Environment  Health  Techniques

Streptomyces and Lentzea strains from soils in Argentina 1

Research Paper
Isolation and characterization of indigenous Streptomyces
and Lentzea strains from soils containing boron compounds
in Argentina

Norma Beatriz Moraga1,2, Hugo Ramiro Poma1, María Julia Amoroso3,4 and
Verónica Beatriz Rajal1,2,5

1
Instituto de Investigaciones para la Industria Química (INIQUI), CONICET-UNSa, Salta, Argentina
2
Facultad de Ingeniería, Universidad Nacional de Salta, Salta, Argentina
3
Planta de Procesos Industriales y Microbiológicos (PROIMI), CONICET, Tucumán, Argentina
4
Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Tucumán, Argentina
5
Fogarty International Center, University of California in Davis, Davis, CA, USA

The Salta Province – in the northwest of Argentina – is the main worldwide producer of
hydroboracite and leads in exports of boron mineral and its derivatives in Latin America. In
addition to the natural presence of boron compounds in the soils, there are others contaminated
due to the boron mining industry. Although some bacteria are known to require boron for their
growth or to be capable of storing boron, no studies have been published about Streptomyces or
Lentzea genera’s capacity to tolerate high boron concentrations, or about their metabolic capacities
in boron contaminated environments. The results of this research show the isolation and molecular
characterization of eight strains belonging to the actinobacteria phylum collected from different
soils contaminated with high boron concentration in Salta state. The boron tolerance assays, which
show that three of the strains were able to tolerate up 60–80 mM boron, demonstrate the potential
capability of this group of bacteria to grow and maybe to remove boron from the environment.
They appear to be promising, considering that these microorganisms are infrequent pathogens, are
metabolically versatile and many Streptomyces can synthesize boron containing metabolites.

Abbreviations: ADH – arginine dihydrolase; ADI – adipic acid; ARA – L-arabinose; CAP – capric acid;
CIT – trisodium citrate; ESC – esculin; GEL – gelatin; GLF – D-glucose fermentation; GLU – D-glucose; GNT –
potassium gluconate; MAL – D-maltose; MAN – D-mannitol; MIC – minimum inhibitory concentration; MLT –
malic acid; MM – minimal medium; MNE – D-mannose; NA – nutrient agar; NAG – N-acetyl-glucosamine; NB
– nutrient broth; NO3
– nitrate reduction into nitrites; PAC – phenylacetic acid; PCR – polymerase chain
reaction; PNPG – 4-nitrofenil-bD-galactopiranoside; SCA – starch casein agar; SI – sequence identities;
TRP – L-tryptophane; URE – urea

Keywords: Actinobacteria / Boron-tolerance / Streptomyces, Lentzea / Boron contaminated soils

Received: November 15, 2012; accepted: January 19, 2013

DOI 10.1002/jobm.201200714

Introduction that some animals and unicellular eukaryotes including

humans [3] also require boron, but the concentrations
Boron (B) is a metalloid, whose importance as a nutrient needed vary widely according to the species. Cyanide
for some plant growth has long been known [1]. In the bacteria [4] and Bacillus boroniphilus sp. nov. [5] are the only
last decade, its essentiality in the structure of plants and bacteria where boron has been determined to be essential
cell walls was clearly demonstrated [2]. Evidence suggests for growing. Boron has been reported to stimulate
nitrogen fixation mechanisms in Azotobacter [6] and also
intervenes in mediation mechanisms of Quorum Sensing
Correspondence: Verónica Beatriz Rajal, Instituto de Investigaciones
para la Industria Química (INIQUI), CONICET-UNSa, Avda. Bolivia 5150, in Vibrio harveyi [7].
Salta 4400, Argentina Boron is toxic for cells when found in greater than
E-mail: [email protected]
Phone: þ54 387 4255410
threshold values. Due to the toxic effect on some
Fax: þ54 387 4251006 microorganisms, boron is used in the treatment of

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2 Norma Beatriz Moraga et al.

vulvovaginitis caused by Candida and Saccharomyces [8], as (1997–2000), revealed a high content of soluble boron and
a food preservative [9] and as a cockroach pesticide [10]. salinity in soil, thus showing that the land was not
There are many metabolites containing boron synthe- suitable for human, farming, and domestic animal
sized by bacteria, like aplasmomycins [11], tartorlon A breeding activities [16].
and B [12], boromycines [13] and borophycin [14]. Since Anthropogenic boron contamination in Salta has three
the 19th century this element has been used on modern essential sources: (1) borate extraction, (2) borate
lifestyles. There is over 200 boron bearing minerals, but transportation, and (3) industrialization/refining process.
boron industrial exploitation is feasible for only five of The current treatment used to reduce the environmental
them, which are found in very few places in the world. contamination produced by the industrialization of
Borate deposits are located in the Puna in the central boron products involve direct waste disposal in situ,
Andes. This region encompasses about 100,000 km2 in recycling of processes streams, waste reutilization, and
the northwestern Argentine, where Salta Province is the re-engineering of operations and processes [17].
located (Fig. 1). This province is ranked as the first Latin Few microorganisms have been studied for their boron
American borate producer, the first world producer of tolerance: Bacillus boroniphilus sp. nov. [5], Gracilibacillus
hydroboracite (116,099 tons in 2010), and the third world boraciitolerans sp. nov. [18], Lysinibacillus boronitolerans [19],
producer of borates [15]. Boron recovery may be the Lysinibacillus parviboronicapiens [20], Bacillus, Rhodococcus
longest-standing mining activity in Argentina. This sp., Rhodococcus erythropolis, and Microbacterium folio-
intense industrial development has an important envi- rum [21] as Gram-positive bacteria and Chimaereicella
ronmental impact, fundamentally on water and soils boritolerans sp. nov. [22] and Variovorax boronicumulans sp.
which are hardly recoverable. A case is Baradero, where a nov. [23] as Gram-negative, among others Pseudomonas,
firm worked until 1993 producing borax and boric acid, Shewanella, and Variovorax [21] less tolerant.
leaving behind an important environmental burden. Actinobacteria are the most abundant group of
This area, (46,620 m2) is surrounded by the densely bacteria in soils (90%) and show primary biodegradative
inhabited neighborhoods. Studies conducted in the area activity, secreting extra cellular enzymes to metabolize

Figure 1. Boron mineral deposits and anthropogenically contaminated locations in the Province of Salta, Argentina. The boron anthropogenically
contaminated area, Baradero, as a zoom.

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 Streptomyces and Lentzea strains from soils in Argentina 3

recalcitrant molecules [24]. Amongst actinobacteria in let stand for 30 min for the solids to settle. Three
soil, there are examples of different strategies, from successive tenfold dilutions from the previous solution
cycles of rapid proliferation and sporulation to mainte- were poured in Petri dishes containing a general
nance the population by prolonged slow growth and medium, nutrient agar (NA; Britania), and a specific
scavenging. This versatility gives them a great deal of for flora present in soils, starch casein agar (SCA; 1%
potential for biotechnological applications [25]. starch, 0.1% hydrolyzed casein, 0.05% K2HPO4, 1.5%
The aim of this work was to characterize boron- agar, pH 7  0.5) and incubated at 30 °C for 7 d. The
tolerant actinobacteria from soils naturally containing colonies obtained were isolated in SCA. Pure cultures
high concentrations of boron and from those anthro- were stored at 4 °C in SCA tubes until used.
pogenically contaminated. To the best of our know-
ledge, there is no specific published information Characterization of bacteria
about Streptomyces or Lentzea tolerant to high boron Only 50 out of the 127 cultures initially isolated were
concentrations. characterized by their ability to grow at 30 °C in nutrient
broth (NB) with NaCl added to final concentrations of 0.5,
1, 2.5, 5, 7.5, 10, 15, and 20% w/v; pH was adjusted to
Materials and methods 7  0.2 prior to sterilization. Bacterial growth with and
without NaCl was carried out to analyze if they were
Soil sampling and physicochemical characterization halotolerant [30, 31]. In order to know their ability to
Three locations from the Province of Salta were selected. grow at different temperatures, 0.1% v/v inocula of fresh
Baradero, located in an urban area, presents anthropo- cultures in NB of the 50 isolates were cultured in NB at 4,
genic contamination with boron compounds, Tincalayu 15, and 30 °C, for 24 and 48 h.
and Animaná, which are naturally high in boron content Fresh cultures (NB, 24 h) were Gram stained. Salmonella
(Fig. 1). The samples from Tincalayu were obtained sp. and Staphylococcus aureus were used as controls.
randomly from three different sectors (A, B, and C) of an The identification of the Gram negative isolates (33)
exploitation mine. The third site, Animaná (Fig. 1), was was carried out using the commercial kit API® 20 NE
also sampled randomly from three different sectors of (BioMérieux, France). The other 17 isolated strains were
the river side (D, E, and F). In each case 10 g of soil from also tested to elucidate and/or confirm some information
each sector were taken from the 0–15 cm deep surface related to their capacity of assimilating and degradating
layer, considering it to be the depth, where most of some compounds. This standardized system combines
microorganisms should be concentrated. eight conventional assays and 12 assimilation tests of
A systematic sampling of Baradero soil was conducted different carbohydrates.
on a rectangular grid [26] (zoom in Fig. 1), following the
recommendations for homogeneous areas below five Boron tolerance studies
hectares [27]. The total area of 46,620 m2 was divided Eight actinobacteria isolates were selected in order to
into nine 70 m  74 m sectors of 5180 m2, and each of further study boron-tolerance by different methods. A
them was subdivided into five equidistant points to take qualitative screening assay was carried out in Petri dishes
the 10 g samples. Thus, 50 g of soil was obtained per containing SCA. Rectangular troughs were cut in the
sector in only three (S1, S2, S3) of the nine sectors center of plates and filled with 600 ml of sterile boric acid
according to Álvarez-Manilla et al. (2002) [28] for this type solutions. Five different concentrations were tested: 80,
of analysis. 150, 340, 390, and 440 mM. Isolates were inoculated by
The soil samples from Baradero, Tincalayu, and streaking 3 cm with a sterile stick perpendicularly to the
Animaná were analyzed for water content, organic troughs. Microbial growth was used as the qualitative
matter content, pH, B2O3, and total boron [29]. The parameter of boron tolerance. Three categories depend-
concentrations of boron were measured and those values ing on the length of growth were defined: limited (1 cm),
were used later as a reference to study the tolerance. partial (2 cm), and total (3 cm). The inoculated plates
were incubated at 30 °C for 7 d [32].
Microorganisms: isolation and preservation For semi-quantitative screening, 50 ml of seven H3BO3
Each soil sample was placed on a 3 cm thick plastic and solutions (80, 150, 210, 270, 340, 390, and 440 mM) were
all of the elements larger than 2 cm were hand picked. used to fill the wells of Petri dishes containing SCA
The sample was quartered; 10 g were taken and added to medium previously inoculated with 200 ml of a spore
90 ml of 1% sodium hexametaphosphate sterile solution, suspension (4  106 CFU ml
1) of the strain to be tested.
used as extracting agent. It was vortexed 10 min and then Sterile water was used as a control. After 24 h incubation

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4 Norma Beatriz Moraga et al.

at 30 °C the growth inhibition diameter was measured. resis in a 2% w/v agarose gel, stained with ethidium
The strains were considered tolerant to that boron bromide, and then visualized using the program Kodak-
concentration when the diameter of inhibition was less EDAS. The 100 base pair DNA Ladder (Promega) was used
than 7 mm, and non-tolerant when it was higher, as a molecular weight marker. The amplified fragments
according to an arbitrary criteria [32]. were shipped to Korea to be purified and sequenced.
The minimum inhibitory concentration (MIC) was also
determined as an indication of boron tolerance. Different Sequencing of 16S rDNA and phylogenetic analyses
concentrations of boric acid solutions (5, 10, 15, 20, 30, DNA sequencing on both strands was performed by
40, 50, 60, 70, and 80 mM) in NB were inoculated with a Macrogen® Korea (Macrogen, Seoul, Korea) on an ABI
standardized number of cells (from a spore suspension) 3730 XL automatic DNA sequencer, using the same
grown in minimal medium (MM, composition in g/L: primers as used for PCR amplification.
glucose, 10.0; L-asparagine, 0.5; K2HPO4, 0.5; Sequences belonging to the same species or closely
MgSO4.7H2O, 0.20; FeSO4.7H2O, 0.01; pH 7) at 30 °C, related species were selected by comparing 16S rDNA
250 rpm, for 6 h. The tubes were then incubated for 72 h sequences of isolated strains with the database of
at 30 °C. The MIC was determined as the lowest nucleotides sequences deposited at the NCBI web server
concentration of boric acid preventing growth. (www.ncbi.nlm.nih.gov), through Basic Local Alignment
To prepare the spore suspension, 10 ml of sterile water Tool (BLAST) program (http://www.ncbi.nlm.nih.gov/
were added to three Petri dishes with fresh cultures blast/) [34]. With this procedure the percentages of
grown in SCA at 30 °C for 5 d. The agar was carefully sequence identities (SI) among the strains were obtained.
scraped with a loop to release the spores. The spore The sequences of 16S rDNA were aligned using
suspension obtained was filtered through a compacted CLUSTALW software [35]. The alignments were used to
cotton bed and the filtered was then centrifuged at calculate phylogenetic distances using the Kimura two-
5400 rpm. The supernatant was discarded and the pellet parameter model [36]. The phylogenetic tree for the data
resuspended in 1 ml of sterile water. The spore sets was inferred from the neighbor-joining method [37]
concentration was determined by colony count. using the software MEGA version 5 [38]. In the
phylogenetic tree, defined clusters for phylogenetic
Isolation of chromosomal DNA similarity coefficients were compared to the classifica-
DNA from the eight selected strains was isolated using a tions proposed by Williams [39] for the Streptomyces group
commercial kit (DNeasy Blood & Tissue Kit from in the Bergey’s Manual of Determinative Bacteriology
QIAGEN), with some variations in the conditions (1994).
described by the manufacturer’s protocol. Briefly,
biomass as spore suspension was collected from 5-d pure
cultures from Petri dishes of SCA. Proteinase K (Promega) Results
was added to achieve a final 70 mg ml
1 concentration
and the mixture was incubated at 55 °C for 1 h. Then, Soil physicochemical characterization
560 ml of Buffer AL was added and the resulting solution The physicochemical characterization of soils obtained at
was incubated at 85 °C for 15 min. DNA was precipitated S1, S2, S3 from Baradero, Tincalayu (A, B, and C), and
by adding 560 ml of ethanol and mixing 5 s. Finally, to Animaná (D, E, and F) were determined (Table 1). The soil
avoid the inhibition of enzymatic reactions, 8 ml of in the central sector of Baradero (S2) was very compact,
RNase H was added and the mixture was incubated at 37 ° sandy, and dry but not as much so as that in sector S1.
C for 20 min. Sector S3 differed from both of them as it was more
humid and sandy with incipient vegetation. The sample
PCR amplification of 16S rDNA obtained in this latter sector was used as control of low
Total genomic DNA from the eight strains was used as the boron contamination from Baradero. As expected,
template for polymerase chain reaction (PCR) amplifica- samples from the exploitation mine in Tincalayu, showed
tions. The amplifications were performed in 25 ml the greater boron content values, especially in sectors A
reaction volume using universal oligonucleotide primers and C. Animaná samples seemed rather uniform.
(Sigma): 63 Forward (50 -CAGGCCTAACACATGCAAGTC-30 )
and 1389 Reverse (50 -ACGGGCGGTGTGTACAAG-30 ) [33]. Microorganisms: isolation and characterization
PCR reactions of 16S rDNA were performed in an From the 127 strains isolated initially, 50 isolates
automated thermal cycler GeneAmp® PCR 9600 (Applied were selected so as to leave behind unicellular and
Biosystems). PCR products were detected by electropho- filamentous fungi. From the isolates, 33 were identified

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 Streptomyces and Lentzea strains from soils in Argentina 5

Table 1. Physicochemical characterization of soils from Baradero (sectors S1, S2, and S3), Tincalayu (A, B, and C), and Animaná
(sectors D, E, and F).

Baradero Tincalayu Animaná

Parameters S1 S2 S3 A B C D E F
Organic matter content (%) 2.36 3.44 2.78 3.58 3.22 3.71 0.25 1.18 1.06
Humidity at 110 °C (%) 5.49 5.83 6.06 12.53 2.2 7.61 0.20 0.68 0.78
pH 8.22 8.72 7.28 ND ND ND 8.06 8.23 8.13
pH in KCl 8.25 8.80 7.03 9.13 9.03 9.00 7.90 7.85 7.83
[B2O3] (g per 100 g soil) 1.08 2.72 0.56 10.38 0.84 5.24 2.21 2.26 2.37
Btotal (g per 100 g soil) 0.34 0.84 0.17 3.20 0.26 1.62 0.69 0.70 0.74
ND, not determined.

as Gram-negative rods and most of them were later in any culture before 48 h of incubation at 4 °C.
classified as Pseudomonas. The 17 remaining isolates were Conversely, all the strains grew in 48 h at 30 °C, even
Gram positive and eight of them seemed to belong to the with 5% w/v NaCl. Strain 128 was able to grow until 7.5%
actinobacteria phylum, based upon macro and micro- of NaCl at 72 h, while strain 053 could grow until 10%
scopic morphology, mycelium color, spore formation, NaCl at 48 h.
colony consistency, and the distinctive “earthy” odor Even though the kit API® 20 NE system (BioMérieux)
because of the geosmin present [40]. could not be used to identify the Gram positive micro-
Subsequent studies were only performed with the organisms, the results provided important physiological
eight aforementioned strains isolated: 048 and 050 from and biochemical information for the eight selected
sector S2, 002 from S1, 053 from S3, 128 and 130 from A, strains (Table 4). Conventional tests for NO3
(nitrate
and 132 and 133 from sectors E and F, respectively, in reduction into nitrites), TRP (indol formation from
Animaná. Another strain, 129, was also isolated and even L-tryptophan), GLF (D-glucose fermentation), and ADH
sequenced from sector C in Tincalayu soil. However, it (presence of enzymes such as arginine dihydrolase) were
was not considered for further studies as it was negative, as expected for actinobacteria. All the strains
determined to be Sphingomonas sp. (HQ538728), which were ESC (hydrolysis of esculin) positive. Regarding the
does not belong to the actinobacteria phylum. assimilation tests, all the strains used D-glucose (GLU) and
Even though the microorganisms were able to grow conversely none of them used capric acid (CAP). Strains
properly in NB, later cultures were carried on only with 002 and 128 were the only ones that did not use
SCA because distinctive features (Table 2) were observed trisodium citrate (CIT). Also strain 128 was the only strain
for each colony facilitating their identification and purity that did not use D-mannose (MNE).
control. All of the strains produced extensively branch-
ing, primary mycelium transformed during the life cycle Boron tolerance screening
into aerial mycelium bearing and to typical spore. Four The tolerance to boron (20–440 mM) was analyzed for
strains (002, 048, 128, and 133) showed diffusible the eight actinobacteria. Initially, the qualitative
pigments in SCA. screening showed that the microorganisms were able
Growth in NB was assessed at three different temper- to grow in all of the boron concentrations tested,
atures (4, 15, and 30 °C) and eight different NaCl although not always closer to the trough. The semi-
concentrations at 30 °C (Table 3). No growth was detected quantitative assay was then carried out in SCA. An

Table 2. Macroscopic characteristics observed for the isolated strains when grown in starch casein agar at 30 °C.

Strains
Property 002 048 050 053 128 130 132 133
Mycelium color R Y W Y R W W R
Spore color W G G W W G G G
Colony appearance RU RU RU SW RU RU RU RU
Pigmentation þ þ

þ

þ
R, red; Y, yellow; W, white; G, gray; þ, positive;
, negative; RU, Rugose; SW, smooth warty.

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6 Norma Beatriz Moraga et al.

Table 3. Culture of the isolated strains in nutrient broth with and without NaCl at different temperatures after 24 h.

Without NaCl With NaCl in different % w/v, at 30°C

a
Strain 4 °C 15 °C 30 °C 0.5% 1% 2.5% 5% 7.5% 10% 15% 20%
002

(
) b
þ þ þ þ þ




048 þ
(þ)b þ þ þ
(þ)b
(þ)b




050 þ þ þ þ þ þ þ




053 þ þ þ þ þ þ þ
(þ)b
(þ)b


128
þ þ þ þ þ þ
(þ)c



130 þ þ þ þ þ þ þ




132

(
)b
(
)b þ þ þ þ




133 þ
(þ)b þ þ þ þ þ




þ, positive;
, negative.
a
Result correspond to 4 days.
b
Results in parenthesis correspond to 48 h.
c
Results in parenthesis correspond to 72 h.

Table 4. a. Assimilation tests of different carbohydrates with API® 20 NE (BioMérieux; GLU, D-glucose; ARA, L-arabinose; MNE,
D-mannose; MAN, manitol; NAG, N-acetil-glucosamine; MAL, D-Maltose; GNT, potassium gluconate; CAP, capric acid; ADI, adipic acid;
MLT, malic acid; CIT, trisodium citrate; PAC, phenil acetic acid) and b. Conventional tests with API® 20 NE (BioMérieux; NO3, nitrate
reduction; TRP, indol formation from L-triptophane; GLF, D-glucose fermentation; ADH, presence of arginine dihydrolase enzyme; URE,
presence of urease enzyme; ESC, esculine hydrolysis ferric citrate; GEL, gelatine hydrolysis; PNPG, 4-nitrofenil-bD-galactopiranoside
hydrolysis).

a. Carbohydrates assimilationa b. Conventional testsb

Strain ARA MNE MAN NAG MAL GNT ADI MLT CIT PAC URE GEL PNPG
002 þ þ

(þ) þ (þ) (þ)



þ
048 (þ) þ þ (þ) þ þ
(þ) þ (þ) þ
þ
050 þ þ þ þ þ (þ)
þ þ (þ)
þ

053 þ þ þ þ þ þ
þ þ þ
þ þ
128




þ





(þ)
130
þ





þ

þ (þ)
132
þ (þ)
þ þ (þ) þ þ

þ

133 (þ) þ þ þ


þ þ
(þ)
þ
þ, positive;
, negative; (þ), weakly positive.
a
All the strains used GLU and none of them used CAP.
b
Tests for NO3, TRP, GLF, and ADH were all negative, as expected for actinobacteria. All the strains were ESC positive.

inhibition zone of 7 mm in diameter was arbitrarily Phylogenetic analyses

designated as the criterion to determine boron resis- Only one product of amplification was obtained by PCR,
tance of the tested strains. All isolated strains were able as verified by electrophoresis in agarose gel. The
to grow at 80 mM, while only 50 and 13% of them grew amplified fragments were sequenced at Macrogen® and
at 210 and 440 mM, respectively. According to the the results obtained were used to elucidate the taxonomic
inhibition criteria adopted, all the strains were B- position of the eight isolated strains as explained before.
tolerant up to 270 mM (Fig. 2). Interestingly, strain 048 The 16S rDNA sequences obtained for isolated strains
was able to grow without any inhibition over the entire were compared to 49 sequences belonging to the same or
range of boric acid concentrations used, while strain closely related species, available through the public
128 showed tolerance even to 440 mM of B. Higher databases. The sequences were uploaded to NCBI
concentrations of boric acid were not used due to GenBank and their accession numbers are given in
solubility limitations. parentheses (Fig. 3).
The MICs determined after 72 h were 20 mM for 002 The generated phylogenetic tree was divided into six
and 132, 30 mM for 130, 40 mM for 133, 50 mM for major clusters on the basis of their evolutionary
050, 60 mM for 053, 70 mM for 128, and 80 mM distances calculated through the neighbor-joining
for 048. method. The first cluster was further divided into two

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 Streptomyces and Lentzea strains from soils in Argentina 7

Figure 2. Semi-quantitative assay of boron tolerance measured as

the diameter of the inhibition zone. The horizontal line indicates the
arbitrary limit used to define boron tolerant (below) and non-tolerant
(above) strains.

sub-clusters. In sub-cluster Ia, the isolated strain 048

(HQ538723) was closely associated to S. achromogenes
(99.4%) and to S. nogalater while strain 133 (HQ538729)
was associated to S. griseosporeus (98.5%). In sub-cluster Ib,
the isolated strain 053 (HQ538724) was closely associated
to S. albogriseolus (100%) and to S. thermoluteus and the
isolated strain 002 (HQ538731) was closely associated to S.
iakyrus (100%). Most of the species located in cluster I
were incorporated in the largest cluster-group A, and
widely distributed in different clusters (19, 23, 12, 18, 15,
16, etc.) according to the proposed classification. In
cluster II, the isolated strain 050 (HQ538725) was closely
associated to S. fradiae (100%). The species located in
cluster II were incorporated in the cluster-group G, and
located only in the cluster 68. In cluster III, the isolated
strain 130 (HQ538727) was closely associated to S.
polychromogenes (100%), the species located in cluster III
were incorporated in the cluster-group F and only in the
cluster 61. In cluster IV, the isolated strain 128
(HQ538726) was closely associated to S. lincolnensis
(99.8%), the species located in the cluster IV are
incorporated in the cluster-group A and located in
clusters 18 and 19. In cluster V, the isolated strain 132
(HQ538730) was associated to species of the genus Lentzea
(97.6 - 97.8%). In cluster VI, the isolated strain 129
(HQ538728) was closely associated to with Sphingomonas
melonis (99.1%).

Figure 3. Phylogenetic tree of actinobacteria species inferred by

the neighbor-joining method [37], using Kimura’s evolutionary
distance [36] and based on the comparison of nearly complete 16S
rDNA sequence of 49 organisms. Accession numbers of 16S rDNA
sequences are given in parentheses. The label • shows the isolated
strains in this study. Figure 3.

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8 Norma Beatriz Moraga et al.

Discussion 128, both isolated from heavily contaminated soils, had

even higher B-tolerance, up to concentrations of 440 mM
The eight strains selected belong to the actinobacteria boron. The level of B-tolerance is similar to that reported
phylum, a physiologically diverse bacterial group that by Ahmed et al. [5, 18] for other Gram-positive bacteria.
has the soil as their natural habitat. Previous studies have Regarding the MIC, the strains 048, 053, and 128 showed
demonstrated that Gram-positive bacteria with high the highest B-tolerance (80, 60, and 70 mM, respectively).
G þ C content, i.e., actinomycetes, dominated the Miwa and Fujiwara [21] reported some microorganisms,
community in non-contaminated soils as well as including three actinobacteria, as high boron tolerant
contaminated soils [41]. after they showed ability to grow at concentrations of
Actinobacteria are known for their ability to produce 0.1 mM of boric acid in liquid media. Taking that, the
antibiotics and metabolites, and to remove heavy metals. strains isolated look very promising, since they are able
From the eight isolates obtained in this work, strain 132 to grow in the presence of at least 600 times higher
from Animaná was identified as Lentzea sp., while the concentrations than the reported ones.
other seven strains were Streptomyces sp. (Fig. 3) according The sensibility of the studied strains was considerably
to the phylogenetic tree based on 16S rDNA sequences. higher in liquid medium. As Majzlik et al. [50] suggest the
Some of the strains can be associated with contaminated differences in values of MIC on agar and liquid medium
soils, i.e., Lentzea with heavy-metal contaminated are likely to be caused by completely different environ-
soils [42]. Others were reported for specific abilities to ments. These authors propose that in liquid medium,
degrade some compounds, such as S. iakyrus and bacteria are able to create a suitable environment around
parathion [43] and S. albogriseolus and latex [44], to the cells (synthesis of peptides, proteins that are released
produce antibiotics such as S. achromogenes and strepto- into their immediate surroundings) and internal defense
zotocin, rubradirin [45], and tomaymycin [46], S. mechanisms are altered even at low concentrations.
ambofaciens and spiramycin [47], S. lincolnensis and Same authors pose another feasible hypothesis based on
lyncomicine [48], and to produce enzymes, such as S. the nature of agar as a complex organic molecule that
fradie with keratolytic activity [49]. However to the best of contains binding sites that can react or form complex
our knowledge there are no reports about their capacity with boric acid in this case, thereby reducing its effective
to grow in soils or media with high boron concentrations. concentration. We also think that diffusive effects that
Only few microorganisms have been reported previ- are involved in the solid medium may cause this
ously as having boron tolerance. Three of them were difference. In liquid medium cells are in intimate contact
actinobacteria. For Microbacterium foliorum intracellular with boric acid while in agar not.
boron concentration was less than 0.4 nmol per gram of Finally, strains 048 and 128 look promising to deal
dry weight and less than 0.7 nmol per gram of dry weight with environmental contamination with boron. Whether
for both Rhodococcus sp. and Rhodococcus erythropolis [21]. the bacteria are using the boron for the production of
Among others, Gram-positive bacteria Lysinibacillus parvi- some metabolite or just capturing and accumulating it
boronicapiens, can tolerate up to 6% w/v NaCl, but only internally will need to be elucidated through further
50 mM boron [20]. Lysinibacillus boronitolerans can tolerate studies evaluating the boron mass balance during
up to 150 mM boron [19], while Bacillus boroniphilus sp. growth.
nov. [5] and Gracilibacillus boraciitolerans sp. nov. [18] can
tolerate over 450 mM of boric acid. G. boraciitoloerans
actually requires boron to grow. Conclusions
Gram-negative bacteria are also able to tolerate high
boron concentration such as Chimaereicella boritolerans sp. Eight strains of actinobacteria isolated from soils
nov. up to 300 mM B [22] and Variovorax boronicumulans sp. obtained in the Province of Salta in Argentina showed
nov. could accumulate boron intracellular [23]. the ability to grow in the presence of boron compounds.
The results obtained in this work from the qualitative Three of them, showed the greatest tolerance to the
screening about boron tolerance were neither clear nor highest concentrations analyzed. This is the first study
consistent. The growth was subjected to a great about Streptomyces and Lentzea genera that are tolerant to,
uncertainty since the true boron concentration at or able to grow in, high boron concentrations.
different points in the agar gel was unknown. The Further studies will be conducted to determine the
semi-quantitative results instead showed that all the feasibility of using these boron-tolerant strains for
isolates were able to grow in concentrations up to biotechnological applications. In that case the fate of
270 mM. They also evidenced that the strains 048 and the boron will have to be studied and elucidated.

ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim www.jbm-journal.com J. Basic Microbiol. 2013, 00, 1–10
 Streptomyces and Lentzea strains from soils in Argentina 9

Acknowledgments [12] Irschik, H., Schummer, D., Gerth, K., Höfle, G. et al., 1995.
The tartrolons, new boron-containing antibiotics from a
myxobacterium, Sorangium cellulosum. J. Antibiot., 48(1),
This project was partially supported by National Institute 26–30.
of Health Grant # D43 TW005718 funded by the Fogarty [13] Kohno, J., Kawahata, T., Otake, T., Morimoto, M. et al.,
International Center and the National Institute of 1996. Boromycin, an anti-HIV antibiotic. Biosci. Biothech-
Environmental Health Sciences. It was also funded by nol. Biochem., 60(6), 1036–1037.
the Consejo de Investigaciones de la Universidad Nacional [14] Arai, M., Koizumi, Y., Sato, H., Kawabe, T. et al., 2004.
de Salta (Salta, Argentina) through the research projects Boromycin abrogates bleomycin-induced G2 checkpoint. J.
Antibiot., 57, 662–668.
N° 1669 and 1798. Norma Moraga and Ramiro Poma are
recipients of graduate fellowships from Consejo Nacional [15] Secretaría de Minería de Salta, Ministerio de Desarrollo
Económico, 2011. Crisol Ediciones, Salta.
de Investigaciones Científicas y Técnicas. The authors
[16] PNUMA: Programa de las Naciones Unidas para el Medio
would like to thank Dr. Jerold Last for his help with the Ambiente Oficina Regional para América Latina y el Caribe,
English corrections and Oscar Gamboni for his technical 2004. Resumen de Prensa. Boletín del 29 de Septiembre de
assistance. 2004. 5–6.
[17] Flores, H.R., 2004. El Beneficio de los Boratos. Historia,
Minerales, Yacimientos, Usos, Tratamiento, Refinación,
Propiedades, Contaminación, Análisis Químico. Crisol
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