: 1 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract The obligate marine actinobacterium Salinispora arenicola was successfully cultured from temperate sediments of the Pacific Ocean (Tosa Bay, offshore Kochi Prefecture, Japan) with the highest latitude of 33°N ever reported for this genus. Based on 16S rRNA gene sequence analysis, the Tosa Bay strains are of the same phylotype as the type strain S. arenicola NBRC105043. However, sequence analysis of their 16S-23S rRNA intergenic spacer (ITS) revealed novel sequence variations. In total, five new ITS sequences were discovered and further phylogenetic analyses using gyrase B and rifamycin ketosynthase (KS) domain sequences supported the phylogenetic diversity of the novel Salinispora
isolates. Screening of secondary metabolite genes in these strains revealed the presence of KS1 domain sequences previously reported in S. arenicola strains isolated from the Sea of Cortez, the Bahamas and the Red Sea. Moreover, salinosporamide biosynthetic genes, which are highly homologous to those of Bahamas-endemic S. tropica, were detected in several Tosa Bay isolates, making this report the first detection of salinosporamide genes in S. arenicola. The results of this study provide evidence of a much wider geographical distribution and secondary metabolism diversity in this genus than previously projected. Keywords Marine actinomycetes Salinispora arenicola Secondary metabolism Salinosporamide
Electronic supplementary material The online version of this article (doi:10.1007/s10482-013-0067-2) contains supplementary material, which is available to authorized users.
Introduction K.-S. Goo Evaluation and Support Organization for Young Researchers, Kochi University, Kohasu, Oko-cho, Nankoku, Kochi 783-8505, Japan M. Tsuda Center for Advanced Marine Core Research, Kochi University, Monobe-otsu 200, Nankoku, Kochi 783-8502, Japan D. Ulanova (&) Oceanography Section, Science Research Center, Kochi University, IMT-MEXT, Kohasu, Oko-cho, Nankoku, Kochi 783-8505, Japan e-mail: [email protected]
Actinobacteria have been isolated from a large variety of samples collected worldwide (Okazaki 2006; Ward and Bora 2006; Valverde et al. 2012), with some genera showing preferential occurrence in specific environments (Mincer et al. 2002; Zhi et al. 2007). However, their global distribution patterns, as well as the environmental factors in different locations that affect biodiversity, are poorly understood. For example, some terrestrial species were found in marine environments, but it is believed that they were washed out from the shoreline. Further studies on these marine
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species revealed higher tolerance of salinity and some differences in their metabolite production, suggesting possible adaptation of these species to the marine environment (Okazaki 2006). Nevertheless, such ambiguities over terrestrial/marine origins often complicate environmental studies. In recent years, marine actinobacteria have been extensively isolated and studied for the presence of bioactive natural compounds that are useful for clinical purposes (Ward and Bora 2006; Bull and Stach 2007; Blunt et al. 2013). Compared to their terrestrial counterparts, marine actinobacteria are relatively underexplored, and thus there exists the potential of discovering novel metabolites with as yet unknown activities (Zotchev 2012). The genus Salinispora contains the first reported obligate marine actinomycetes, which have a strict requirement for seawater in order to survive and proliferate (Jensen et al. 1991; Mincer et al. 2002). To date, only three Salinispora species have been described, namely Salinispora arenicola, Salinispora tropica and Salinispora pacifica (Maldonado et al. 2005; Ahmed et al. 2013). Although these three species share more than 99 % identity in their 16S rRNA gene sequences, they are distinguished into different species due to them having less than 60 % of DNA–DNA relatedness (Maldonado et al. 2005; Ahmed et al. 2013). Nonetheless, they can be differentiated by their 16S rRNA gene sequences (Freel et al. 2012). So far, five phylotypes based on signature nucleotide positions of 16S rRNA gene sequences have been described for S. arenicola, 15 for S. pacifica and only one for S. tropica (Freel et al. 2012; Vidgen et al. 2012). Salinispora species are widely found in tropical and subtropical marine sediments, showing species-specific biogeographical distributions (Jensen and Mafnas 2006). At present, S. arenicola has been reported to have a more diverse distribution, where strains of this species have been isolated from the Bahamas, several locations in the tropical and subtropical Pacific Ocean, and the Red Sea (Jensen and Mafnas 2006; Freel et al. 2012). On the other hand, S. pacifica was only reported in seven out of the 12 locations studied, mainly in the Pacific regions and the Red Sea (Freel et al. 2012). As for S. tropica, it appears to be endemic to the Bahamas (Mincer et al. 2002). Interestingly, no Salinispora species has been cultivated from temperate regions, leading to the hypothesis that low temperature affects
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Salinispora viability (Jensen and Mafnas 2006). However, Salinispora strains have been successfully isolated from deep-sea sediment samples obtained from the Bahamas at a depth of 1,100 m with temperatures near 4 °C (Mincer et al. 2005). Therefore, it is very likely that there are other factors affecting Salinispora viability and distribution in temperate regions. This hypothesis is supported by culture-independent methods, where the DNA of Salinispora species has been detected in temperate marine environments (San Diego, 32°N) and deep-sea sediments of 5,699 m, although attempts to cultivate them from these samples remain unsuccessful (PrietoDavo et al. 2013). The most significant aspect of studying this actinobacterial genus is the diverse range of secondary metabolites it produces. Extensive screening studies of Salinispora species have revealed species-specific production of secondary metabolites and geographical production patterns. The profiling of over 30 S. arenicola strains from six geographically distinct locations has revealed that all of them produce rifamycin, staurosporine and saliniketal. Similar profiling studies of several Bahamas-derived S. tropica strains have revealed that they produce salinosporamide and sporolide. In addition to the core chemotype, some S. arenicola strains were found to specifically produce compounds unique to certain locations (Jensen et al. 2007). These results, combined with the analysis of the Salinispora genome, led to the conclusion that secondary metabolism is a major speciation factor for this genus and that secondary metabolite profiles could be location-specific (Udwary et al. 2007; Penn et al. 2009). However, it remains unclear how these location-specific metabolites influence Salinispora species diversity, geographical distribution and their adaption to the local environment. Nevertheless, there lies the possibility of strains isolated from new locations that produce yet unknown secondary metabolites with new bioactivities. In this study, we report the successful isolation of Salinispora strains from offshore sediments of Tosa Bay (Kochi Prefecture, Japan) at the highest latitude of 33°N recorded so far. These strains were identified as S. arenicola based on physiological properties and 16S rRNA gene sequence analysis. However, detailed sequence analysis of the 16S-23S rRNA intergenic region, housekeeping and secondary metabolic genes revealed variations between the isolates, despite their
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conserved 16S rRNA sequence. Interestingly, phylogenetic analysis with deposited genome sequences of S. arenicola showed the relatedness of some Tosa Bay strains with isolates from other reported locations, especially regarding their secondary metabolism.
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filamentous hyphae were detected by eye and/or using a microscope (SZX7, Olympus). Thereafter, colonies were transferred to fresh culture plates of the same medium until pure cultures were obtained. Manipulation of type strains
Materials and methods Sampling of marine sediment and selective actinobacterial isolation Sediment samples were collected in January 2012 from Tosa Bay, off Kochi Prefecture, Japan, at depths of 10, 30, 50, 80 and 100 m below seawater surface (33°27.110 N, 133°28.500 E; 33°26.060 N, 133°29.240 E; 33°24.260 N, 133°30.090 E; 33°21.350 N, 133°31.230 E; 33°18.530 N, 133°32.320 E, respectively), using a Smith-McIntyre sediment sampler (Smith and McIntyre 1954). Sediment samples were stored at 10–20 °C and processed within one week using methods described by Jensen et al. (2005). Briefly, three pretreatment methods were used: 1. Dry method: About nine grams of each sample were dried in a biosafety cabinet for four to five hours. After drying, sediment samples were ground and inoculated by a plate stamping technique (Mincer et al. 2002); 2. Dry and heat method: Dried sediment samples were added to sterile deep seawater (DSW, Kochi Prefectural Deep Seawater Laboratory) in 1:4 dilution and heated for 6 min at 55 °C. After heating, samples were mixed by vortexing and 50 lL of each sample were inoculated; 3. Wet method: One gram of wet sediment was added to 9 mL of sterile DSW and one gram of glass beads, following by shaking at room temperature at 180 rpm for 1 h. Ten and 100 times dilution of samples were inoculated onto plates using sterile spreaders. Seven high-nutrient cultivation media (AMM, Bennet’s, ISP1, starch-casein agar, non-sporulation agar, Mueller–Hinton agar, Middlebrook agar), as well as six low-nutrient media (SMC, SMP, SNC, SPC, STC, SMY) were used (Jensen et al. 2005; Maldonado et al. 2009). All media were prepared using DSW. Cycloheximide (100 mg L-1) or nystatin (50 mg L-1) was added to suppress fungal growth and some media were also supplemented with rifampicin (5 mg L-1) to reduce the growth of fast growing bacteria. All culture plates were incubated at 28 °C for up to 4 months. Actinobacterial-like colonies forming
Type strains of S. arenicola (CNH643/ATCC BAA917) and S. tropica (CNB440/ATCC BAA916) were obtained from NBRC (Biological Resource Center, National Institute of Technology and Evaluation, Japan) and are referred as NBRC105043 and NBRC105044 in this study, respectively. They were cultivated according to supplier’s recommendations and used as a positive control to validate gene amplification conditions. Low-temperature sensitivity, seawater requirement and rifampicin resistance For testing of seawater requirement and rifampicin resistance, isolates were cultivated on AMM media prepared using DSW or distilled water with or without addition of rifampicin (20 mg L-1) for 2–3 weeks at 28 °C. For testing of viability after prolonged lowtemperature incubation, selected strains were first cultivated at 28 °C for 3 weeks on two sets of AMM agar plates. Thereafter, one set of plates was transferred to 4 °C. Every week, isolates from the 28 and 4 °C plate sets were transferred to fresh AMM agar and cultivated at 28 °C. Viability of strains was visually estimated after 2–3 weeks of cultivation. BOX-PCR Genomic DNA of marine isolates was extracted by lysozyme treatment, followed by phenol:chloroform extraction and ethanol precipitation (Kieser et al. 2000). The final BOX-PCR reaction mixture contained 19 Ex TaqÒ buffer (TaKaRa), 0.5 mM dNTPs, 10 % (v/v) DMSO, 0.5 lM BOX-A1R primer (Lanoot et al. 2004), 0.2 mg mL-1 BSA, 2.5 U Ex TaqÒ DNA polymerase (TaKaRa) and 100 lg mL-1 genomic DNA. The cycling condition used was initial denaturation at 95 °C for 5 min, followed by 35 cycles of 92 °C for 30 s, 53 °C for 30 s and 68 °C for 6 min, and final extension at 68 °C for 16 min. The PCR products were subjected to agarose gel electrophoresis using 2 % (w/v) agarose gels prepared in 19 TAE (Tris–Acetate–
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EDTA) buffer and electrophoresed at 50 volts for 60 min. Subsequently, the agarose gels were stained using SYBRÒ Safe DNA stain (Invitrogen). Amplification of 16S rRNA and 16S-23S rRNA intergenic spacer sequences 16S rRNA gene sequencing was used to determine the identity of the marine isolates that morphologically resembled members of the family Micromonosporaceae. The amplification of nearly complete 16S rRNA gene sequences was performed using the primers 28F and 1492R (Lane 1991; Jensen and Mafnas 2006). The amplification reaction mixture contained 19 Ex TaqÒ buffer (TaKaRa), 0.5 mM dNTPs, 5 % (v/v) DMSO, 3 lM of each primer, 2.5 U Ex TaqÒ DNA polymerase (TaKaRa) and *100 lg mL-1 genomic DNA. The cycling condition used was initial denaturation at 94 °C for 1 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 90 s, and final extension at 72 °C for 5 min. Amplification of PCR products was detected using agarose gel electrophoresis and the amplimers subjected to DNA sequencing using the ABI PRISMTM dye terminator cycle sequencing kit and ABI PRISMTM 3100 Genetic Analyser, according to manufacturer’s recommendations. The same primers for gene amplification were used for sequencing, including additional primers 530R and 936R (Jensen and Mafnas 2006). For the amplification of the 16S-23S rRNA intergenic spacer (ITS), the reaction mixture contained 19 Ex TaqÒ buffer (TaKaRa), 0.5 mM dNTPs, 5 % (v/v) DMSO, 3 lM of each primer, ITS_F and ITS_R (Table S1), 2.5 U Ex TaqÒ DNA polymerase (TaKaRa) and *100 lg mL-1 genomic DNA. The cycling condition used was initial denaturation at 95 °C for 1 min, followed by 30 cycles of 95 °C for 30 s, 65 °C for 30 s and 72 °C for 30 s. Amplification of PCR products was detected using agarose gel electrophoresis and subjected to DNA sequencing as described above, using the same amplification primers. Amplification of gyrB sequence Gene amplification of gyrase B subunit (gyrB) was based on the primers gyr33F, gyr611F, gyr662R and gyr1300R reported by Jensen and Mafnas (2006), which included M13 primer sequences on 50 end. The reaction mixture contained 19 Ex TaqÒ buffer
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(TaKaRa), 0.5 mM dNTPs, 5 % (v/v) DMSO, 0.6 lM of each primer, 2.5 U Ex TaqÒ DNA polymerase (TaKaRa) and 100 lg mL-1 genomic DNA. The cycling condition used was initial denaturation at 95 °C for 2 min, followed by 30 cycles of 95 °C for 30 s, 65 °C for 30 s and 72 °C for 30 s. Amplification of gene products was detected using agarose gel electrophoresis and subjected to DNA sequencing as described above, using M13 primers. Detection of ketosynthase sequences Gene amplification of ketosynthase (KS) sequences, i.e., KS1-4 and the rifamycin polyketide synthase KS domain, was based on the specific primers reported by Edlund et al. (2011). The reaction mixture contained 19 Ex TaqÒ buffer (TaKaRa), 0.5 mM dNTPs, 5 % (v/v) DMSO, 0.6 lM of each primer, 2.5 U Ex TaqÒ DNA polymerase (TaKaRa) and 100 lg mL-1 genomic DNA. The gene amplification cycling condition reported by Edlund et al. (2011) was also adopted. Amplification of gene products was detected using agarose gel electrophoresis and subjected to DNA sequencing as described above. Detection of S. tropica-specific secondary metabolite gene clusters Representative genes from S. tropica-specific secondary metabolite gene clusters were amplified using the primers listed in Table S1. The reaction mixture contained 19 Ex TaqÒ buffer (TaKaRa), 0.5 mM dNTPs, 5 % (v/v) DMSO, 0.6 lM of each primer, 2.5 U Ex TaqÒ DNA polymerase (TaKaRa) and 100 lg mL-1 genomic DNA. The gene amplification cycling condition was initial denaturation at 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 64–72 °C for 30 s, depending on the primer pairs (Table S1), and 72 °C for 30 s. Amplification of gene products was detected using agarose gel electrophoresis and subjected to DNA sequencing as described above. Sequence manipulation and phylogenetic analysis DNA sequences were manipulated using Geneious R6 sequence analysis software (Biomatters Ltd). DNA sequences were aligned and sequence similarities were
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calculated using the Geneious alignment method. Phylogenetic trees were constructed using MEGA5 software (Tamura et al. 2011). All sequences were verified by at least two independent DNA amplification and sequencing experiments. New sequence data have been deposited in the GenBank database and their accession numbers are provided in the phylogenetic trees and Supplementary Table S2. Sequence comparison was also performed using the BLAST program (Basic Local Alignment Search Tool from National Center for Biotechnology Information) in both nucleotide collection and whole-genome shotgun contigs databases to search for homologous sequences, up to 11th June 2013.
Results Isolation and identification of Salinispora species In this study, three methods were used to pretreat the Tosa Bay sediments collected at five different depths. A total of 13 culture media, comprising seven highnutrient and six low-nutrient media, were used to isolate actinobacteria from these sediments. 101 actinobacterial-like colonies were isolated and the majority of these colonies (80 %) were obtained using the dry-stamping method. The most effective media for isolation were high-nutrient Bennet’s, AMM and ISP1 culture media. Interestingly, a higher yield of actinobacterial-like colonies per gram of processed sediment was obtained from deeper sediments (30–100 m) (Table 1). Among the 101 actinobacterial-like colonies isolated, 38 colonies possessed the typical characteristics of the family Micromonosporaceae with orange Table 1 Yield of Salinispora isolates from marine sediments of Tosa Bay
Depth (m)
pigmentation and no aerial mycelium formation. Partial 16S rRNA analysis revealed that six of them belong to the genus Micromonospora, three to the genus Nocardia and the majority (29 colonies) to the genus Salinispora. Subsequently, the 16S rRNA gene sequences of the Salinispora isolates were fully sequenced to reveal their species identities. All the sequences obtained were identical to the 16S rRNA gene sequence of the S. arenicola type strain NBRC105043 (Jensen and Mafnas 2006). Neither other phylotypes of S. arenicola, nor those of strains of S. pacifica and S. tropica were detected. Subsequently, the Salinispora isolates were tested for seawater requirement, low-temperature sensitivity and rifampicin resistance, in order to further validate their identities. As five of the 29 isolates lost their viability after several rounds of sub-culturing, only 24 isolates were used for further analysis. All 24 isolates failed to grow on culture media without seawater, indicating an obligatory requirement of seawater for growth, which is a typical characteristic of the genus Salinispora. Furthermore, selected isolates showed reduced viability after prolonged storage at 4 °C (up to 9–23 weeks), similar to previously reported Salinispora strains (Jensen and Mafnas 2006). All strains grew well on culture media supplemented with a high concentration of rifampicin, thus confirming the identity of these isolates to be S. arenicola since only this species, out of the three known Salinispora species, is rifampicin-resistant. Phylogenetic analyses of Salinispora isolates In order to reduce clonal replicates of Salinispora isolates, BOX-PCR using BOX-A1R primer, which has proved to be useful method for the whole-genome
No. of Salinispora colonies
Mean no. of Salinispora colonies per gram of sediment
No. of actinobacterial colonies
Mean no. of actinobacterial colonies per gram of sediment
10
0
0
11
0.55
30
7
0.35
18
0.9
50
7
0.35
31
1.55
80
9
0.45
27
1.35
100
6
0.6
14
1.4
Total number of isolates
29
–
101
–
123
212
level analysis and reduction of Salinispora clonal isolates (Vidgen et al. 2012), was performed to compare the Salinispora isolates on a whole-genomic level. From the 24 isolates analysed, ten different BOX profiles were obtained (Fig. S1). For a more comprehensive analysis of the interspecies diversity observed in the BOX-PCR results, the 16S-23S rRNA ITS sequence of the isolates were amplified, sequenced and compared with those deposited in GenBank database. To date, only five ITS sequences of S. arenicola have been reported (Freel et al. 2012) and they are highly homologous to each other, differing only at two positions—G9 and T218 (nucleotide numbering based on the S. arenicola CNS205 ITS sequence) are deleted in the type strain CNH643, and T218 is deleted in the strain CNH964. The rest are identical to that of strain CNH964. Strain NBRC105043, which is clonal to the type strain CNH643, was used as a control in our analysis. Contrary to the sequence deposited in GenBank (AY371897), the ITS sequence of strain NBRC105043 does not have the G9 deletion, suggesting there are only two S. arenicola ITS phylotypes instead of the initial three sequence variations. This sequence was verified by triplicate sequencing experiments from independent DNA isolations and PCR amplifications. More importantly, the S. arenicola isolates from Tosa Bay possessed several differences in the ITS sequence that have not been reported previously (Table 2). Only two isolates had the same ITS sequence as the type strain NBRC105043 and CNH964, where they are referred to as ITS group 0 in this study. The remaining 22 isolates were divided into five different ITS groups. Phylogenetic analysis of all Salinispora species ITS sequences showed a close relationship of these new sequences with those of other S. arenicola strains reported earlier (Fig. S2). Additionally, analysis of yet-to-be-annotated sequences deposited in the whole-genome shotgun contigs GenBank database revealed that some previously reported Salinispora strains possess ITS sequences identical to the Tosa Bay isolates. For example, strains CNS991 and CNH962 have the same ITS sequences as ITS groups 3 and 4, respectively. Nevertheless, the ITS sequences of ITS groups 1, 2 and 5 were found to be unique to the Tosa Bay Salinispora isolates. In order to further validate the ITS sequence diversity, another common phylogenetic marker, gyrase subunit B gene (gyrB), was used (Jensen and
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Mafnas 2006; Peeters and Willems 2011). Nearly completed gyrB sequences were amplified, sequenced and compared. The isolates of the same ITS group were found to possess the same gyrB sequences, which differed from other groups (Table 3). Although ITS group 0 has the same ITS sequence as the type strain NBRC105043, there was some nucleotide sequence variations observed in their gyrB sequences (Table 3). Similarly, sequence comparison of gyrB sequences of ITS groups 3 and 4 with those of strains CNS991 and CNH962 deposited in the whole-genome shotgun contigs GenBank database revealed distinct sequence variations, respectively (Fig. S3). However, all these nucleotide differences do not translate into amino acid differences from the type strain NBRC105043. Interestingly, gyrB sequences of ITS groups 0, 1 and 2 clustered, with high bootstrap values, with sequences of strains from the Sea of Cortez that belong to 16S rRNA phylotype B (Fig. S3) (Jensen and Mafnas 2006). To reflect the 16S rRNA and ITS diversity of the Tosa Bay isolates, as well as to provide an improved support for the separation of ITS groups 1 and 2, a phylogenetic tree of concatenated 16S rRNAITS-gyrB sequence was constructed (Fig. 1). This concatenated tree supports the notion that the Tosa Bay Salinispora isolates are phylogenetically different from other reported strains, despite small variations in the three sequences analysed. Ketosynthase gene analysis Several location-specific sequences coding for newtype ketosynthase domains (designated as KS1–KS4) have been reported in S. arenicola, where these domains are presumably involved in the biosynthesis of as yet unknown polyketide compounds (Edlund et al. 2011). Since secondary metabolism is an important speciation factor in the genus Salinispora, homologues of these sequences were also probed in this study to determine the relatedness of Tosa Bay isolates to reported strains. For this and further analyses, we used one representative strain from each ITS group, designated as JP00–JP05. Each representative strain was screened using the specific primers and gene amplification procedures reported by Edlund et al. (2011). Interestingly, S. arenicola strains JP00, JP01, JP02 and JP03 were found to possess KS1 sequences, which were mainly detected previously in S. arenicola strains isolated from the Sea of Cortez, the Bahamas and the Red Sea. These KS1
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213
Table 2 Sequence variations in 16S-23S rRNA ITS of S. arenicola isolates ITS group
Representative strain (GenBank accession number)
Number of isolates
218a
231
312–314
338–339
349
–
CNS205 (HQ642901)b
–
T
T
CTG
GG
T
–
CNH964 (AY371900)
–
D
–
CNH962 (ARKS01000023)
–
D
–
CNS991 (ARBB01000003)
–
D
–
NBRC105043
–
D
0
JP00
2
D
1
JP01
10
D
C
2
JP02
4
D
C
3
JP03
2
D
4
JP04
2
D
5
JP05
4
D
a
Nucleotide numbering based on S. arenicola strain CNS205 16S-23S rRNA ITS
b
Data were obtained from whole-genome shotgun GenBank database using BLAST
sequences (575 bp) of JP strains were identical to that of the type strain NBRC105043, which was isolated from the Bahamas. However, KS2, KS3 and KS4, reported from Guam, the Sea of Cortez and the Bahamas, were not detected in any of the JP strains using the specific primers and gene amplification conditions reported by Edlund et al. (2011) (Table S3). In this study, we also analysed a ketosynthase sequence associated with rifamycin biosynthesis (Wilson et al. 2010). Rifamycin has been reported to be produced by all S. arenicola isolated so far and so it is considered a core metabolite specific for this species. The sequences for comparison were based on the S. arenicola CNS205 Sare1247 gene (FJ844623). The Sare1247 partial sequence of Salinispora JP strains and the type strain NBRC105043 were amplified and compared with S. arenicola strain CNS205 (Table S4). Similarly to the ITS and gyrB sequences, all partial Sare1247 sequences of JP strains varied from that of strain CNS205 and the type strain NBRC105043. Although strain JP00 has the same ITS sequence as the type strain NBRC105043, its rifamycin ketosynthase domain sequence differed and thus, further supported the phylogenetic difference between ITS group 0 isolates and the type strain. Detection of S. tropica secondary metabolite biosynthetic gene clusters in S. arenicola isolates Currently, two genomic sequences of Salinispora species are available, i.e., S. tropica CNB440 and S.
D GGTGG
GGTGG GGTGG D C
arenicola CNS205 (Udwary et al. 2007; Penn et al. 2009). Out of the 30 secondary metabolite gene clusters in S. arenicola, 12 clusters were homologous with S. tropica (Penn et al. 2009). We were curious as to whether the new phylogenetically distinct JP strains could possess secondary metabolite gene clusters that have been previously reported as unique for S. tropica. Seven S. tropica gene clusters reported to be absent in S. arenicola CNS205, i.e., pks1, sal (salinosporamide gene cluster), lom (lomaiviticin gene cluster), spo (sporolide gene cluster), slm (salinilactam gene cluster), sid3 and sid4 (Penn et al. 2009; Kersten et al. 2013), were selected. The presence of representative genes from each cluster was analysed by PCR detection. No representative genes of the pks1, lom, spo, slm, sid3 and sid4 gene clusters were detected. Interestingly, salRIII sequence was detected in four of the six representative JP strains with identities of 94 and 90 % with the S. tropica CNB440 salRIII gene and protein sequences, respectively (Table 4). To further validate these findings, specific primers targeting salA, salI, salJ, salL and salZ, which spanned across the sal cluster in S. tropica CNB440, were designed and screened against these strains. Interestingly, nearly all these sequences were present in the four strains that tested positive for salRIII, except for salJ (Table 4). Next, salA sequences of JP strains were used to search for homologues in the GenBank whole-genome shotgun contigs database in order to screen yet-to-beannotated sequences of various S. arenicola strains. Interestingly, three S. arenicola strains (CNP105,
123
T A
A
C
C
T C
C G
C C T
T
JP02 JP03
JP04
A
T JP01
T JP05
A NBRC105043/CNH643
Only nucleotide variation at nucleotide position 916 leads to amino acid difference between strain CNS205 (Ala306) and other strains (Thr306). The nucleotide and amino acid numberings are based on S. arenicola strain CNS205 gyrB (Sare0007) sequence numbering
T C G
T
T
T A A A
A
A A
A C
C T JP00
G C G CNS205
123
A T
C
T
T
C
C C
G
C
G C T C G G
A C
T C C
C
T
C
1,377 1,338 1,332 1,296 1,224 1,221 1,197 966 924 916 813 696 636 561 555 528 426 396 Strain\nucleotide no.
Table 3 Sequence variations in gyrB gene of S. arenicola isolates
C
Antonie van Leeuwenhoek (2014) 105:207–219 1,446
214
CNP193 and CNY280) were found to harbour salA homologues. Phylogenetic analysis of salA sequences showed distinct branching of S. arenicola salA sequences from those of S. tropica and S. pacifica, with higher homology to S. tropica sequence (Fig. 2). Similar to the 16S rRNA-ITS-gyrB concatenated tree (Fig. 1), salA sequences from strains JP00, JP01 and JP02 clustered with sequences of strains CNP105 and CNP193 from the Sea of Cortez, whereas strains JP03 and CNY280 showed distinct branching that was well supported by bootstrap analysis.
Discussion The distribution patterns of Salinispora species have been studied extensively during the last decade, which makes this genus a model for studying marine bacterial biogeography and specialisation (Freel et al. 2012). Various methods and cultivation media were adopted in this study to isolate Salinispora species from Tosa Bay marine sediments. Similar to the observation reported by Jensen et al. (2005), the majority of the Salinispora isolates were obtained using the dry-stamping method. One possible explanation could be that the bacterial cells were tightly attached to sediment particles, such that common separation methods based on sediment washing with water were less effective for their isolation. Another observation was that a higher yield of actinobacterial colonies, including Salinispora isolates, per gram of processed sediment was obtained from deeper sediments. This result could be due to the size of sediment particles; shallower sediments consisted of larger sand particles, which could be more difficult to homogenise by grinding, as compared to the fine particles in deeper sediments. Another explanation could be due to the overgrowth of fast-growing bacteria, which are more abundant in shallow sediments close to the shore, as previously reported by Becerril-Espinosa et al. (2013). Among the 29 Salinispora isolates, 24 of them were isolated from AMM, Bennet’s and Mueller–Hinton culture media, which indicated the need for enriched cultivation media to isolate Salinispora species from this region, in contrast to those reported by Jensen et al. (2005), which were mostly isolated using lownutrient media. In this study, viable Salinispora strains were isolated from offshore marine sediments of Japan at
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215
Fig. 1 Neighbour-joining phylogenetic tree generated from concatenated 16S rRNA, ITS and gyrB sequences (2,620 bp total). Bootstrap values above 60 %, calculated from 1,000 resamplings, are shown for neighbour-joining (NJ) and
maximum likelihood (ML) methods on the respective nodes. The two-letter code after the strain numbers denotes the location of strains reported previously by other groups. BA the Bahamas, FJ Fiji, PA Palau, RS the Red Sea, SC Sea of Cortez
Table 4 Screening of selected S. tropica sal genes in S. arenicola JP strains ST salA (Strop1024)a
ST salI (Strop1015)
ST salJ (Strop1013)
ST salL (Strop1026)
ST salRIII (Strop1032)
ST salZ (Strop1043)
S. tropica NBRC105044
?
?
?
?
?
?
S. arenicola NBRC105043
-
-
-
-
-
-
JP00
?
?
-
?
?
?
JP01 JP02
? ?
? ?
-
? ?
? ?
? ?
JP03
?
?
-
?
?
?
JP04
-
-
-
-
-
-
JP05
-
?
-
-
-
-
CNP105 (ARGZ01000046)
b
?
?
-
?
?
?
CNP193 (ARGY01000047)
?
?
-
?
?
?
CNY280 (ARHH01000035 and ARHH01000052)
?
?
-
?
?
?
a
Gene identifier of S. tropica CNB440 showed in parentheses was selected for screening of sal gene cluster
b
Data were obtained from whole-genome shotgun GenBank database using BLAST
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Fig. 2 Neighbour-joining phylogenetic tree generated from salA KS domain sequences (623 bp). Bootstrap values above 50 %, calculated from 1,000 resamplings, are shown for neighbour-joining (NJ) and maximum likelihood (ML) methods
on the respective nodes. The two-letter code after the strain numbers denotes the location of strains reported previously by other groups. BA the Bahamas, FJ Fiji, GU Guam, PA Palau, SC Sea of Cortez
the highest latitude (33°N) recorded so far for members of this genus, compared to the last reported highest latitude of 28°N at Los Angeles Bay, Gulf of California (Fig. S4) (Becerril-Espinosa et al. 2013). Contrary to the unsuccessful attempt to isolate Salinispora species from offshore temperate sediments of San Diego (32°N) (Prieto-Davo et al. 2013), it is surprising that Salinispora was successfully isolated from Tosa Bay, even though it is one degree higher in latitude than San Diego. One possible reason for the successful isolation of Salinispora in Tosa Bay could be due to the flow of the Kuroshio Current, which is considered a warm current with an annual mean sea surface temperature of 22 °C. This is as opposed to the
California Current, which is colder with an annual mean sea surface temperature of 17 °C (Fig. S4) (Yashayaev and Zveryaev 2001). Since Salinispora strains have been reported to be cultivable at 10 °C (Maldonado et al. 2005), sediment temperature is likely not the sole factor that affects the survival of Salinispora. It could be an interplay of various biological factors, such as microbial community composition, or physical factors, such as nutrient, pressure, salinity, etc., which determine the presence and survival of any organism. Hence, other environmental and culturing studies have to be carried out in order to elucidate and determine the impact of each of these factors.
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Based on 16S rRNA gene sequence analysis, the Tosa Bay isolates were classified as S. arenicola of the type strain phylotype. This classification was further supported by phenotypic characteristics, such as rifampicin resistance and the positive detection of a rifamycin biosynthetic gene. We did not detect any S. tropica colony, which was expected since this species appears to be endemic to the Bahamas region (Maldonado et al. 2005; Jensen and Mafnas 2006). However, it was surprising that no S. pacifica strains were isolated, given that many have been found in different regions of the Pacific Ocean (Freel et al. 2012). This is probably due to a number of reasons, such as the overgrowth of other microorganisms in culture media, a low abundance of S. pacifica strains in this region or the small sample size. One of the challenges in identifying Salinispora species is the low diversity of its 16S rRNA gene sequence. This is particularly true as horizontal gene transfer is very common in this genus (Penn et al. 2009) and thus identification based on the conserved 16S rRNA gene sequence does not reflect the true diversity of the various strains isolated. The diversity of 16S rRNA gene sequences of S. arenicola strains isolated hitherto is relatively conserved as compared to that of S. pacifica. The majority of S. arenicola strains isolated, including the Tosa Bay strains, belong to the type strain phylotype, while the remaining are divided into four phylotypes A–D (Freel et al. 2012). However, we have observed diversity within the Tosa Bay strains using BOX-PCR. Using 16S-23S rRNA ITS sequencing and analysis of the housekeeping gene gyrB, five new ITS variants were identified in this study and the concatenated 16S rRNA-ITS-gyrB sequence analysis clearly demonstrated the Tosa Bay isolates to be phylogenetically distinct from other known S. arenicola strains. Secondary metabolism is an important aspect in the study of Salinispora. In fact, it has been proposed as a speciation marker because despite having highly similar 16S rRNA gene sequences, the genomes of the three Salinispora species are very diverse due to the presence of different secondary metabolite biosynthetic gene clusters. Since rifamycin production is a core chemotype of S. arenicola, the ketosynthase sequence associated with rifamycin biosynthesis was compared and sequence variations were observed between each representative strain, as well as the type strain. This further supports the suggestion that the
217
Tosa Bay Salinispora isolates are phylogenetically distinct from each other and the type strain. Interestingly, a location-specific KS1 sequence reported by Edlund et al. (2011) was detected in four strains of the Tosa Bay isolates. This KS1 sequence was only detected previously in strains isolated from the Sea of Cortez, the Red Sea and the Bahamas, but not in Guam and Palau, both of which are geographically closer to Tosa Bay (Edlund et al. 2011). Therefore, this result has important implications with respect to the biogeographical distribution and secondary metabolite evolution of S. arenicola. Although the other three ketosynthase sequences were not detected in the Tosa Bay isolates, the possibility of sequence variations resulting in unsuccessful gene amplification could not be ruled out. An earlier study by Jensen and co-workers (2007) suggested that salinosporamide is uniquely produced by S. tropica. However, recent studies on S. pacifica revealed the production of a salinosporamide analogue in some strains (Eustaquio et al. 2011; Freel et al. 2011). This is hardly surprising since phylogenetic studies have indicated a closer relationship between S. tropica and S. pacifica, rather than with S. arenicola. In this study, the salinosporamide gene cluster was detected for the first time in S. arenicola. The selected genes used in this study span across the whole sal cluster in S. tropica. Thus, these results suggest that an almost complete S. tropica sal cluster is present in S. arenicola strains, including the salL gene, which is absent in S. pacifica. Although salJ was not detected in JP strains, it is possible that the salJ gene was displaced by a transposition event in the Tosa Bay strains. This is suggested by the presence of an annotated transposase sequence upstream of salJ in the sal cluster of S. tropica (Eustaquio et al. 2009). It is also possible that the detection primers were not specific enough to amplify the salJ sequences. Preliminary RT-PCR analysis detected the presence of salL transcript in S. arenicola strain JP00, indicating the expression of this cluster during broth cultivation in Bennet’s medium (data not shown). Therefore, it is very likely that the sal gene cluster is functional in Tosa Bay strains. Complete sequence analysis, coupled with further studies on the production and structural analysis of salinosporamide in these S. arenicola strains will provide more insights into the structural relationship of salinosporamide produced by S. tropica.
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Importantly, the discovery of sal genes in S. arenicola refutes the earlier hypothesis that salinosporamide is produced only by the closely related S. tropica and S. pacifica (Freel et al. 2011). Perhaps, salinosporamide biosynthesis is a common characteristic of all Salinispora species and is not unique to S. tropica and S. pacifica, as previously thought. Yet, the phylogenetic analysis of salA from all three Salinispora species in this study suggested a separate evolutionary trajectory between S. tropica and S. pacifica, much earlier than with S. arenicola, which contradicts the description of S. tropica and S. pacifica as sister taxa (Freel et al. 2012). Since S. arenicola and S. tropica have been reported to co-occur in the Bahamas (Freel et al. 2012; Maldonado et al. 2005), it seems to be more likely that a horizontal gene transfer had occurred between both species. This hypothesis is supported by a sequence comparison study of staD genes of staurosporine biosynthesis from S. arenicola and S. pacifica strains, which also suggested interspecies recombination (Freel et al. 2011). Furthermore, sequence searches in the whole-genome shotgun contigs database using BLAST revealed the presence of sal genes in three other S. arenicola strains (CNP105, CNP193 and CNY280), indicating a higher occurrence of sal gene clusters in S. arenicola, and not just in the Tosa Bay strains. Although an earlier secondary metabolite profiling study of Salinispora species did not reveal that strains CNP105 and CNP193 produce salinosporamide (Jensen et al. 2007), this observation could have been due to silencing of the sal genes under the specified culturing conditions. Therefore, with more genome sequences of S. arenicola made available, perhaps more strains may be revealed to possess the sal gene cluster, which in turn allows a more thorough investigation of their evolution. In summary, this study has provided novel insights into the evolution and biodiversity of the unique marine genus Salinispora. The distribution of secondary metabolic genes in this genus may be more diverse or random than previously thought. Hence, continuing efforts in isolating them from new regions may unravel new clues to further strengthen the current understanding of their biogeographical distribution and secondary metabolic potential. Acknowledgments The authors thank C. Ihara for technical assistance, T. Nishisaka for assistance during sediment collection,
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Antonie van Leeuwenhoek (2014) 105:207–219 Z. Imoto and T. Hashimura of Usa Marine Biological Institute, Kochi University, for helping with sediment collection using research vessel Ms. Neptune, and Kochi Prefectural Deep Seawater Laboratory for providing deep seawater. This work was performed through Program to Disseminate Tenure Tracking System of the Ministry of Education, Culture, Sports, Science and Technology, The Japanese Government.
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