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Diversity of cultivable soil ultramicrocells

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Environmental Microbiology and Environmental Microbiology
Journal:
Reports

Manuscript ID: EMI-2011-0033

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Manuscript Type: EMI - Research article

Journal: Environmental Microbiology

Date Submitted by the

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13-Jan-2011
Author:

Complete List of Authors: Silbaq, Fauzi; Mar Elias Educational Institutions, Research
Nevo, Eviatar; University of Haifa, Institute of Evolution
Hadi, Bahaa; University of Haifa, Institute of Evolution
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Azaizah, Hassan; The Galilee Society, R&D Center

bacteria, microbial communities, uncultured microbes, growth and

Keywords:
survival
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2 Diversity of cultivable soil ultramicrocells

4 Fauzi Silbaq*, Bahaa Hadieh, Eviatar Nevo, Hassan Azaizah, and Jeries Jedoun.

5 Mar Elias Educational Institutions and Mar Elias Campus, Ibillin, Galilee 30012, Israel
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6 Dr. Fauzi S. Silbaq

7 849 Marble Drive

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8 Fort Collins, CO 80526

9 Phone: 970-223-0699
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10 E-mail : fsilbaq@gmail.com
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11 Eviatar Nevo

12 Institute of Evolution, International Graduate Center of Evolution, University of Haifa, Haifa

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13 31905, Israel. Email: nevo@research.haifa.ac.il

14 Jeries Jedoun and Hassan Azaizah

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15 The Galilee Society R&D Center (Associated with Haifa University), P.O. Box 437, Shefa-Amr
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16 20200, Israel. Email: hazaizi@gal-soc.org, jeries@gal-soc.org

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1 Summary

2 Ultramicrocells (UMC) are reduced forms of microorganisms, usually associated with reductive

3 cell division during starvation or stress, which can pass through a 0.2 µm filter. In this study, we

4 aimed to cultivate and examine the phylogenetic diversity of ultramicrocell isolates in soils and

5 to shed light on soil bacteria communities, the majority of whose members have yet to be
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6 cultivated or identified phylogenetically. Soil ultramicrocells that pass through 0.2 µm filters
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7 were cultivated on low-nutrient agar and identified on the basis of their 16S rDNAs sequences.

8 All cultivated ultramicrocells formed large cells after secondary cultivation. A total of 76
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9 independent ultramicrocells isolates identified in this study were closely related to uncultured

10 and cultured bacteria of different phyla. Seventy-five percent (75%) of the isolates were
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11 affiliated with Proteobacteria (alpha, beta, and gamma groups at 31.6%, 49.1%, 19.3%,

12 respectively), 21.1% were affiliated with Actinobacteria, and 3.9% were affiliated with
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13 Firmicutes. A total of 23 isolates were related to uncultured bacteria, and 16 isolates had been
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14 categorized as unclassified at the genus level. Furthermore, the cultivable ultramicrocell diversity
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15 show similar diversity to the total soil bacterial diversity that was studied by molecular-approach

16 methods and to soil dwarf, uncultured bacteria (0.45 µm filtrate).

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17 Keywords: Soil ultramicrocells, starved bacteria, 0.2 µm filtrate, diversity, “unculturable”

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1 Introduction

2 Bacteria have diverse metabolic capacities that allow them to exploit the wide range of energy

3 sources in soils. However, very few members of soil communities are cultivatable using standard

4 microbiological media. Estimates are that only about 1% of the 109 bacterial cells in each gram of

5 soil form visible colonies, using standard cultivation methods (Amann et al., 1995), and that each
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6 gram of soil contains fewer than 100 cultivable species (Dunbar et al., 1999). As a result, recent
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7 analyses of the microbial diversity in soil and other environments have primarily relied on culture-

8 independent molecular methods (DeSantis et al., 2007; Schloss and Handelsman, 2006). Estimates
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9 of bacterial diversity reveal high diversity in soils (Torsvik et al., 1990; Curtis et al., 2002),

10 Recently, Roesch et al., (2007) used a high throughput DNA Pyrosequencing and statistical
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11 inference to assess bacterial diversity, and they found that the number of bacterial species, based on

12 sequence diversity could be as high as 52,000 per gram of soil. Genetic surveys of soil bacteria
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13 have also revealed large differences among soil samples in terms of diversity. Researchers have
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14 correlated bacterial diversity with numerous environmental factors including latitude, temperature,
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15 soil pH, anthropogenic factors, and sample size (Ellis et al., 2003; Lipson and Schmidt, 2004; Gans

16 et al., 2005; Fierer and Jackson, 2006, Kakirde et al. 2010).

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17 Although, soils are rich with bacterial diversity, the proportion of bacteria from the soil samples
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18 that are cultivable on standard plate-counting media varies between 0.08 and 2.2%. Metal

19 contamination appears to have a particularly negative effect on soil culturing. Ellis et al., (2003)

20 found that Metal contamination did not have a significant effect on the total genetic diversity

21 present but appeared to affected the cells physiological status, so that the number of bacteria

22 capable of responding to laboratory culture.

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1 The frequent discrepancy between direct microscopic counts of active bacterial numbers

2 and numbers of “culturable” bacteria from environmental samples is just one of several indications

3 that current information on cultivation requirements is limited and restricts our knowledge on the

4 diversity of microorganisms in nature. Christensen et al., (1999) counted total active soil bacteria

5 (collected on membrane filtration, with a pore size of 0.2 µm) by fluorescence in situ hybridization
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6 with an rRNA oligonucleotide probe and uncovered 4.8 × 108 active bacteria per gram of dry soil.

7 The majority (68-77%) of actively growing bacteria were members of the smallest size class (cell
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8 width, 0.25 to 0.5 µm), but the active and growing bacteria still represented only approximately 5%

9 of the total bacterial population as determined by DAPI (4',6-diamidino-2-phenylindole) staining

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10 (Bae and Casida, 1973; Bakken and Olsen, 1987; Christensen et al., 1999). The calculated average
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11 percent viability of dwarf cells was only 0.2% for cells with diameters below 0.4 µm, and only 10–

12 20% of the viable cells with diameters below 0.4 µm increased their diameter to >0.4 µm prior to
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13 growth (Bakken and Olsen,1987).

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14 The majority (72%) of active soil microbes therefore, appear to exist in situ as
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15 uncharacterized cells less than 0.3 µm in diameter (Bae et al., 1972). These starved bacteria,

16 ultramicrocells, and ultramicrobacteria; have been collectively identified as bacteria that pass
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17 through a 0.45 µm membrane filter (Rutz, and Kieft, 2004) or “dwarf” bacteria. While the majority

18 of viable but “unculturable” soil bacteria fall under this category, most of the traditional methods of
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19 collecting cells by filtering examined only those captured on the filters and excluded most

20 ultramicrocells that passed through the filter pores (De Fede and Sexstone, 2001; Rutz and Kieft,

21 2004). Dwarfing or rapid size decrease of bacteria under starvation appeared to be an active process

22 which occurred more rapidly when the cells were in an early stage of logarithmic growth at the

23 onset of starvation (Humphrey et al., 1983). Interfaces, such as solid-water interfaces found in soil,

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1 lack a sufficient amount of nutrient or stress trigger cells to become smaller in size (Kjelleberg et

2 al., 1982; Sonia et al., 2004; Amako et al., 2008, Lee and Veeranagouda, 2009) to increase their

3 surface/volume ratio and packing density. Dwarfing is reversibly inhibited by low temperature and

4 low pH but was not inhibited by chloramphenicol, an inhibitor of protein synthesis (Humphrey et

5 al., 1983).
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6 Our study focused on the cultivation of soil UMCs, which are thought to be an adaptation to
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7 nutrient limitation and which had not been previously identified (Torrella and Morita 1981; Bakken

8 and Olsen 1987, Eguchi et al., 2001, Weart et al., 2007, Lee and Veeranagouda, 2009). Bacterial
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9 cells with a width smaller than 0.2 µm developed microcolonies on agar media at slow rates. Those

10 microcolony isolates, which might not be adapted to grow at faster rates, and hence had been
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11 overgrown by the larger cell size fraction of bacteria (>0.2 µm filterate). UMCs developed

12 microcolonies upon isolation on Alpha agar, but not on Nutrient agar, and a normal size colony
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13 upon second cultivation on rich media. Microcolonies of soil bacteria were observed by Ferrari et
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14 al., (2005), who concluded that the missing cultivable majority may have a growth strategy that is
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15 not observed when traditional cultivation indicators are used. In addition, a differential response of

16 size-fractionated soil bacteria in BIOLOG® Microtitre plates suggest that small cells are
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17 metabolically distinct from large cells, and small cells primarily utilized carbohydrates and

18 carboxylic acids, compared to a broad range of substrate utilization by large cells (De Fede and
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19 Sexstone, 2001).

20 In this study, we describe and classify a diverse fraction of “culturable” UMCs that pass

21 through 0.2 µm pore-size filters and grow to a larger size upon secondary cultivation on rich media.

22 Often this fraction of UMCs is overgrown by dwarf bacteria fractions with the bigger diameter

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1 (bacteria that pass through 0.4 µm pore-size filters) and higher average percent viability.

2 Previously, some of us have cultivated and identified UMCs from biofilm of drinking water system

3 walls using low-nutrient agar media (Silbaq, 2009). This medium seems to favor the growth of

4 UMCs. Small cells with volumes conforming to the definition of UMC have routinely been

5 observed in soil samples, and most are not yet isolated and identified. Here we apply similar
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6 approaches to cultivate UMCs from soils. This group of bacteria might be indicator species for

7 determining the impact of natural and anthropogenic changes on soil communities.

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8
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9 Results

10 Cultivable soil UMCs

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11 A total of 76 independent culturable UMCs were considered for this study. The UMCs were

12 isolated at two different seasons: 44 (ST1-1 to ST7-4 isolates) different UMCs were isolated in
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13 May, and 32 (WST1-1 to WST7-5 isolates) different UMCs were isolated in November (Table 1).
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14 Soil UMCs were isolated on Alpha agar as microcolonies (Fig. 1). The size of the first isolates of
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15 UMCs colonies were very small microcolonies and could be detected mainly on alpha agar, by light

16 microscope under magnification. However, with the passage of isolates from Alpha agar into
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17 Nutrient agar, the isolates grew at a faster rate and developed normal-sized colonies. Beside the

18 bacterial cell size on rich media, UMCs isolates seem to be normal in size and shape, as estimated
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19 by a light microscopy with Gram staining.

20 UMCs members of the phylum Proteobacteria

21 Fifty-seven isolates represented 75% of the total isolates and were affiliated with the phylum

22 Proteobacteria. Of these 31.6% were Alphaproteobacteria, 49.1% were Betaproteobacteria, and

23 19.3% were Gammaproteobacteria (Table 1 and Fig. 2). UMC members of the class

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1 Alphaproteobacteria comprise the most unclassified and uncultured UMCs. This group consists of

2 31.6% (18 isolates) of total Proteobacteria: 16.7% were affiliated with the order Rhodospirillales,

3 22.2% affiliated to Caulobacterales, 22.2% affiliated to Rhizobiales and 38.9% affiliated to

4 unclassified Alphaproteobacteria. Group ST3-2, ST6-1, and WST6-5 corresponded to isolate that

5 were cultivated from underground water (Silbaq, 2009), which were confidently classified in the
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6 order Rhodospirillales. Isolate ST2-7 was identified in family Brucellaceae. Cluster WST7-1 and

7 WST3-4, cluster ST7-4, WST7-3 and WST3-1, ST2-11, and ST1-12 were identified only at the
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8 class level (Fig. 2). Our results showed the abundance of unclassified and unidentified

9 Alphaproteobacteria at the order level. Isolates ST7-4, WST7-3, and WST3-1 should be studied in
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10 more details in order to be identified and categorized. However, all three isolates show similarities
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11 to the 16S rRNA gene sequence around 94%-99% to Phaeospirillum sp. (Table 1). The Table 1 and

12 Fig. 2 show that 11 out of 18 (61%) isolates are unclassified at phylum and genus levels.
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13 Ultramicrocell members of the class Betaproteobacteria consisted of 49.1% (28

14 independent isolates) of the total Proteobacteria. The majority (71.4%) were assigned to the order
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15 Burkholderiales, with 28.6% isolates affiliated to the order Rhodocyclales. However, the
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16 Betaproteobacteria classd ominate most of the isolates and most of the uncultured members of
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17 Proteobacteria. Most of the classification limits were at the family level of Rhodocyclaceae

18 isolates and at the genus level (Alcaligenes) of the Alcaligenaceae family. Isolate WST3-6 showed
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19 92% similarity to the 16S rRNA gene sequence of Aquabacterium parvum isolated from the

20 drinking water system (Kalmbach et al., 1999) and 89% confidence of classification to be affiliated

21 to the genus Aquabacterium including four species.

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1 The most interesting was the abundance of the Azospira and Alcaligenes isolates that had

2 not been cultured before. The genus Azospira included just one species that was identified as

3 Azospira oryzae by Reinhold-Hurek and Hurek (2000). It seems that Azospira species are abundant

4 in UMCs in the soil and can be easily be cultivated on Alpha agar. However, colony development

5 might be very slow and grow in the microcolony stage; most of the microcolonies can be seen
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6 using a microscope with relatively high magnification (x200-400). It is important for some

7 oligotrophic isolates, such as Azospira, to be given the chance to grow without competition
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8 pressure by faster growers and by those who were removed according to size selection (0.2 µm

9 filtration) in this study. As a result of this and the previous study on water UMCs, the fractionation
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10 of bacterial populations, by ultrafiltration or by any other harmless methods, should be applied in

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11 order to cultivate bacterial isolates from as complex a system as soil.

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13 Ultramicrocell members of the class Gammaproteobacteria consisted of 19.3% (11

14 independent isolates) of the total Proteobacteria, of there were 81.8% affiliated with the order
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15 Xanthomonadales, and 18.2% were affiliated with the order Pseudomonadales. Just one isolate,
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16 WST1-4, out of 11 was assigned as unclassified Pseudomonas (Table 1 and Fig. 2). However, the
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17 order Xanthomonadales consisted mainly of isolates affiliated to the genus Stenotophomonas and to

18 genus Silanimonas. All isolates affiliated to the genus Silanimonas showed high homology to the
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19 16S rRNA gene sequence of uncultured bacteria. Surprisingly, the genus includes just one species,

20 Silanimonas lenta, isolated from a hot spring (Lee et al., 2005). Many isolates (WST5-2, ST5-2,

21 and WST6-2) of this class showed the highest similarities with the 16S rRNA gene sequence level

22 in previously isolated UMCs clones from a flow system model with PVC (polyvinyl chloride) pipes

23 (Silbaq, 2009). Those isolates were isolated from the walls of flow system biofilm that had been

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1 developed in situ and, hence, showed the potential of those UMCs to attach and grow in

2 oligotrophic environments, such as chlorinated drinking water.

4 UMCs members of the phylum Firmicutes

5 Firmcutes isolates consisted of 3.9% of the total isolates (3 independent isolates). All isolates were
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6 affiliated to the order Bacillale. Two isolates, ST1-8 and WST1-1, were classified as Staphylococci

7 with complete (100%) confidence of classification, and the third isolate ST1-6 was classified as
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8 Bacillus with 100% confidenc. BLAST search showed 100% identities (665/665) to environmental

9 isolates, Bacterium 8-gw1-3 (accession number DQ990033) and Bacillus pumilus in the NCBI
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10 database (Tiquia et al., 2008) (Table 1, Fig. 2).

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11 UMCs members of the phylum Actinobacteria

12 Sixteen different UMC isolates belonged to the order Actinomycetales (Table 1 and Fig. 3). A
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13 majority of 68.8% UMC isolates were affiliated with the suborder Micrococcineae, 18.8% were

14 affiliated with the suborder Corynebacterineae, and 12.5% were affiliated with the suborder
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15 Propionibacterineae. Nine of the UMC isolate sequences from soil were identical to sequences
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16 isolated from drinking water; most of these isolates had not been previously cultivated or identified
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17 (Silbaq, 2009). The isolates were classified into the genera Aeromicrobium, Leucobacter, Kocuria,

18 Microbacterium, Arthrobacter, Brachybacterium, and Rhodococcus.

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20 DISCUSSION

21 Cultivation of soil UMCs

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1 UMC cultivation shows that direct cultivation of 0.2 µm filterable cells from soil, is feasible and

2 supports the idea that most “unculturable” bacteria consisted mainly of “as-yet-uncultured” species

3 (Gest, 1993). Although the estimation of percent viability of UMC might be below or similar to

4 dwarf’s (>0.4 µm filterate), the calculated average percent is only 0.2% of total cultivable soil

5 bacteria (Bakken and Olsen, 1987). Our findings dealt with low amounts of isolates for each gram
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6 soil, and the small amount of cultivable isolates hindered the comparison of bacterial species

7 richness between summer and winter isolates. Similar low counts have documented by Vybiral et
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8 al., (1999) and support the conclusion that most of these bacteria were starvation forms at the time

9 of isolation and not ultramicrobacteria as defined by Schut et al., (1993). Recently, cultivable UMC
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10 cell populations, capable of passing through 0.2 µm filters had been identified in many environment
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11 samples (Miteva and Brenchley, 2005; Silbaq, 2009). In general, our results support the conclusion

12 that cultivation methods are critical in microbial diversity studies because they can detect
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13 organisms undetected by molecular techniques (Donachie et al., 2007; Davis et al., 2005; Joseph et

14 al., 2003; Sait et al., 2002).

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15 UMC affiliations
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16 A total of seventy-six independent isolates of UMCs (Table 1) isolated and identified in this

17 study were closely related to uncultured and cultured bacteria of different phyla. Seventy-five
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18 percent of the isolates were classified as Proteobacteria (Alpha, Beta, and Gamma groups

19 consist of 31.6%, 49.1%, 19.3%, respectively), 21.1% were classified as Actinobacteria and

20 3.9% as Firmicutes (Fig. 2, 3). The results show a high percentage of cultivates were classified

21 in Actinobacterial phylum (21%), much higher than previously described (5%) by Pyrosequncing

22 (Roesch et al., 2007), using universal 16S RNA primers. Universal 16S rRNA gene primers are

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1 not capable of amplifying this gene from all bacteria within an environmental sample. Some

2 Actinobacteria may share 100% sequence similarity to universal primers but remain undetected

3 (Farris and Olson, 2007). Our results of UMC cultivates did not support the suggestion that the

4 population of small bacteria consists mainly of gram-positive organisms (Lindahl et al., 1997),

5 determined by the amounts and types of phospholipid fatty acids composition of indigenous
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6 dwarf soil bacteria (<0.4µm). In addition, the cultivation results support conclusions that culture-

7 based approaches are still a powerful qualitative tool that can be used in parallel with molecular
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8 methods and show a total of 23 cultivates were related to “uncultured” bacteria, and 16 cultivates

9 were categorized as unclassified above the genus level (Table 1).

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10
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11 The abundance of cultivable UMC affiliated to Proteobacteria

12 The UMC cultivates (Table 1, and Fig. 2) show similar phylogenetic diversity to the data that was
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13 obtained by microcultivation (Ferrari et al., 2005), cultivation (Davis et al., 2005; Joseph et al.,

14 2003; Sait et al., 2002; Axelrood et al., 2002), and molecular methods (Fierer and Jackson, 2006).
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15 In addition, our results show similar diversity to the semiarid soil dwarf uncultured bacteria (0.45
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16 µm pore-size filtrate) studied by molecular approaches and cultivation (Iizuka et al., 1998; Rutz
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17 and Kieft, 2004). However, not a single cultivable representative of Alphaproteobacteria was

18 isolated by Ferrari et al., (2005) and Axelrood et al., (2002). Ferrari and his colleagues (2005)
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19 applied a novel cultivation method of total soil bacteria captured by 0.2 µm pores size filter,

20 including all bacteria that could not pass through the 0.2 µm, and excluded most of the UMC

21 population. Isolation and phylogenetic analysis of aerobic copiotrophic “ultramicrobacteria” (that

22 passed through a 0.45 µm membane filter) from urban soil showed that the "ultramicrobacteria"

23 isolates were classified into three major groups: Betaproteobacteria and Gammaproteobacteria and

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1 the Actinobacteria (Iizuka et al., 1998; Axelrood et al., 2002). Again, not a single cultivable

2 representative of Alphaproteobacteria was isolated. On the other hand, semiarid soil intrinsic dwarf

3 bacteria (0.45 µm pore-size filtrate and that retained their small size, also termed

4 ultramicrobacteria) had been subjected to DNA extraction, PCR using universal bacterial and

5 archaeal 16S rRNA gene primers, and cultivation into R2B medium. The data showed that intrinsic
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6 dwarf (that retained dwarf size) Proteobacteria dominated the enrichment cultures and were

7 identified as Alpha- and Betaproteobacteria (Rutz and Kieft, 2004), but no representative of the
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8 Gammaproteobacteria group were cultivated. The lack of any representative of

9 Alphaproteobacteria (Iizuka et al., 1998; Axelrood et al., 2002; Ferrari et al., 2005) or
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10 Gammaproteobacteria (Rutz and Kieft, 2004) could be as the result of many reasons, such as
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11 overgrowth of this group of bacteria by other faster growing isolates or by the selective biases of

12 the medium that had been used such as incubation time, temperature, and the development of
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13 microcolonies, which is not visible by the naked eye, and hence had been not recognized.

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15 The abundance of cultivable UMC affiliated to Actinobacteria

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16 The abundance of cultivable UMCs affiliated to Actinobacteria phyla in soil seem to be higher than
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17 previously estimated (Fig 3. and Table 1). Similar results were obtained from drinking water

18 UMCs, (63.5% Proteobacteria, 32.7% Actinobacteria, and 3.8% Firmicutes). However, the
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19 distribution of the different groups of Proteobacteria in water environments were 45.5% Alpha,

20 33.3% Beta, and 21.2% Gamma, and the Actinobacteria related isolates were more abundant in

21 water environments at the expense of the Proteobacteria phylum (Silbaq, 2009). Sixteen

22 Actinobacterial isolates affiliated to subclass Actinobacteridae. The phylogenetic tree (Fig. 3)

23 showed that the some of the UMCs isolates belong to new clusters and sub clusters

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1 phylogenetically related and bordered between clusters acII and the newly proposed acSTL cluster

2 (Allgaier and Grossart, 2006). Soil UMCs identified in this study showed similar affiliation to tap

3 water UMCs isolates (Silbaq, 2009).

4 In conclusion, we have cultivated and identified a number of diverse soil UMCs, most of which

5 were not previously known to be in minute forms. UMCs were members of the Alpha, Beta,
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6 Gamma-proteobacteria, Actinobacteria and Firmicutes. Many of the sequences represented
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7 unclassified microorganisms. The most profound results were the percentage of the unclassified

8 and uncultured affiliated UMCs isolated in this study on minimal medium such as Alpha agar
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9 plates. Microbial diversity is controlled primarily by soil variables (Fierer and Jackson, 2006).

10 Thus, minimal changes in cultivation strategy can result in the isolation of globally distributed, but
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11 previously uncultured, phylogenetically novel soil bacteria. It is tempting to speculate that much

12 difference in the results between different research groups could be, to some extent, due to the use
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13 of different methods, to the type of media used in each study, and the nonexistence of optimal
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14 media able to support the growth of vast bacterial diversity existing in soil. The same can apply to
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15 the lack of ultramicrobacteria or intrinsic dwarf bacteria smaller than 0.2 µm in diameter in our

16 results. It seems that Alpha agar support mainly UMCs, but not other dwarf bacteria. During
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17 cultivation of soil UMCs (this work) and water UMCs, we had encountered bacterial isolates that

18 ceased growing after the first isolation; hence, no data had been obtained about this group of
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19 isolates.

20 Experimental Procedures

21 Soil samples

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1 Soil samples of 200 g each were collected from the upper layer (1-10 cm deep) of Evolution

2 Canyon, in Lower Nahal Oren, Mt. Carmel, Israel (32º43’ N; 34º58’ E) (Nevo, 1995, 2001). In

3 order to collect the highest diversity, seven (200 g each) samples were collected from different sites

4 during summer (May), and another seven samples had been collected during winter (November).

5 Isolation of UMCs
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6 A total of fourteen different samples of 200 g of soil were incubated with 500 ml of 100mM

7 NaCl each. The mixtures had been incubated overnight at room temperature with mild shaking.
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8 Sand particles were allowed to settle as sediment for 30 min. The supernatant of the soil mixture

9 was filtrated through 0.45 and subsequently through 0.2 µm pore-size filter membranes.
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10 Different samples of the 0.2 µm filtrates were spread over different solid media such as Nutrient
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11 agar, various strengths of nutrient agar, R2A agar (Reasoner and Geldreich 1985), and Alpha

12 agar. Colony forming units were estimated from each medium with careful attention to the
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13 colonies morphological diversity.

14 The plates were incubated at room temperature (22°C) in darkness for at least three weeks. Isolated
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15 colonies of UMCs, had observed mainly on Alpha agar after three weeks of incubation.
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16 Microcolonies were transferred to a Nutrient agar (15 g l-1 agar, 6 g l-1 Peptone Mix, 0.5 g l-1 Beef
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17 extract, 0.5 g l-1 Soy bean meal, 2 g l-1 yeast extract, and 5 g l-1 NaCl). Isolates that were collected

18 during summer were labeled ST1-1 to ST7-4, and winter isolates were labeled WST1-1 to WST7-5
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19 (Table 1). UMCs with different colonial morphology were transferred on Alpha and Nutrient agar

20 plates for further study.

21 Microscopy

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1 The inoculated Alpha agar plates were observed periodically with light microscope (Axiovert 40

2 CFL, Carl Zeiss), with Objectives LD "Plan-Neofluar" 40x/0.60 korr, and Objectives LD "Plan-

3 Neofluar" 63x/0.75 korr, (Fig. 1).

4 Genomic DNA extraction from UMCs.

5 Bacterial chromosomal DNA was extracted from each one of the UMC isolates using QIAamp
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6 DNA Mini Kit (QIAGEN GmbH, Hilden, Germany), following the manufacturer’s protocol.

7 PCR reactions
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8 Amplification and sequencing of the 16S rDNA gene of the isolates were performed at a final

9 volume of 50 µl. All PCR reactions contained the following reagents: A 50-100 ng of
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10 chromosomal DNA was added to PCR-MIX - 0.25 units of Vent DNA polymerase (BioLabs) x10
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11 buffer, 2.5 mM of each dNTPs, 200 ng of each primer. Primer FP1 5'-

12 AGAGTTTGATCCTGGCTCAG-3' (27F; Tanner et al., 1998) corresponding to E. coli positions 1-

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13 27, and RP1 5'-CCGGGTTACCTTGTTACGAC-3', corresponding to E. coli nucleotide positions

14 1513 to 1491. DDW was added to a final volume of 50 µl. The PCR reaction was performed on an
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15 automated thermal cycler (Perkin-Elmer/Cetus). The two PCR primers used for the PCR reaction
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16 were universal bacterial primers. Twenty-seven cycles of PCR amplification were conducted. Each
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17 cycle included an initial denaturing step at 94 °C for 45 s followed by a 40 s annealing step at 47 °C

18 and a 1.5 min extension step at 72 °C. The amplification cycles were preceded by a single
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19 denaturing step at 94 °C for 3 min prior to the first cycle and included a final 72 °C extension step

20 for 8 min.

21 ARDRA (Amplified rDNA Restriction Analysis)

22 All PCR products were analyzed by ADRA before sequencing. Isolates with the same pattern of

23 rDNA restriction, were considered as the same isolate. 17.5 µl of the PCR product was taken and

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1 added to ARDRA mix (2 µl 10x buffer and 2 units of restriction enzyme Hae III). The mixture was

2 incubated at 37 oC for 3 hours. The DNA fragments were loaded on 2.5% agarose gel and

3 electrophoresed at 100 V for 60-90 min. and stained with ethidium bromide at final concentration

4 of 0.2 µg ml-1.

5 PCR product purification and sequencing

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6 The PCR products were purified using the QIAquick Spin Kit (QIAGEN GmbH, Hilden,

7 Germany) and sequenced directly using an ABI Prism Big Dye Terminator Cycle Sequencing
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8 Ready Reaction Kit (Applied Biosystems). The PCR products of the DNA templates were

9 sequenced with ABI 377 DNA sequencer (Applied Biosystems) using the FP1 or RP1 primers.
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10 Phylogenetic analysis
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11 The 16S rRNAs encoding gene of the UMCs had been amplified and sequenced. In case of

12 complete sequence identity between the isolated clones, only one representative was included in the
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13 results. Sequence files chromatograms were edited and the final sequences were compared to

14 GenBank sequences using the basic local alignment search tool (BLAST). At the same time,
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15 Chimera detection and a phylogenetic classification of all the clones was done online at Ribosomal
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16 Database Project II, release 9 (RDP II) (http://rdp.cme.msu.edu/index.jsp) in which each of the
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17 resulting 16S rDNA query sequence is assigned to a set of hierarchical taxa and given a member of

18 the genera with the highest probability.

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19 All phylogenetic analyses of the sixty-two nonredundant 16S rDNA gene sequences of the

20 isolated UMCs were performed on the Phylogeny.fr platform and “Advanced mode” (Dereeper et

21 al., 2008): Sequences were aligned with the Muscle program. The phylogenetic tree was

22 reconstructed using the maximum likelihood method implemented in the PhyML program

23 (http://www.phylogeny.fr/version2_cgi/alacarte.cgi). The HKY85 substitution model was selected

16
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1 assuming an estimated proportion of invariant sites and 4 gamma-distributed rate categories to

2 account for rate heterogeneity across sites. The gamma shape parameter was estimated directly

3 from the data. Reliability for an internal branch was assessed using the bootstrapping method (100

4 bootstraps). Graphical representation and edition of the phylogenetic tree were performed with

5 TreeDyn (http://www.treedyn.org/). Sixty-two nonredundant sequences obtained in this study (out

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6 of a total of seventy-six isolates) were deposited in EMBL Nucleotide Sequence Database under

7 accession numbers: AM500653 to AM500714.

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8
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9 Acknowledgments
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10 The authors thank Dr. Scott Kelley (San Diego State University, San Diego, U.S.A), and Dr. Mary

11 Stomberger (Colorado Staete University, Fort Collins, U.S.A.) for critical reading of the
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12 manuscript. We extend special thanks to the Israel Science Foundation (Grant Agreement Number:

13 01-18-00440) for support in initiating this research. Many thanks also to Dr. Gertrud Steinbruck
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14 and colleagues of North-Rhine, Westphalia, Germany, for their support towards completion of this
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15 study. A special thanks to E. Nevo, Institute of Evolution, University of Haifa for facilitating the
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16 research and his cooperation in assisting me in this study.

17 Footnote
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18 NB: Author’s former address - The Regional Center for Research & Development GS, P.O. Box

19 437, Shefa Amr 20200, Israel, an affiliate of Haifa University.

20

21

22

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22 964.

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Soil microcolonies. Ultramicrocells pass through 0.2µm pore-size filter and were grown on Alpha
agar for 21 days at room temperature. Arrows indicates different microcolony types of
ultramicrocells under microscope (magnification of x200).
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254x190mm (96 x 96 DPI)

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Phylogenetic diversity of Proteobacteria and Firmicutes isolates. Phylogenetic relationship of 16S

rRNA gene sequences of cultivable soil ultramicrocells affiliated to Proteobacteria and Firmicutes
phyla. Phylogenetic tree represents the diversity of soil ultramicrocells isolated from soil and their
affiliation to different phyla, with published references sequences from the GenBank database as
indicated by their accession number. Bootstrap values at the main branching points are given. The
scale bar corresponds to 10 base substitutions per 100 nucleotide positions.
200x333mm (600 x 600 DPI)

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Phylogenetic diversity of Actinobacteria. Phylogenetic relationship of 16S rRNA gene sequences of

soil ultramicrocells affiliated to Actinobacteria phylum. Phylogenetic classification of ultramicrocells
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among known freshwater Actinobacterial isolates references. Bootstrap values at main branching
points are given. The analysis was performed on the Phylogeny.fr platform and “Advanced mode”
(Dereeper et al., 2008).
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200x156mm (600 x 600 DPI)

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Table 1.

Soil RDP II Closest relative species by sequence 16S rRNA Accession

Isolates Classification identity sequence number
[Confidence] Identities
ST1-6 Bacillus [63%] Bacterium 8-gw1-3 a 665/665 (100%) AM500655

ST2-7 Brucella [61%] Ochrobactrum pseudogrignonense b 743/744 (99%) AM500665

ST5-4 Micrococcaceae [77%] Uncultured soil bacterium clone 689/797 (86%) AM500680
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c
W4Ba99
ST6-1 Rhodospirillaceae [78%] Phaeospirillum fulvum strain PVC30 866/868 (99%) AM500682
d

Phaeospirillum sp. PVC30 d

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ST3-2 Rhodospirillaceae [69%] 670/677 (98%) AM500671
ST3-5 Azoarcus [45%] Beta proteobacterium 579/583 (99%) AM500673
HTCC379e
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ST7-3 Azoarcus [27%] Beta proteobacterium 571/573 (99%) AM500686
HTCC379 f
ST7-4 Rhizobiales [59%] Phaeospirillum fulvum 444/445 (99%) AM500687
isolate S3 e
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WST1-4 Pseudomonas [79%] Pseudomonas sp. S7-42 800/823 (97%) AM500690

g

WST3-1 Rhizobiales [35%] Phaeospirillum fulvum e 819/828 (98%) AM500695

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WST3-4 Brevundimonas [76%] Uncultured clone Alpha 887/893 (99%) AM500698

proteobacteriumSM2A11 h
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WST5-5 Arthrobacter [41%] Arthrobacter dextranolyticus 603/624 (96%) AM500705

i

WST6-1 Sterolibacterium [48%] Uncultured beta proteobacterium- 939/943 (99%) AM500706

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bacterium HOClCi25 k
WST6-5 Rhodospirillaceae [59%] Phaeospirillum fulvum and 879/892 (98%) AM500710
Phaeospirillum sp. PVC30 d
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WST7-1 Brevundimonas [49%] Uncultured alpha proteobacterium j 774/785 (98%) AM500711

WST7-3 Sphingomonadales [26%] Uncultured bacterium l 781/822 (94%) AM500713

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a
Tiquia, et al., 2008; b Kaempfer, et al., 2007; c Lueders, et al., 2006; d Silbaq, 2009;e Baek, et

al., 2003; f Kyselkova et al., 2008; g Frapolli, et al., 2007; h Bonheyo, et al., 2003;; i Hatada et

al., 2004; k Williams, et al., 2004; l Noguera, et al., 2008.

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Table 1. Cultivable soil ultramicrocells below the confidence threshold (80%) of classification.

Their phylogenetic affiliations had been determined on the bases of identities of 16S rRNA

encoding gene sequences. The phylogenetic affiliations and classification done by RDP II and

closest relative species 16S rRNA encoding gene had been obtained by BLAST NCBI databank.
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