diversity of soil microorganisms: …...endospore forming bacteria were isolated by katherine...

DIVERSITY OF SOIL MICROORGANISMS: INTERACTIONS BETWEEN NOVEL ISOLATES

ALICJA DABROWSKA University of Warwick

M I C R O B I A L D I V E R S I T Y 2 0 1 5

M a r i n e B i o l o g i c a l L a b o r a t o r y • 7 M B L S t r e e t • Wo o d s H o l e , M A

AbstractAlthough soil microbes are important not only for the environment, but also biotechnology, there is a dearth of in-formation about the interactions between them. Thirty seven different microorganisms were isolated from the boat launch area near the Woods Hole Aquarium using a novel medium. The isolates were characterised in terms of col-ony morphology on different media types and ten of them were successfully identified based on 16S rRNA and rpoB sequences. 60% of the identified microorganisms belonged to the class of Actinobacteria. Two of the isolates cleared MnO₂ plates suggesting a possibility of extracellular electron transfer. Identified bacteria were streaked against one another and endospore forming bacteria in three different set-ups to test for possible interactions. A number of differ-ent interactions was observed. One isolate, Streptomyces sp. isolate 7, not only showed a variety of phenotypes on different media, but also seemed to interact with several organisms. Further research is needed to describe the mechanisms of interactions between these soil microorganisms.

IntroductionSoil microorganisms play many essential environmental roles - from degrading carbon compounds to biogeochemical cycling of sulphur and other chemical elements. They are also involved in a range of interactions with macroorgan-isms - mechanisms of plant-microbe mutualism, but also pathogenesis and parasitism, have been well studied in re-cent years (Lugtenberg et al., 2002; Newton et al., 2010). However, there is a dearth of information about interactions between soil microorganisms.

Most of the work that has been done on just soil microbes is focusing on natural products. With 80% of the antibiotics in use today being sourced from Streptomyces sp. and even more compounds waiting to be discovered (Watve et al., 2001), it is essential to understand their biology. However, it is also important to appreciate that they share their envi-ronment with other microorganisms and the interactions that occur in natural settings are probably one of the major reasons why they are capable of producing such a wide range of compounds. Studying these interactions is not only important for answering basic biological questions, but could also help us improve our understanding of natural product biosynthesis.

The aim of this study was to screen for interactions between novel soil isolates. To do this, a novel enrichment method for Actinobacteria was assessed, new cultures were characterised using several different methods and three different types of interaction plates were tested. Isolates of special interest were described in more detail and will be studied further in the near future.

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MethodsIsolation

Soil samples were collected from the boat launch area near the Woods Hole Science Aquarium (41.526459 N, 70.674094 W). With the aim of enriching for Actinobacteria, a calcium carbonate treatment method was used (El-Nakeeb & Lechevalier, 1962) with the following modifications: soil was dried overnight in 30ºC oven and incubation with calcium carbonate was for one day only. Serial dilutions were spread on cellobiose histidine phenylalanine (CHiP) medium plates (Appendix A). Plates were incubated at 30ºC until colonies appeared. Thirty seven unique colonies were picked and restreaked on fresh CHiP plates several times until a pure culture was obtained.

Endospore forming bacteria were isolated by Katherine Hargreaves and Yunji Wu Davenport from the same soil sample by two rounds of incubating pasteurised soil dissolved in taurine medium (Appendix A) at 30ºC until visible turbidity and plating on 5YE plates. These cultures were used for interactions assays only.

Culturing

Pure cultures were streaked on 5YE (5g yeast extract per 1 litre of water, 1.5% agar), CHiP and SWC plates (Appendix A). For DNA extraction, proteomics and streaking interaction plates, 5 ml of liquid 5YE medium were inoculated and incubated at 30ºC, 250rpm, for 48h. Cultures were routinely examined under a transmitted light microscope to de-scribe colony morphology and culture purity. Microscope photographs were taken using the Zeiss SteREO Discovery V12 and Axio Imager A2 microscopes.

DNA extraction

Aliquots of 2 ml of all cultures in liquid were centrifuged (4000 rpm) for 5 minutes and the pellet was resuspended in 300 µl of lysis buffer (appendix A) and 100 µl of 20 mg ml⁻¹ lysozyme. After incubation at 37ºC for 30 minutes, the lysates were transferred to first wells of Promega Maxwell RSC Blood DNA Kit cartridges and inserted into the Promega Maxwell RSC instrument according to manufacturers protocol. Samples were eluted into 50 µl of DNase free water. The concentration of DNA was measured using QuantiFluor ONE dsDNA System with Quantus Fluo-rometer.

PCR

To amplify genomic DNA, PCR was performed using Promega GoTaq Hot Start Green Master Mix and appropriate primers on MJ Research PTC-200 Thermal Cycler in 50 µl volumes. For 16S rRNA, 8F and 1492R primers were used (Edwards et al., 1989; Frank et al., 2008), while for identification of Actinobacteria rpoB primers were used (Guo et al., 2008). 5 µl of PCR product was run on a 1% agarose TAE gel with a Promega BenchTop 1kb DNA Ladder and visual-ised using SYBR Safe DNA Gel Stain and GE ImageQuant LAS 500 system. Samples which gave bands of appropriate molecular weight were cleaned using Promega Wizard PCR Preps DNA Purification System (following the Direct Purification of DNA from PCR Amplifications Without a Vacuum Manifold protocol) and sent for sequencing.

MALDI-ToF

Seven CHiP plates of pure cultures (isolates 6, 11, 15, 18, 23, 27 and 36) were sent for analysis to Dr Amanda Bulman, Senior Applications Scientist at Bruker Daltonics for MALDI-ToF analysis. Samples were processed according to standard Biotyper tube extraction protocol with positive and negative controls.

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Spectrometry

Colony absorbance spectra were collected using the Spectral Evolution SR-1900 Extended Range Spectroradiometer. At least 5 spectra were collected for a single colony, high colony density region and clear agar control for every sam-ple. For isolates presenting various colony morphologies on different media, measurements were repeated for every medium.

EET tests

To test the isolates for a possibility of extracellular electron transfer, MnO₂ CHiP, 5YE and SWC plates were prepared by pouring 8 ml of the liquid medium mixed at 45ºC with 1 ml of freshly synthesised MnO₂ over a thin layer of the same medium. 10µl of every liquid culture was spotted on a MnO₂ plate (4 spots per plate). After 48 hours aerobic incubation at 30ºC, the plates were moved to an anaerobic chamber and checked daily for 10 days.

Interactions assays

Three types of interactions plates were prepared (Figure 1). Twenty seven strains were used for streak 5YE, SWC and CHiP plates (4 plates of 6 isolates per plate, one plate with two mucoid isolates and one plate with a mucoid isolate and a negative control). 10 µl of eighteen cultures (selected based on successful DNA extraction) were spotted on plates with lawns of 5 endospore forming bacteria isolated from the same soil sample (9 spots per plate). Two types of cross plates were prepared: with 5 endospore forming bacteria cultures streaked in the middle against 15 strains streaked across the full length of the plate and streaked without touching the endospore (5 cultures per plate) and 5 isolates identified as Streptomyces sp. streaked against the other 5 identified soil microorganisms.

Figure 1 Different types of interactions assays: streak plate (left), drop plate (centre) and cross plate (right).

Results & DiscussionCharacterisation of isolates

Twenty seven isolates growing on three different media plates and in 5YE liquid were characterised in terms of col-ony morphology, growth rates, diffusible pigment production and hyphae formation (Table 1 - selected data; Figure 2). Good quality MALDI-ToF spectra were obtained for 7 isolates (Appendix B), but a search against all standard Bruker database entries did not produce any similarity scores above the standard threshold value. However, looking at the top spectrum score (although below the threshold) for isolates 15 and 23, the genus was identified correctly (Burkholderia sp. and Rhizobium sp. respectively; Appendix B). This is a very encouraging result and taking into ac-

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count how time and cost effective this method is, it could soon become a real alternative to the traditional PCR and sequencing approach.

Ten isolates were successfully identified by searching their 16S or rpoB sequences against BLASTn nucleotide collec-tion (identities ≥90% with query coverage ≥80% and low absorbance background noise in sequencing data) (Table 2). CHiP medium was designed to enrich for Actinobacteria and it seemed to work quite well with six out of ten identi-fied isolates belonging to the Actinomycetales order. This percentage could be different upon identification of all iso-lates. However, based on colony morphology, it appears that a significant proportion of unidentified isolates could also belong to the same group (especially Streptomyces sp. - e.g. isolates 10, 27, 34). Despite the fact that the soil envi-ronment from which the sample was collected is regularly exposed to marine water, less than half of the isolates could grow on SWC medium. Increasing salt concentration in the CHiP medium could select for Streptomyces sp. (four out of five grew well on SWC), while stopping the growth of mucoid organisms (isolate 14, Burkholderia sp. and Rhizobium sp.) which made isolation of pure cultures difficult (possible cause of failed or low quality sequencing data).

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Figure 2 Colony morphologies of the 27 isolates on 5YE (left), SWC (centre) and CHiP (right) plates"

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Table 1 Bacteria isolated from the boat launch soil sample.

I S O L AT E NUMBER

COLONY MORPHOLOGY

GROWTH ON CHIP PLATES

HYPHAE FORMATION

SPREADING PIGMENTS

1 after 40 hours no no




7 after 24 hours yes no


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I S O L AT E NUMBER

COLONY MORPHOLOGY


HYPHAE FORMATION

SPREADING PIGMENTS

10 after 40 hours no yes, grey




16 after 24 hours yes yes, yellow

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I S O L AT E NUMBER

COLONY MORPHOLOGY


HYPHAE FORMATION

SPREADING PIGMENTS



20 after 40 hours yes yes, grey

22 after 40 hours yes, white and grey yes, yellow-orange



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I S O L AT E NUMBER

COLONY MORPHOLOGY


HYPHAE FORMATION

SPREADING PIGMENTS

25 after 40 hours yes, white and grey no

27 after 40 hours no yes, grey

28 after 48 hours no yes, orange-grey


30 after 40 hours yes no

31 after 48 hours yes, white and grey no

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I S O L AT E NUMBER

COLONY MORPHOLOGY


HYPHAE FORMATION

SPREADING PIGMENTS



34 after 40 hours no yes, orange

36 after 40 hours no yes, orange

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Table 2 Identification of isolates 3, 6, 7, 15, 20, 22, 23, 25, 32 and 33 based on 16S rRNA and rpoB sequences

I S O L AT E NUMBER

IDENTIFICATION GENE IDENTITY QUERY COVERAGE

CLOSEST MATCH

3 Rhodococcus sp. rpoB 98% 93% Rhodococcus erythropolis BG43

6 Sphingomonas sp. 16S rRNA 98% 95% Sphingomonas sp. NBRC 101705

7 Streptomyces sp. rpoB 98% 96% Streptomyces sp. HI-17

15 Burkholderia sp. 16S rRNA 91% 81% Burkholderia sp. KN-9

20 Streptomyces sp. rpoB 94% 95% Streptomyces avermitilis MA-4680

22 Streptomyces sp. rpoB 95% 97% Streptomyces nodosus ATCC 14899

23 Rhizobium sp. 16S rRNA 98% 92% Rhizobium sp. UT 6-10

25 Streptomyces sp. rpoB 96% 96% Streptomyces incarnatus NRRL 8089

32 Luteibacter sp. 16S rRNA 99% 94% Luteibacter sp. UR 6-04

33 Streptomyces sp. rpoB 95% 97% Streptomyces incarnatus NRRL 8089

Isolate 7 (Streptomyces sp.) showed an interesting change in colony colour depending on the type of media used for growth (Figure 3). Pigment production is a characteristic feature of Streptomyces sp., they are often used as “biofacto-ries” of dyes (Chakraborty et al., 2015). The pH, temperature, salinity, carbon and nitrogen sources could affect the amount of pigment produced and thus its lighter or darker colour (Kiruthika & Boominathan, 2015). Colony absor-bance spectra of the isolate grown on different media show absorbance peaks at the same wavelength, which sug-gests that the same pigment could be produced in all three conditions, but at different concentrations. It would be useful to extract the pigments and try to identify them by absorbance spectra or mass spectrometry. Moreover, when grown in 5YE liquid medium, the isolate has two different types of colonies - free floating colonies that settle at the bottom of a tube when not shaken and colonies that stick to the sides of a tube and are difficult to remove (Figure 4). These two well-defined morphological characteristics make this isolate an interesting system for studying production of pigments and adhesive substances by Streptomyces sp. by genetic approaches such as transposon or chemical mu-tagenesis.

Figure 3 Different colony morphologies of isolate 7 on 5YE (left), SWC (centre) and CHiP (right) plates

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Figure 4 Two types of isolate 7 colonies - free floating and sticky.

Two isolates (6 and 1) appeared to clear MnO₂ CHiP plates in anaerobic conditions (Figure 5). Isolate 6 maintained its colour and the droplet was increasing in size with time, while isolate 1 lost its colour on MnO₂ SWC and CHiP plates and did not seem to be growing. This suggests that the clearance by isolate 1 could be caused by release of chemical compounds due to death and breakdown of colony, not active extracellular electron transport. Isolate 6, identified as Sphingomonas sp., is known to be able to degrade a wide range of compounds in both aerobic and anaerobic condi-tions (Kudlich et al., 1996). Further research is needed to establish whether this strain is actually capable of extracellu-lar electron transport or the clearance is again a by-product of cell lysis.

Figure 5 Clearing of MnO₂ by isolates 6 (top row) and 1 (bottom row) on 5YE (left), SWC (centre) and CHiP (right) plates

Interactions between soil microorganisms

Three major types of interactions were observed: neutral (two isolates growing together), negative (at least one of the organisms is growing poorly) and positive (growth of at least one of the organisms is enhanced) (Figure 6). Most or-ganisms did not show any visible changes in morphology when grown together or in close contact with another or-ganism. In some cases, pigment production was visible, but no growth inhibition or enhancement of the other organ-ism could be noticed (Figure 7).

Isolate 7 shows a number of different visible interactions with many other isolates and endospore forming bacteria. Apart from producing a yellow pigment when grown together with several other organisms, it also appears to pro-duce white rings only when close to endospore forming bacteria isolate E2 (Figure 8). These rings do not look like hyphae, compared to the interaction with isolate 32, and are absent in the interaction with endospore forming bacte-ria E3 (Figure 9).

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Rhodococcus sp. (isolate 3) and Burkholderia sp. (isolate 15) seem to influence hyphae production of Streptomyces sp. isolate 7 (Figure 10). In the case of isolate 3, if Streptomyces sp. colony is covered by the Rhodococcus sp. isolate, it can produce hyphae that extend out of the other organism’s surface. When isolate 7 is not fully covered by the second organism, it seems to inhibit the growth of its competitor and produce less hyphae (compared to a pure culture) or no hyphae at all. In contrast, when grown with Burkholderia sp., the Streptomyces sp. isolate has enhanced hyphae pro-duction. Further research is needed to establish the mechanism of these interactions, whether they are mediated by metabolites, competition for nutrients or another unknown factor.

A study looking at the interactions between Streptomyces coelicolor and Bacillus subtilis established that surfactin pro-duced by the endospore former can inhibit the formation of aerial hyphae and spores by Streptomyces sp. (Straight et al., 2006). Further studies like this are needed to understand other types of effects that different genera of microbes can have on the organisms they usually share the environment with.

Figure 6 Examples of neutral (left), negative (centre) and positive (right) interactions.

Figure 7 Pigment production by isolate 20 when streaked close to isolate 10 (left) and by isolate 7 when streaked close to isolate 15 (right).

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Figure 8 White ring structure present at the side of isolate 7 closest to endospore forming bacteria isolate E2

Figure 9 Changes in colony morphology when only half of a colony is in direct contact with another microorganism: changes in colony edge (left; endospore forming bacteria isolate E2 in contact with isolate 6), formation of hyphae rings (centre, isolate 7 in contact with isolate 32 and right, isolate 7 in contact with endospore forming bacteria isolate E3).

Figure 10 Differences in hyphae formation when isolate 7 is grown in close contact with other microorganisms: hy-phae rings coming out of a lane of isolate 3 (left), hyphae production from underneath isolate 3 and visible inhibition with no hyphae production (centre), enhanced hyphae formation when grown with isolate 15 (right).

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Conclusions and future workAs a wide variety of microorganisms inhabit soil, different types of interactions between them can be envisaged. By sharing the same environment, including nutrient resources, microbes have to engage in competition, cooperation, direct and indirect modifications of the surrounding area. Despite their importance for the environment and biotech-nology, interactions between soil bacteria tend to be overlooked by both plant biologists and bioengineers.

The novel medium for isolating Actinobacteria seemed to work well, with 60% of identified isolates belonging to this class. Identification of more isolates would give more suggestions for modifications of the medium to further im-prove its efficiency. Also, soils from different environments should be used as an inoculum, to test how reproducible this result is.

MALDI-ToF seems to be a quick and reliable way of identification of microorganisms, including soil isolates. How-ever, its success as an alternative to traditional sequencing approaches will depend on building a large, publicly available database of good quality spectra.

Studying extracellular electron transport using MnO₂ plates poses several major questions, including how to separate the effect of cell lysis and consequent release of a wide variety of compounds influencing manganese dioxide from the actual activity of a cell looking for final electron acceptors. MnO₂ plates are useful as a quick screen for looking for bacteria potentially capable of extracellular electron transport, but further tests have to be done to explain the cause of clearance.

A wide variety of microbe interactions can be observed using a simple screen such as co-plating of isolates. Cross plates proved to be most useful for assessing changes in morphology, drop plates - for checking for inhibition of growth and streak plates - release of pigments. Although time consuming and possible on a limited scale only, this method gives an opportunity to identify which strains are not visibly neutral towards each other and should be stud-ied further by more advanced methods such as proteomics, transcriptomics or metabolomics.

Isolate 7, Streptomyces sp., displayed not only an interesting phenotype in both liquid and on plates, but also seemed to interact with a range of organisms, including endospore forming bacteria, Burkholderia sp. and Rhodococcus sp. iso-lates. It would be very interesting to study the mechanisms of these phenomena in the future.

An advantage of this study was its time scale - less than a month from isolation of the microorganisms to interactions tests. It would also be interesting to compare this study with a similar one done using microbes that have been main-tained in a laboratory in pure cultures for years to see whether and, if so, how adaptation to the lab environment can influence the potential of microbes to interact.

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AcknowledgementsI would like to thank Jared Leadbetter and Dianne Newman for invaluable discussions about my project ideas, help, inspiration, motivation and, above all, the opportunity to take part in this course. I would also like to thank Scott Dawson and Srijak Bhatnagar for their help with DNA extraction and sequencing, Kurt Hanselmann for colony ab-sorbance spectra and MALDI-ToF, Tim Enke for Matlab data analysis.

I am also very grateful to Amanda Bulman (Bruker Daltonics) for running some of the isolates on MALDI-ToF, Scott Saunders (Caltech) for synthesising MnO₂ and his help with MnO₂ plates and Mak Saito, Dawn Moran and Matt McIlvin (WHOI) for the unique opportunity to learn their novel protein extraction method for LCMS analysis of bac-terial proteomes.

Many thanks to the (best!) group 4: Katherine Hargreaves, Lorenzo Lagostina, James Russell and Yunji Wu Daven-port for isolating endospore-forming bacteria, fun sampling trips and all the good times. I would also like to thank all other course participants, TAs, faculty, visiting lecturers and sponsor companies.

I would also like to acknowledge and thank for the support of Abigail Salyers Scholarship Fund, Amgen Scholars Alumni Support Scheme and my PhD supervisors: Dave Scanlan, Chris Corre and Joseph Christie-Oleza - without their support I would not have been able to attend this course. As always, many thanks to my husband, parents and friends for patiently listening to “fascinating microbe!” stories and surviving the constant stream of “beautiful bacte-ria!” photos.

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References1. Chakraborty, I., Redkar, P., Munjal, M., Sathish Kumar, S.R., Bhaskara Rao, K.V. (2015) Isolation and characterisa-

tion of pigment producing marine actinobacteria from mangrove soil and applications of bio-pigments. Der Pharmacia Lettre 7 (4), 93-100

2. Edwards, U., Rogall, T., Blöcker, H., Emde, M., Böttger, E.C. (1989) Isolation and direct complete nucleotide de-termination of entire genes: characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Research 17, 7843-7853

3. El-Nakeeb, M.A., Lechevalier, H.A. (1962) Selective isolation of aerobic Actinomycetes. Applied Microbiology 11, 75-77

4. Frank, J.A., Reich, C.I., Sharma, S., Weisbaum, J.S., Wilson, B.A., Olsen, G.J. (2008) Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Applied Environmental Microbiology 74 (8), 2461-2470

5. Guo, Y., Zheng, W., Rong, X., Huang, Y. (2008) A multilocus phylogeny of the Streptomyces griseus 16S rRNA gene clade: use of multilocus sequence analysis for streptomycete systematics. International Journal of Systematic and Evolutionary Microbiology 58, 149–159

6. Kiruthika, P., Boominathan, M. (2015) Isolation, purification and characterisation of biologically active melanin from marine Streptomyces. American Journal of Biological and Pharmaceutical Research, 2 (2), 75-80

7. Kudlich, M., Bishop, P.L., Knackmuss, H.J., Stolz, A. (1996) Simultaneous anaerobic and aerobic degradation of the sulfonated azo dye Mordant Yellow 3 by immobilized cells from a naphthalenesulfonate-degrading mixed culture. Applied Microbiology and Biotechnology 46, 597-603

8. Lugtenberg, B.J.J., Chin-A-Woeng, T.F.C., Bloemberg, G.V. (2002) Microbe-plant interactions: principles and mechanisms. Antonie van Leeuwenhoek 81 (1-4), 373-383

9. Newton, A.C., Fitt, B.D.L., Atkins, S.D., Walters, D.R., Daniell, T.J. (2010) Pathogenesis, parasitism and mutualism in the trophic space of microbe-plant interactions. Trends in Microbiology 18 (8), 365-373

10. Straight, P.D., Willey, J.M., Kolter, R. (2006) Interactions between Streptomyces coelicolor and Bacillus subtilis: Role of surfactants in raising aerial structures. Journal of Bacteriology 188 (13), 4918-4925

11. Watve, M.G., Tickoo, R., Jog, M.M., Bhole, B.D. (2001) How many antibiotics are produced by the genus Strepto-myces? Archives of Microbiology 176, 386-390

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Appendix ATable A1 Composition of Cellobiose Histidine Phenylalanine (CHiP) medium

COMPONENT AMMOUNT PER 1 LITRE

Freshwater base (100x, see Table A4) 10 ml

MES (1M, pH 5.5) 10 ml

Histidine 1 g

Cellobiose 10 g

Phenylalanine 0.1 g

Sodium thiosulphate (1M) 0.2 ml

Potassium phosphate (100mM, pH 7.1) 1 ml

Trace elements mix (see Table A6) 1 ml

Multivitamin mix (see Table A7) 1 ml

Cycloheximide (50mg ml⁻¹ 100% ethanol) 1 ml

Agar (for plates) 15 g

Table A2 Composition of Taurine medium for endospore formers


Freshwater base (100x, see Table A4) 10 ml

1M NH₄Cl 10 ml

150 mM KH₂PO₄ 10 ml

1M Na₂SO₄ 1 ml

1M MOPS pH 7.2 10 ml

Trace elements mix 1000x 1 ml

Taurine 1 g

Table A3 Composition of Sea Water Complete (SWC) medium


Seawater base (100x, see Table A5) 997 ml

Bacto tryptone 5 g

Yeast extract 1 g

Glycerol 3 ml

HCl required for pH 7.0

Agar (for plates) 15 g

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Table A4 Composition of freshwater base 100x


NaCl 100 g

MgCl₂·6H₂O 40 g

CaCl₂·2H₂O 10 g

KCl 50 g

Table A5 Composition of seawater base 100x


NaCl 400 g

MgCl₂·6H₂O 60 g

CaCl₂·2H₂O 3 g

KCl 10 g

Table A6 Composition of trace elements mix 1000x


FeSO₄·7H₂O 2100 mg

H₃BO₃ 30 mg

MnCl₂·4H₂O 100 mg

CoCl₂·6H₂O 190 mg

NiCl₂·6H₂O 24 mg

CuCl₂·2H₂O 2 mg

ZnSO₄·7H₂O 144 mg

Na₂MoO₄·2H₂O 36 mg

NaVO₃ 3 mg

Na₂WO₄·2H₂O 3 mg

Na₂SeO₃·5H₂O 6 mg

20 mM HCl 1 ml

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Table A7 Composition of multivitamin mix 1000x


Riboflavin 100 mg

Biotin 30 mg

Thiamine HCl 100 mg

L-Ascorbic acid 100 mg

d-Ca-pantothenate 100 mg

Folic acid 100 mg

Nicotinic acid 100 mg

4-aminobenzoic acid 100 mg

Pyridoxine HCl 100 mg

Lipoic acid 100 mg

NAD 100 mg

Thiamine pyrophosphate 100 mg

Cyanocobalamin 10 mg

10 mM MOPS pH 7.2 1000 ml

5M NaOH until dissolved (5-10 drops)

Table A8 Composition of lysis buffer

COMPONENT AMMOUNT PER 0 .2 LITRE

Sucrose 20.52 g

1M Tris HCl 5 ml

0.5M EDTA 10 ml

RNase 2 U

H₂O 185 ml

HCl required for pH 7.5

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Appendix B

Figure B1 MALDI-ToF spectra for isolates 6, 11, 15, 18, 23, 27, 36 (from top to bottom)

Figure B2 MALDI-ToF database search results for isolate 15 (identified by 16S rRNA sequence as Burkholderia sp.)

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Figure B3 MALDI-ToF database search results for isolate 23 (identified by 16S rRNA sequence as Rhizobium sp.)

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diversity of soil microorganisms: …...endospore forming bacteria were isolated by katherine...

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