Isolation and characterization of novel halophilic
prokaryotes from the Dead Sea and from
experimental mesocosms containing mixtures of
Dead Sea and Red Sea water
M. Sc. Thesis
Submitted to the Inter-Faculty Graduate
Biotechnology Program
of the Hebrew University of Jerusalem
for the degree of
"Master of Science in Biotechnology"
Natalie Vaisman
1.12.2009
This work was supervised by Prof. Aharon Oren
Department of Plant and Environmental Sciences
The Institute of Life Sciences
The Hebrew University of Jerusalem
I’d like to thank my adviser, Prof. Aharon Oren, for teaching, advising, helping,
supporting and encouraging. You were the most important piece not only in this work
but also in my Aliah process and adaption Israel. I’ll be forever grateful;
Thanks to my parents, who proved that distance is inexistent when you love someone.
You were the most present people in my life, despite the ocean between us;
Thanks to my friends and family in Brazil, who kept being important and present in my
life as they always used to be; and to the creators of internet, skype, facebook, orkut,
etc., who made it possible.
To the friends I made in Israel, who were almost as a family. I think that it already says
what you mean to me. I won’t write names so I won’t forget anyone.
To my lab colleagues: Lily Mana, for the long talks, laughs, recipes, and, of course, help
with the experiments; Danny Ionescu, for teaching me molecular biology and diving;
Rahel Elevi Bardavid and Yafit Sorek, who were more than simple lab colleagues; to
Atalya Moncaz, who recently joined the group but already helped me with the abstract
in Hebrew;
To the Christian Organizations, who fed me for 2 years;
To the Yael Piton Fund for financial support;
To the State of Israel, which gave me the opportunity to live this amazing experience.
Am Israel Chai!
ABSTRACT
The Dead Sea is rapidly drying out. During the 20th century, its level has dropped more than
20 m, and during the past decade, the level has decreased approximately 1 m per year on the
average. The lake is now supersaturated with NaCl, and massive precipitation of halite from
the water column has led to a decrease in the Na+ concentration, concomitant with an increase
in Mg2+. The lake’s chemistry became thus more and more dominated by divalent cations,
making the lake an ever more extreme environment for microbial life. However, even under
these harsh conditions the lake has been proven to be the habitat of several microorganisms,
including members of the domain Bacteria, unicellular algae, fungi, viruses, and especially
Archaea.
To counteract the drying out of the lake and to restore the water level to a desired
elevation, the construction of a water carrier between the Red Sea and the Dead Sea (the ‘Peace
Conduit’) has been proposed. Simulation experiments are being performed on the grounds of
the Dead Sea Works at Sedom, Israel, to provide information on the microbiological properties
of the Dead Sea when the ‘Peace Conduit’ plans will be implemented and massive quantities of
Red Sea water will enter the Dead Sea and lower the salinity of the upper water layers.
Samples were collected from the Dead Sea and from the experimental ponds at Sedom
for the isolation and characterization of new organisms. Two strains belonging to the genus
Halorhabdus (Archaea) were isolated from the Dead Sea and were partially characterized. The
finding of this isolates confirm the results of Bodaker et al. (2009), who detected the presence
of Halorhabdus in the lake through a molecular approach.
A strain isolated from an experimental mesocosm containing 80% Dead Sea water and
20% Red Sea water was characterized as a novel organism, Salisaeta longa. Its closest relative
is the rod-shaped red colored Salinibacter ruber, a member of the Bacteroidetes branch of the
Bacteria, but physiologically resembling the Halobacteriales in many properties. Salisaeta
longa consists of very long (15-30 µm) rods and is less halophilic than Salinibacter ruber,
although it is highly magnesium-tolerant. Both Salisaeta and Salinibacter accumulate
intracellular K+ in high concentrations as haloadaptation mechanism.
Microscopic examination of pond samples containing Dead Sea-Red Sea water ratios
different from that where S. longa was originally isolated showed very long rods similar to S.
longa cells. Samples were plated on S. longa optimum medium, and five strains which
presented typical S. longa colonies and cells were isolated. The 16S RNA gene of the strains
showed 99% similarity with S. longa.
A pair of primers was designed to specifically amplify part of the 16S rRNA gene of
Salisaeta. The targeted fragment could be amplified from S. longa S4-4T, from the five isolates
previously mentioned, and also from DNA extracted directly from two of the experimental
ponds. No amplification was obtained when DNA from Salinibacter ruber, Escherichia coli or
other environmental samples were used as templates in the PCR reaction. The detection limit
of the developed method was 104 Salisaeta cells per sample.
Denaturing gradient gel electrophoresis was performed with partial 16S rRNA genes of
Archaea and of Bacteria amplified from the experimental ponds containing different Dead SeaRed Sea water ratios (from 80 to 40% Dead Sea water), in order to analyze how it affects the
microbial community of the ponds. The archaeal community changed significantly according
to the water mixture, presenting the greatest diversity when 35% Red Sea water was added to
the Dead Sea water. The bacterial community couldn’t be properly analyzed due to problems
with the initial PCR. However, from the samples for which amplification was obtained (ponds
containing 70 or 60% Dead Sea water), only one band was visualized on the denaturing gel.
The band from the pond containing 60% Dead Sea water-40% Red Sea water was sequenced
and presented 99% similarity with Salisaeta longa.
INDEX
Abstract
1. Introduction...........................................................................................................................1
1.1. The Dead Sea......................................................................................................................1
1.2. Life in the Dead Sea: the halophiles...................................................................................1
1.3. The “Peace Conduit” and the experimental ponds at the Dead Sea Works.......................4
2. Aims of the project................................................................................................................6
3. Materials and Methods..........................................................................................................7
3.1. Buffers................................................................................................................................7
3.1.1. TBE 10X (Tris/Borate/EDTA) buffer.............................................................................7
3.1.2. TAE 50X (Tris/Acetic Acid/EDTA) buffer....................................................................7
3.1.3. Lysis buffer......................................................................................................................7
3.2. Growth Media.....................................................................................................................7
3.2.1. S medium.........................................................................................................................8
3.2.2. S Lev medium..................................................................................................................8
3.2.3. Sg medium.......................................................................................................................8
3.2.4. LB medium......................................................................................................................9
3.3. Sampling and enrichment...................................................................................................9
3.3.1. Experimental ponds samples...........................................................................................9
3.3.2 Dead Sea samples.............................................................................................................9
3.4. Isolation and characterization of strains...........................................................................10
3.4.1. Morphological observation............................................................................................10
3.4.2. Growth in different conditions......................................................................................10
3.4.3. Production of hydrolases...............................................................................................11
3.4.4. Nitrate reduction test.....................................................................................................11
3.4.5. Indole production test....................................................................................................12
3.4.6. Catalase and oxidase production tests...........................................................................12
3.4.7. Antibiotics resistance test..............................................................................................12
3.4.8. Growth stimulation and acid production from sugars...................................................13
3.4.9. Polar lipids extraction....................................................................................................13
3.4.10. Thin layer chromatography (TLC) and polar lipids analysis......................................14
3.4.11. Fatty acids extraction and analysis..............................................................................14
3.4.12. Pigments extraction and analysis.................................................................................15
3.4.13. Intracellular K+ concentration measurement...............................................................15
3.4.13.1 Protein measurements................................................................................................16
3.4.14. Glycerol degradation analysis.....................................................................................16
3.4.15. Dihydroxyacetone production analysis.......................................................................16
3.4.16. Growth under anaerobic conditions............................................................................17
3.5. DNA extraction from pure cultures..................................................................................17
3.6. DNA extraction from the experimental ponds samples...................................................17
3.7. Polymerase Chain Reaction (PCR) .................................................................................18
3.7.1. Universal PCR for Eubacteria.......................................................................................18
3.7.1.1. PCR for 16S rRNA gene sequencing.........................................................................18
3.7.1.2. PCR for DGGE applications......................................................................................18
3.7.2. Universal PCR for Archaea...........................................................................................18
3.7.2.1. PCR for 16S rRNA gene sequencing.........................................................................19
3.7.2.2. PCR for DGGE applications......................................................................................19
3.7.3. PCR specific for Salinibacter ruber..............................................................................19
3.7.4. PCR specific for Salisaeta longa...................................................................................20
3.8. Electrophoresis in agarose gels........................................................................................20
3.9. Designing a primer set specific for Salisaeta longa.........................................................20
3.10. DNA cloning and sequencing.........................................................................................21
3.10.1. PCR products...............................................................................................................21
3.10.2. DGGE bands................................................................................................................21
3.10.3. PCR products cloning and plasmid extraction............................................................21
3.11. Analysis of the DNA sequences.....................................................................................22
3.12. Determination of the detection limit of the primer set specific for S. longa .................22
3.13. Denaturing gradient gel electrophoresis (DGGE)..........................................................22
4. Results.................................................................................................................................23
4.1. Isolation and characterization of strain S4-4....................................................................23
4.2. Comparison between Salisaeta longa and Salinibacter ruber.....................................24
4.3. Specificity of the designed pair of primers for S. longa...................................................26
4.4. Detection of S. longa in situ.............................................................................................27
4.5. Calculation of the detection limit of primers set specific for S. longa.............................27
4.6. Characterization of the microbial community structure using DGGE............................28
4.7. Isolation and characterization of haloarchaeal strains S21 and S22................. ...............29
5. Discussion............................................................................................................................31
6. References...........................................................................................................................36
7. Articles and abstracts based on this thesis..........................................................................40
‫תקציר‬
1. INTRODUCTION
1.1. THE DEAD SEA
The Dead Sea is a unique, athalassohaline, salt-saturated lake with extremely high
divalent cation concentrations, located at the lowest point of the Syrian–African Rift
Valley, on the border between Israel and Jordan (Elevi Bardavid et al., 2007a). Its main
sources of water input are the Jordan River, bringing water from Lake Kinneret, and
winter rain floods.
From the early 1900s the water balance has been negative due to climate changes
and anthropogenic intervention as diversion of freshwater for irrigation and drinking
water, leading to the increase in the salinity of the upper water layers. During the 20th
century, the Dead Sea level has dropped by more than 20 m, and during the past decade,
the level has dropped approximately 1 m per year on the average (Gavrieli et al., 2002;
Dvorkin et al., 2007). This drop in water level is causing severe problems to local
infrastructure, tourism, and industrial activities (Oren et al., 2004).
Currently the water column is supersaturated with NaCl, and massive amounts of
halite have precipitated to the bottom in recent years. As a result, the ionic composition
of the brines has changed dramatically, being more dominated by divalent cations
(Bodaker et al., 2009). The last ionic values reported were 1.98 M Mg2+, 1.54 M Na+,
0.47 M Ca2+, 0.21 M K+, 6.48 M Cl-, 0.08 M Br- and 0.004 M SO42-, in 2007 (Dr. Ittai
Gavrieli, Geological Survey of Israel, personal communication).
The water activity (aw) of the brines is now around 0.67, near the lowest level
known to support life (Oren, 2008). Due to the precipitation of halite, the total salt
concentration has remained approximately constant at around 340 g l-1 and the pH of the
water is about 6.
1.2. LIFE IN THE DEAD SEA: THE HALOPHILES
Environments with NaCl concentrations approaching saturation are often populated by
dense communities of microorganisms, called halophiles. As a result of the lack of
predation and the often high nutrient levels, densities of 107-108 cells ml-1 and higher
are not unusual in those habitats. Many halophilic microorganisms have a high content
of carotenoid pigments, conferring a bright red color to the waters of hypersaline
environments (Oren, 2002; Oren, 2008).
The Dead Sea, however, is a very harsh environment even for the microorganisms
best adapted to life at high salt concentrations. Not only it contains the highest salt
concentration of all natural lakes inhabited by living organisms and a very low water
activity, but the unusual high concentration of divalent cations in the waters (Mg2+ and
Ca2+) is also inhibitory even to the microorganisms adapted to life in the lake (Oren,
1999a).
In spite of the hostility of the environment, the Dead Sea is inhabited by a variety of
microorganisms, as was first demonstrated by Benjamin Elazari Volcani (Wilkansky)
more than 70 years ago (Wilkansky, 1936; Elazari-Volcani, 1940; Oren, 1999a), when
the salinity of the lake was much lower than at present. However, only from 1980
onwards has a systematic monitoring of the biological communities and processes in the
Dead Sea been performed.
The biota of the Dead Sea includes the unicellular green alga Dunaliella, the sole
primary producer in the lake, and red halophilic Archaea belonging to the family
Halobacteriaceae (Fig. 1). Species first reported from the Dead Sea include Haloferax
volcanii,
Haloarcula
marismortui,
Halorubrum
sodomense
and
Halobaculum
gomorrense. Aerobic members of the Bacteria were also found, including novel species
as
Chromohalobacter
marismortui,
Halomonas
halmophila,
Chromohalobacter
israelensis and Salibacillus marismortui. Anaerobic bacteria have been obtained from the
sediments, such as Halobacteroides halobius, Sporohalobacter lortetii, Orenia
marismortui and Selenihalanaerobacter shriftii (Oren et al., 2004). Protozoa (Volcani,
1944), fungi (Oren, 2003) were also isolated from the lake and virus-like particles were
observed by electron microscopy (Oren et al., 1997).
In the years 1980-1982 and 1992-1995, dense microbial blooms were observed in the
entire Dead Sea, following exceptionally rainy winters. The dilution of the upper water
layers caused the formation of a pycnocline at a depth varying between 5 and about 15 m,
turning the holomictic regime of the lake into a meromictic one (Gavrieli et al., 1999).
During these blooms, the algal density reached values up to 9x103 and 1.5x104 Dunaliella
cells ml-1 in 1980 and 1992, respectively (Oren et al., 1995; Oren, 1999a). Concomitant
with the algal blooms, red halophilic Archaea rapidly develop in high numbers - 2x107
and 3.5x107 Archaea ml-1 in 1980 and 1992, respectively - at the expense of organic
material produced by Dunaliella (Oren & Gurevich, 1995; Oren, 1999a). These archaeal
blooms imparted a red color to the entire lake and ended with the termination of the
meromictic state and the renewed overturn of the water column (Oren & Anati, 1996).
a
b
c
d
Fig. 1 - The biota of the Dead Sea. a - the unicellular algae Dunaliella parva; b - the archaeon Haloferax
volcanii; c - the aerobic bacterium Chromohalobacter marismortui; d - the anaerobic bacterium
Sporohalobacter lortetii.
But not only dilution of the upper water layers is necessary for a microbial bloom to
occur in the Dead Sea; phosphate, the limiting nutrient in the lake, must also be available
(Oren & Shilo, 1985; Oren et al., 2004). Those conditions have not been fulfilled since
the 1992-1995 microbial bloom, and the lake has become ever more extreme biotope due
to the continuing drying out accompanied by a dramatic increase in the
divalent/monovalent cation ratio (Bodaker et al., 2009). Nevertheless, the Dead Sea still
supports the life of small Archaea and Bacteria communities, as demonstrated by
Bodaker et al. (2009), but conditions have probably become too extreme for active
growth. Dunaliella, in the other hand, has not been seen in the water column during the
past 12-13 years.
1.3. THE PEACE CONDUIT AND THE EXPERIMENTAL PONDS AT THE
DEAD SEA WORKS (SEDOM)
To counteract the drying out of the Dead Sea and to restore the water level to a desired
elevation, the construction of a water carrier between the Gulf of Aqaba (Red Sea) and
the Dead Sea has been proposed. The idea has been discussed many times in the past,
but only after the peace treaty between Israel and Jordan was signed in 1994, the
implementation project – the ‘‘Peace Conduit’’ – could become real (Oren, 1999a; Oren
et al., 2004).
The difference in elevation of about 416 m between the two seas will enable the use
of the water carrier for seawater desalination by reverse osmosis. Introduction of
seawater from the Gulf of Aqaba (about 40 g/l salts), whether or not concentrated in the
reverse osmosis process, into the Dead Sea (>340 g/l total dissolved salts) will probably
involve significant dilution of the upper water layers of the Dead Sea and lead to the
formation of a stratified water column (Oren et al., 2004).
Hence, future implementation of the ‘‘Peace Conduit’’ requires careful planning
and studies on all possible positive and negative effects it may have, including to the
biota of the lake. For this purpose, simulation experiments are being conducted on the
grounds of the Dead Sea Works at Sedom, in experimental ponds (0.9 m3) containing
mixtures of Dead Sea water and Red Sea water (Fig. 2). The ponds have their conditions
periodically altered to evaluate the effects on the microbial community. Some of the
parameters studied were the mixing ratios of the water mixtures; enrichment with low
phosphate concentrations; total water volume naturally lowered by evaporation or
constantly maintained through inflow of fresh water.
Fig. 2 – Experimental mesocosms at the Dead Sea Works, Sedom
Preliminary results showed that, when phosphate is provided, even a moderate
dilution of the Dead Sea (with 15% Red Sea water) can give rise to extensive microbial
blooms. Dramatic biological effects were observed in those ponds that had been filled
with a mixture of 70% Dead Sea water and 30% Red Sea water. Algae and bacteria
started to appear after 1.5–2 months even when no phosphate was added. The water in the
ponds became highly turbid and red-brown colored, mainly because of archaeal
bacterioruberin pigments (Oren et al., 2004).
The dilution of the Dead Sea may also have dramatic effects on its microbial
community by stimulating the growth of microorganisms that do not normally proliferate
in the lake’s natural conditions. A novel organism has already been isolated from the
experimental mesocosms: the unusual flat, gas-vesicle-containing archeon Haloplanus
natans (Elevi Bardavid et al., 2007a).
2. AIMS OF THE PROJECT
The main goal of this project was to isolate and characterize new halophilic
microorganisms directly from the Dead Sea and also from the experimental ponds at the
Dead Sea Works, Sedom.
An additional goal was to develop a molecular methodology for the detection and
identification of a novel organism, characterized during this work.
Finally, attempts were made to analyze the prokaryotic community present in
experimental ponds containing Dead Sea-Red Sea water mixtures at different ratios,
using the denaturing gradient gel electrophoresis (DGGE) technique.
3. MATERIALS AND METHODS
3.1. BUFFERS
3.1.1. TBE 10X (TRIS/BORATE/EDTA) BUFFER
Tris
108 g
EDTA
7.44 g
H3BO3
55 g
H2O q.s.p.
1000 ml
pH 8.0
3.1.2. TAE 50X (TRIS/ACETIC ACID/EDTA) BUFFER
Tris
242 g
EDTA
18.6 g
Glacial Acetic Acid
57.1 g
H2O q.s.p.
1000 ml
pH 8.0
3.1.3. LYSIS BUFFER
NaCl
5.85 g
Tris
12.1 g
EDTA
18.61 g
SDS
10 g
H2O q.s.p.
100 ml
pH 8.0
3.2. GROWTH MEDIA
All media were autoclaved at 121 ºC for 20 min and had 2% agar (w/v) added to
solidify them when necessary.
3.2.1. S MEDIUM (Oren, 1983)
NaCl
125 g
MgCl2.6H2O
160 g
K2SO4
5g
Starch
2g
Yeast extract
1g
Casamino acids
1g
CaCl2.2H2O
0.1 g
H2O q.s.p
1000 ml
pH 7.0-7.2
3.2.2. S LEV MEDIUM
NaCl
100 g
MgCl2.6H2O
50 g
K2SO4
5g
Starch
2g
Yeast extract
1g
Casamino acids
1g
CaCl2.2H2O
0.1 g
H2O q.s.p
1000 ml
pH 7.0-7.2
3.2.3. Sg MEDIUM
NaCl
125 g
MgCl2.6H2O
160 g
K2SO4
5g
Yeast extract
1g
Casamino acids
1g
CaCl2.2H2O
0.1 g
H2O q.s.p
1000 ml
pH 7.0-7.2
After autoclaving, glucose and HEPES (pH 7.0) were added from sterile
concentrated solutions for final concentrations of 0.2% (w/v) and 20 mM, respectively.
3.2.4. LB MEDIUM
Tryptone
10 g
Yeast extract
5g
NaCl
10 g
H2O q.s.p
1000 ml
pH 7.2
3.3. SAMPLING AND ENRICHMENT
3.3.1. EXPERIMENTAL PONDS SAMPLES
Samples were collected in sterile 500 ml bottles in May, 2007 and November, 2008
from ponds containing different mixtures of Dead Sea – Red Sea water at the Dead Sea
Works, Sedom. In May, ponds contained a mixture of 80% Dead Sea and 20% Red Sea
water, and were plated directly on several hypersaline growth media.
Samples collected in November, 2008 were plated on S Lev medium (paragraph
3.2.2) and had DNA extracted as in item 3.6. The water mixtures of the ponds were:
•
Pond #1 - 70% Dead Sea Water – 30% Red Sea Water
•
Pond #2 - 65% Dead Sea Water – 35% Red Sea Water
•
Pond #3 - 60% Dead Sea Water – 40% Red Sea Water
•
Pond #4 - 55% Dead Sea Water – 45% Red Sea Water
•
Pond #7 - 80% Dead Sea Water – 20% Red Sea Water
The other ponds (# 5, 6, 8, 9, and 10) served for other, unrelated experiments.
3.3.2. DEAD SEA SAMPLES (Bodaker et al., 2009)
Samples were collected in February, 2007, at a station 4 km east of Ein Gedi, at the
location of a moored meteorological station (31º 25' N, 35º 26' E), where the depth of
the lake is about 100 m. Water pumped through a hose from a depth of 5 m was
immediately diluted with 10% (vol/vol) of filter-sterilized distilled water to prevent
clogging of the filtration filters due to the crystallization of halite from the NaClsupersaturated brine. About 200 l of water were filtered through glass fiber filters
(Millipore AP2514250; nominal pore size 0.8-8 µm, diameter of the filtered area 11 cm)
before they became clogged with a brownish material.
Filters were cut in pieces, enriched in different hypersaline growth media and
further plated on agar plates containing the correspondent media.
3.4. ISOLATION AND CHARACTERIZATION OF THE STRAINS
Several colonies were randomly selected from those which arose on agar plates
inoculated with Dead Sea samples and experimental ponds samples collected in May,
2007. Preliminary characterization tests – morphological observation (paragraph 3.4.1),
one-dimensional polar lipid TLC (paragraph 3.4.17.1) and 16S rRNA gene sequencing
(paragraphs 3.5, 3.7.1.1, 3.7.2.1, 3.8 and 3.10.1) – were performed on the isolates.
Colonies isolated from experimental ponds samples collected in November, 2008, were
selected based on morphological observation (paragraph 3.4.1) and pigment analysis
(paragraph 3.4.20).
Complete strain characterization was performed on isolates that possibly represented
species not yet described in the scientific literature: strain S4-4, isolated from
experimental pond #4 (May, 2007) and strains S21 and S22, from Dead Sea samples.
The first strain was routinely grown in S Lev medium (paragraph 3.2.2) and the other
two in Sg medium (paragraph 3.2.3), all under aerobic conditions, at 37 ºC, with
shaking (150 rpm).
3.4.1. MORPHOLOGICAL OBSERVATION
Cell morphology was examined using a Zeiss Axiovert microscope equipped with
phase-contrast optics.
3.4.2. GROWTH AT DIFFERENT CONDITIONS
Erlenmeyer flasks containing 40 ml modified S Lev medium (paragraph 3.2.2) were
inoculated with 200 µl of a grown culture and incubated with shaking (150 rpm) at 37 ºC
for one week. Optical density was measured at 600 nm every 2 days in a
spectrophotometer (Spectronic 601, Milton Roy Company, USA). All tests were
performed in duplicates. The modified parameters were:
•
NaCl concentration - 0, 5, 10, 15, 20% (w/v)
•
MgCl2 concentration - 0, 5, 10, 15 and 20% (w/v)
•
pH - 5.5, 6.0, 7.0, 8.0 and 9.0
•
Temperature - 25, 30, 37, 46 and 52 ºC
The pH in the modified media was stabilized by the addition of buffers at a 20 mM
final concentration (PIPES, pH 5.5 and 6.0; HEPES, pH 7.0, 8.0 and 9.0).
3.4.3. PRODUCTION OF HYDROLASES (Holding & Collee, 1971)
Plates containing solid modified S Lev medium (paragraph 3.2.2) were inoculated
with 20 µl of liquid grown cultures, generating spot-shaped colonies. Positive and
negative control microorganisms were included in the experiment. Plates were
incubated at 37 ºC until growth was obtained.
For starch hydrolysis, regular S Lev medium was used. Plates were flooded with
iodine solution (1% (w/v) iodine; 2% (w/v) potassium iodide) and the presence of a halo
around a colony represented positive starch hydrolysis.
Gelatin hydrolysis was tested in plates with 0.4% (w/v) gelatin added. Plates were
flooded with a solution of 15% (w/v) HgCl in 15% (v/v) HCl. The presence of a halo
around a colony represented positive gelatin hydrolysis.
For Tween hydrolysis, plates had 1% (w/v) Tween 20 or Tween 80 added. The
development of an opaque halo around a colony represented positive Tween hydrolysis.
3.4.4. NITRATE REDUCTION TEST (Holding & Collee, 1971)
Tubes containing S Lev medium (paragraph 3.2.2) with 0.5% (w/v) NaNO3 added
were inoculated with 200 µl of liquid grown cultures. Positive and negative control
microorganisms were included in the experiment. Tubes were incubated with shaking
(150 rpm) at 37 ºC until growth was obtained. The formation of gaseous products from
nitrate was detected by the presence of gas bubbles in Durham tubes. The formation of
nitrite was monitored colorimetrically as follows: 1 ml of the culture grown in the test
medium was centrifuged at 10,000 rpm for 4 min in a plastic 1.5 ml tube. Five hundred
microliters of the supernatant were diluted in 4 ml water, followed by the addition of
500 µl of reagent 1 (5 g sulfanilamide in 50 ml concentrated HCl, completed to 500 ml
with distilled water) and 500 µl of reagent 2 (0.5 g N-l-naphtylethylenediamine
dihydrochloride in 500 ml distilled water). The appearance of a pink color indicated
positive nitrite formation. Sodium nitrite was also used as positive control for this
reaction.
3.4.5. INDOLE PRODUCTION TEST (Holding & Collee, 1971)
Tubes containing S Lev medium (paragraph 3.2.2) with 0.01% (w/v) tryptophan
added were inoculated with 200 µl of liquid grown cultures. Positive and negative
control microorganisms were included in the experiment. Tubes were incubated with
shaking (150 rpm) at 37 ºC until growth was obtained. Indole production was detected
by the appearance of a red color on top of the medium after the addition of 500 µl
Kovacs’ reagent (5 g p-dimethylaminobenzaldhyde in 75 ml amylalcohol and 25 ml
concentrated HCl).
3.4.6. CATALASE AND OXIDASE PRODUCTION TESTS (Holding & Collee,
1971)
The presence of catalase was detected when a 1% (v/v) H2O2 solution was dropped
on colonies grown at 37 ºC on S Lev medium (paragraph 3.2.2) with the subsequent
production of bubbles.
Oxidase activity was detected by dropping a 1% (w/v) tetramethyl pphenylenediamine hydrochloride solution on colonies grown on the same medium with
the appearance of a blue color. Positive and negative control microorganisms were
included in all experiments.
3.4.7. ANTIBIOTICS RESISTANCE TEST
S Lev medium (paragraph 3.2.2) was autoclaved before having the following
antibiotics added at a concentration of 40 µg/ml: penicillin G, ampicillin, streptomycin,
novobiocin, bacitracin, rifampicin, anisomycin and neomycin. Erlenmeyer flasks
containing 40 ml medium were inoculated with 200 µl of a grown culture and incubated
with shaking (150 rpm) at 37 ºC for one week. Optical density was measured at 600 nm
to check for growth. The test was performed in duplicate.
3.4.8. GROWTH STIMULATION AND ACID PRODUCTION FROM
SUGARS
S Lev medium (paragraph 3.2.2) without starch and with yeast extract and casamino
acids concentrations lowered to 0.01% (w/v) each had the following sugars added after
autoclaving, from sterile concentrated solutions, for a final concentration of 0.5% (w/v):
glucose, fructose, maltose, sorbitol, manitol and glycerol. Xylose and ribose were
added at a concentration of 0.2% (w/v).
Erlenmeyer flasks containing 40 ml medium were inoculated with 200 µl of a grown
culture and incubated with shaking (150 rpm) at 37 ºC for one week. The pH of the
cultures was measured daily with a pH electrode to detect acid production.
To test for growth stimulation, 20 mM HEPES were added to the medium as a
buffer (pH 7.0). Optical density was measured every 2 days at 600 nm. The tests were
performed in duplicates.
3.4.9. POLAR LIPIDS EXTRACTION (Oren et al., 1996)
Cells were grown in 50 ml S / S Lev / Sg medium (paragraphs 3.2.1, 3.2.2, 3.2.3)
and collected by centrifugation at 10,000 rpm for 10 min (RC5C, Sorvall® Instruments,
Du Pont, USA). Pellets were resuspended in 1 ml distilled H2O and transferred to 15 ml
glass tubes, where 3.75 ml of a methanol-chloroform solution (2:1, v/v) were added.
Extracts were centrifuged for at 5,000 rpm for 5 min after approximately 4 h incubation.
Supernatant was transferred to 15 ml clean glass tubes, followed by the addition 1.25 ml
chloroform and 1.25 ml H2O. After centrifugation at 5,000 rpm for 5 min, the lower
phase was transferred to 10 ml glass bottles and incubated in vacuum until completely
dried.
3.4.10. THIN LAYER CROMATOGRAPHY (TLC) POLAR LIPIDS
ANALYSIS (Oren et al., 1996)
Lipids extracted as in item 3.4.9 were redissolved in 50 µl chloroform and applied
to silica-gel plates (Sigma, 20x20 cm). For one-dimensional TLC, the silica-gel plates
were incubated for approximately 2 h in a chloroform/methanol/acetic acid/H2O
solution (85:22.5:10:4).
When two-dimensional TLC was performed, silica plates were first incubated for
approximately 2 h in a chloroform/methanol/H2O solution (65:25:10). For the second
dimension, plates were turned 90º and again incubated for approximately 2 h in a
chloroform/methanol/acetic acid/H2O solution (80:12:15:4). Plates were air dried before
staining.
Different reagents were sprayed on the dry silica-plates for the staining of polar
lipids, as listed below. Except for the phospholipids staining, plates had to be incubated
at 150 ºC until lipids could be visualized.
•
Total polar lipids - Orcinol Ferric Chloride Spray Reagent (Sigma)
•
Glycolipids - solution 1 (0.5% (w/v) α-naphtol in methanol/H2O solution
(1:1, v/v)), followed by solution 2 (5 ml H2SO4, 95 ml ethanol)
•
Phospholipids - Molybdenum Blue Spray Reagent 1.3% (Sigma)
•
Amino-containing lipids - Ninhydrin Spray Reagent (Sigma)
3.4.11. FATTY ACIDS EXTRACTION AND ANALYSIS (Elevi Bardavid et
al., 2007b)
Cell mass collected from agar plates was transferred to 10 ml glass tubes with
teflon-lined screw caps. The lipids were saponified and esterified in a one step process
using 2 ml of 2% sulfuric acid in dehydrated methanol for 2 h at 80 ºC in a water bath.
The process was terminated by placing the samples in an ice-water bath. Fatty acid
methyl esters (FAME) were extracted with 1.25 ml hexane/methyl-tert-butyl-ether (1:1,
v/v). The tubes were rotated end over end for 10 min, whereafter the upper phase
including the FAME was transferred to a new tube. The procedure was repeated twice
to ensure complete extraction. The pooled extracts were washed with 3 ml 0.012%
NaOH while being rotated end over end for 10 min. The tubes were then centrifuged for
5 min at 3,000 rpm to achieve full phase separation. The upper 2/3 of the upper phase
were collected and stored overnight at 20 ºC before analysis. FAME were analyzed on
a Hewlett Packard G1800B GCMS using a HP-5-MS column, the initial temperature
being 120 ºC, increasing at a rate of 5 ºC per min up to 240 ºC, and then at a rate of 15
ºC per min up to 300 ºC for 20 min. FAME were identified on the basis of their
retention time as compared to authentic standards (Agilent Technologies, Cat.
No.19298–60500),
and
on
the
basis
of
their
mass
spectra
(http://www.lipidlibrary.co.uk/ms/arch_me/index.html;
http://webbook.nist.gov/chemistry).
3.4.12. PIGMENT EXTRACTION AND ANALYSIS (Elevi Bardavid et al.,
2007b)
One milliliter of a grown culture was centrifuged at 12,000 rpm for 7 min (CD2000, Hsiangtai Machinery, Taiwan). Supernatant was removed and 1 ml of a methanolacetone solution (1:1, v/v) was added. After 1 h incubation in the dark, extracts were
centrifuged at 12,000 rpm for 3 min. The supernatant’s absorption spectra were
recorded against the solvent in a Hewlett Packard model 8452A diode array
spectrophotometer.
3.4.13. INTRACELLULAR K+ CONCENTRATION MEASUREMENT (Oren
et al., 2002)
Cells grown in 30 ml S Lev medium at 37 ºC were collected by centrifugation (10
min, 8,000 rpm). Cell pellets were weight and ressuspended in 5 ml distilled water,
followed by sonication twice for 30 sec. One hundred microliters of perchloric acid 70%
(v/v) were added to 0.9 ml of the sonicated cells and extracts were diluted with distilled
water 10 and 100 times. K+ concentration was determined by flame photometry (Evans
Electroselenium Ltd., England) and calculated based on KCl standards measurements.
Final K+ concentrations were calculated per gram of pellet and gram of protein
(paragraph 3.4.21.1), thus enabling comparison with other microbial species.
3.4.13.1. PROTEIN MEASUREMENTS (Lowry et al., 1951)
Different dilutions of the sonicated pellets were boiled for 10 min after the addition
of an equal volume of 4% (w/v) NaOH. After cooling, 5 ml of solution A (2% Na2CO3
(w/v)/4% p-Na-tartarate (w/v)/2% CuSO4.5H2O (w/v), 100:1:0.5) were added and
incubated for 10 min at room temperature. Half a milliliter of Folin reagent (Merck)
diluted in water (1:1, v/v) was added and optical density measured at 660 nm after 40
min incubation in room temperature. Bovine serum albumin (BSA) was used as
standard.
3.4.14. GLYCEROL DEGRADATION ANALYSIS (Elevi Bardavid & Oren,
2008)
Erlenmeyer flasks containing 40 ml S Lev medium (paragraph 3.2.2) with 0, 0.01,
0.05, 0.2 and 0.5% (w/v) glycerol were inoculated with 200 µl of a grown culture and
incubated with shaking (150 rpm) at 37 ºC for one week. Optical density was measured
daily at 600 nm to check for growth.
The colorimetric assay for glycerol was performed on 1 ml samples collected daily
from the growing cultures. Cells were removed by centrifugation (4 min, 12,000 rpm)
and glycerol oxidized to formaldehyde and formic acid with 1 ml periodate reagent (65
mg Na-meta-periodate, 7.7 g ammonium acetate, 90 ml distilled water, 10 ml acetic
acid). After 5 min incubation at room temperature, 5 ml acetylacetone reagent (1 ml
acetylacetone, 99 ml isopropanol) was added to react with formaldehyde, generating a
yellow compound after 20 min incubation at 50 ºC. Absorbance was measured at 410
nm and glycerol concentration calculated based on glycerol standards measurements.
3.4.15. DIHYDROXYACETONE PRODUCTION ANALYSIS (Elevi Bardavid
& Oren, 2008)
Cells were grown and samples collected as described in item 3.4.14. To 0.5 ml
sample, 2 ml resorcinol reagent (2% (w/v) resorcinol in 10 M HCl) were added and the
absorbance measured at 490 nm after overnight incubation at room temperature.
3.4.16. GROWTH UNDER ANAEROBIC CONDITIONS
Closed 100 ml bottles full with Sg medium (item 3.2.3) were inoculated with 1 ml
of grown cultures and incubated at 37 ºC for 2 weeks. Optical density was measured at
600 nm every 3 days in a spectrophotometer. The test was performed in duplicates.
3.5. DNA EXTRACTION FROM PURE CULTURES
Cells were recovered from 1 ml grown cultures by centrifugation (5 min, 8,000 rpm)
and lysed with 0.5 ml lysis buffer (100 mM EDTA, 50 mM EDTA, 100 mM NaCl, 1%
(w/v) SDS) for 10 min at 100 ºC. After 10 min incubation with 250 µl phenol at room
temperature, 250 µl of a chloroform-isoamyl alcohol (24:1) solution were added and
incubated for another 10 min at room temperature. Phases were separated by
centrifugation (10 min, 12,000 rpm) and the upper phase removed to a new plastic tube
with the same volume of the chloroform-isoamyl alcohol (24:1) solution. Phase
separation was performed as described above and the upper phase was removed to a
new plastic tube with the same volume of isopropanol. DNA was precipitated overnight
at -20 ºC or for 40 min at -80 ºC. After 30 min centrifugation at 4 ºC, DNA was washed
with 1 ml 70% (v/v) ethanol by 10 min centrifugation at 4 ºC and air dried before the
addition of 50 µl sterile distilled water.
The DNA amount and of the extracts, as well as their purity, were measured by a
Nanodrop® 1400 device (ThermoScientific) at 260 nm.
3.6. DNA EXTRACTION FROM THE EXPERIMENTAL PONDS SAMPLES
Aproximately 250 ml of each sample was centrifuged at 5,000 rpm for 20 min. The
pellets were tranferred to 2 ml plastic tubes and DNA was extracted as described in
paragraph 3.5.
3.7. POLYMERASE CHAIN REACTION (PCR)
3.7.1. UNIVERSAL PCR FOR EUBACTERIA
PCRs were performed with primers targeting the 16S rRNA gene of Eubacteria. The
size of the amplified fragments varied according to the desired application of the PCR
products.
3.7.1.1. PCR FOR 16S rRNA GENE SEQUENCING
Reactions were performed with primers 27f (5'-AGAGTTTGATCCTGGCTCAG3') and 1492r (5'-GGTTACCTTGTTACGACTT-3'), amplifying a 1,465 bp fragment,
in plastic tubes containing 2X PCR Master Mix (Thermo Scientific, UK), 1 µl of 10
µM primers, 0.5 µl of 25 mM MgCl2, 1 µl (approximately 30 ng DNA) sample, and
completed with distilled sterile H2O for a 20 µl reaction.
The cycle used was: 1x (5 min at 94 ºC); 30x (1 min at 94 ºC; 1 min at 48 ºC; 1 min
at 72 ºC); 1x (5 min at 72 ºC); storage at 4 ºC.
3.7.1.2. PCR FOR DGGE APPLICATIONS
Reactions were performed with primers GM5-GC clamp (5’- CGCCGCCCGCGCG
CGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3’)
and
907r (5’-CCGTCAATTCCTTTGAGTTT-3’), amplifying a 627 bp fragment, in plastic
tubes containing 2X PCR Master Mix (Thermo Scientific, UK), 1.25 µl of 10 µM
primers, 1.25 µl of 25 mM MgCl2, 2.5 µl (approximately 100 ng DNA) sample, and
completed with distilled sterile H2O for a 50 µl reaction.
The cycle used was: 1x (5 min at 94 ºC); 30x (1 min at 94 ºC; 1 min at 56 ºC; 40 sec
at 72 ºC); 1x (7 min at 72 ºC); storage at 4 ºC.
3.7.2. UNIVERSAL PCR FOR ARCHAEA
PCRs were performed with primers targeting the 16S rRNA gene of Archaea. The
size of the amplified fragments varied according to the desired application of the PCR
products.
3.7.2.1. PCR FOR 16S rRNA GENE SEQUENCING
Reactions were performed with primers 21f (5'-TTCCGGTTGATCCTGCCGGA-3')
and 1492r (5'-GGTTACCTTGTTACGACTT-3'), amplifying a 1,471 bp fragment, in
plastic tubes containing 2X PCR Master Mix (Thermo Scientific, UK), 1 µl of 10 µM
primers, 0.5 µl of 25 mM MgCl2, 1 µl (approximately 30 ng DNA) sample, and
completed with distilled sterile H2O for a 20 µl reaction.
The cycle used was: 1x (5 min at 94 ºC); 30x (1 min at 94ºC; 45 sec at 54 ºC; 55 sec
at 72 ºC); 1x (5 min at 72 ºC); storage at 4 ºC.
3.7.2.2. PCR FOR DGGE APPLICATIONS
Reactions were performed with primers 340f-GC clamp (5'-CGCCGCCCGCGCGC
GGCGGGCGGGGCGGGGGCACGGGGGGCCCTACGGGGCGCAGCAG-3')
and
934r (5’-GTGCTCCCCCGCCAATTCCT-3’), amplifying a 600 bp fragment, in plastic
tubes containing 2X PCR Master Mix (Thermo Scientific, UK), 1.25 µl of 10 µM
primers, 1.25 µl of 25 mM MgCl2, 2.5 µl (approximately 100 ng DNA) sample, and
completed with distilled sterile H2O for a 50 µl reaction.
The cycle used was: 1x (5 min at 94 ºC); 30x (1 min at 94 ºC; 1 min at 60 ºC; 40 sec
at 72 ºC); 1x (7 min at 72 ºC); storage at 4 ºC.
3.7.3. PCR SPECIFIC FOR Salinibacter ruber
PCRs were performed with primers EHB4F (Antón et al., 2002) and EHB9R (Peña
et al., 2005), designed to specificaly amplify the partial (550 bp) 16S rRNA gene of
Salinibacter. Reactions occurred in plastic tubes containing 2X PCR Master Mix
(Thermo Scientific, UK), 1 µl of 10 µM primers, 0.5 µl of 25 mM MgCl2, 1 µl
(approximately 30 ng DNA) sample, and completed with distilled sterile H2O for a 20
µl reaction.
The cycle used was: 1x (5 min at 94 ºC); 30x (1 min at 94 ºC; 45 sec at 54 ºC; 45 sec
at 72 ºC); 1x (7 min at 72 ºC); storage at 4 ºC.
3.7.4. PCR SPECIFIC FOR Salisaeta longa
PCRs were performed with primers SaetaF (5'-CCTGCCTTTGAGCGGGGGATAA
CTACG-3') and SaetaR (5'-GATTCGCTACTCCTCCGCGGAGCG-3'), designed to
specificaly amplify the partial (1,100 bp) 16S rRNA gene of S. longa (paragraph 3.9).
Reactions were performed in plastic tubes containing 2X PCR Master Mix (Thermo
Scientific, UK), 1 µl of 10 µM primers, 0.5 µl of 25 mM MgCl2, 1 µl (approximately 50
ng DNA) sample, and completed with distilled sterile H2O for a 20 µl reaction.
The cycle used was: 1x (5 min at 94 ºC); 30x (1 min at 94 ºC; 50 sec at 64 ºC; 55 sec
at 72 ºC); 1x (5 min at 72 ºC); storage at 4 ºC.
3.8. ELECTROPHORESIS IN AGAROSE GELS
All PCR products were applied onto 1.5% (w/v) agarose gels and ran in 1X TBE buffer
(paragraph 3.1.1) at 85 V for approximately 50 min. Gels were stained with ethydium
bromide and bands visualized under U.V. light to confirm the amplification of the
desired fragment. A 100 bp DNA Ladder (Lamda Biotech) was used to indicated the
size of the PCR products.
3.9. DESIGNING A PRIMER SET SPECIFIC FOR Salisaeta longa
The 16S rRNA gene of Salisaeta longa S4-4T (EU426570) was aligned in the BioEdit
sequence alignment editor (Hall, 1999) with the analogous sequences of several
Salinibacter ruber strains deposited in the GenBank website (http://www.ncbi.nlm.nih.
gov/Genbank). The low similarity between both species sequences (88%) excluded the
need of using less similar sequences from other species in the alignment.
The regions from nucleotides 94-120 and 1219-1241 were exclusive to the S. longa
sequence and thus, chosen as forward and reverse primers, respectively. The propensity
to form dimers and hairpin loops, melting temperature and G-C content were analyzed
with the OligoCalc software (Kibbe, 2007).
PCR (section 3.7.4) was performed with DNA extracted from S. longa, S. ruber and
Escherichia coli (paragraph 3.6) to test for specificity of the primers for Salisaeta.
3.10. DNA CLONING AND SEQUENCING
3.10.1. PCR PRODUCTS
PCR products amplified from pure cultures were cleaned up with ExoSAP-IT®
(Usb®, Affimetrix, Inc.) and sequenced with an ABI PRISM 3700 DNA Analyzer. PCR
products amplified from environmental DNA samples were inserted in plasmids and
cloned into E. coli cells as outlined in paragraph 3.10.3 before sequencing.
3.10.2. DGGE BANDS
DGGE bands were excised from the polyacrylamide gel and redissolved in 50 µl
distilled sterile water overnight at 4 ºC. PCR was realized as in section 3.7.1.2 using 1
µl of the redissolved band as template. PCR product was sequenced as in item 3.10.1.
3.10.3. PCR PRODUCTS CLONING AND PLASMID EXTRACTION
PCR products were inserted in plasmid pTZ57R/T with an overnight incubation at
17 ºC, using the InstAclone™ PCR cloning kit (Fermentas Life Sciences).
Transformation to E. coli JM109 cells were performed with heat shock (30 min in ice, 2
min at 42 ºC, 8 min in ice) and incubated in 1 ml LB medium (paragraph 3.2.4) with
shaking (150 rpm) at 37 ºC for cells to start expressing the ampicillin resistance
phenotype. Cells were concentrated with 1 min centrifugation at 10,000 rpm and
removal of 1 ml of the supernatant before plated in LB medium supplemented with 40
mM Xgal, 50 mM ampicillin and 100 mM IPTG. Plates were incubated at 37 ºC for 24
h and white colonies were isolated. Colony PCRs were performed with primers SaetaF
and SaetaR (paragraph 3.7.4) to confirm the presence of the insert in the selected
colonies.
Plasmids containing the insert were extracted with the Wizard® Plus SV miniprep
DNA purification system (Promega) and sequenced as in paragraph 3.10.1.
3.11. ANALYSIS OF THE DNA SEQUENCES
The sequences obtained were aligned with the sequences deposited in the GenBank
database through the Nucleotide BLAST software (Altschul et al., 1997).
3.12. DETERMINATION OF THE DETECTION LIMIT OF THE PRIMER SET
SPECIFIC FOR S. longa
A detection limit experiment evaluated the number of S. longa cells needed in an
environmental sample in order to detect the presence of this species in the environment
with the methodology developed in this work (sections 3.4.2, 3.7.4 and 3.9).
After removal of cells by centrifugation (20 min, 5,000 rpm), water from ponds #3
and #4 was sterilized by autoclaving and distributed in plastic centrifuge tubes (20 ml
per tube). To each tube, 109 cells of Halobacterium salinarum R1 were added, to
simulate the vast archaeal population normally present in the experimental ponds. In
addition, increasing amounts of S. longa cells - 101 to 109 - were added to each tube. No
S. longa cells were added to a control tube.
DNA was extracted as outlined in paragraph 3.6 and PCR carried out as in
paragraph 3.7.4.
3.13. DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE)
DGGE was performed with the Bio-Rad D gene system (Bio-Rad, USA). PCR samples
(sections 3.7.1.2 and 3.7.2.2) were loaded onto 6% (w/v) polyacrylamide gels in 1x
TAE (section 3.1.2). The polyacrylamide gels were made with a 40 to 60% denaturing
agent gradient from 0 and 80% denaturing stock solutions (table below):
Denaturing
solution
0%
80%
40% BIS/
Acrylamide
15 ml
15 ml
TAE 50x
buffer
2 ml
2 ml
Formamide
(deionized)
Urea
32 ml
33.6 g
Distilled
H 2O
q.s.p 100 ml
q.s.p 100 ml
Electrophoresis was performed for 16 h at 60 °C and 80 V. The resulting gels were
stained with SYBR Green stain and photographed.
4. RESULTS
4.1. ISOLATION AND CHARACTERIZATION OF STRAIN S4-4
To characterize the community present in the experimental ponds at the Dead Sea Works,
Sedom, samples were plated on different hypersaline growth media. Twelve different
colonies from those that arose on S medium agar plates were isolated for initial
characterization. Polar lipids were extracted and analyzed by one-dimensional TLC
stained for glycolipids and phospholipids. Strain S4-4, isolated from pond #4, showed a
polar lipids pattern different from that observed in other archaeal and bacterial strains
(Fig. 3 - the lane indicated by the box). Therefore, this strain was selected for further
characterization.
Fig. 3 a - glycolipids analysis; b - phospholipids analysis. 1 - Halobacterium salinarum R1; 2 Haloarcula sp.; 3 - Halorubrum sp.; 4- Haloferax sp.; 5, 7, 8, 9, 10, 13 and 14- Archaeal isolates; 11,
12, 15 and 16 - Bacterial isolates; 6 - Strain S4-4.
S4-4 colonies are big and have the typical orange-red color of Salinibacter ruber.
Microscopic observation revealed Gram-negative, very long rod-shaped cells (15-30 µm,
see Fig. 6d, p. 27). Optimal growth was obtained at 10-12% NaCl (range 5-20%), 5%
MgCl2.6H2O (range 5-20%), pH 6.5-8.5 (range 6.0-9.0) and temperature 37-46 °C (range
25-50 ºC). Therefore, routine growth medium was substituted from S to S Lev, in which
NaCl and MgCl2.6H2O concentrations were adjusted to allow optimal growth. When 5%
(0.25 M) MgCl2.6H2O was replaced by an equivalent concentration of MgSO4, growth
was inhibited. In the absence of NaCl or MgCl2 no growth was obtained.
Starch and gelatin were slightly hydrolyzed and growth was inhibited by Tween.
Nitrate was not reduced; catalase and oxidase were produced, as well as indole from
tryptophan. Glucose, sucrose, maltose and glycerol stimulated growth with acid
formation. No growth stimulation or acid formation was obtained in the presence of
ribose, xylose, mannitol, and sorbitol; fructose caused acidification of the medium, but
didn’t stimulate growth. The isolate exhibited sensitivity to penicillin G, ampicillin,
novobiocin, rifampicin, and was insensitive to streptomycin, neomycin, bacitracin, and
anisomycin.
Polar lipids included three glycolipids and four major phospholipids, one of which
containing an amino group (Fig. 4). The main fatty acids present were 16:0 iso and 16:1
cis 9, followed by 15:0 iso and 15:0 anteiso. Pigment extracts in methanol/acetone
showed an absorption maximum at 478 nm and a shoulder at 506-510 nm.
Fig. 4 - One-dimensional (left panel) and two-dimensional (right panel) chromatogram of polar
lipids extracted from S4-4. In the one-dimensional chromatogram Salinibacter ruber M31T was
included for comparison. P = phospholipid; G = glycolipid; N = amino containing lipid (ninhydrinpositive).
Internal K+ concentrations were 10.1 µmol/mg of protein (137.2 µmol/g of pellet) for
cells grown in medium containing 10% NaCl and 16.5 µmol/mg of protein (190.4 µmol/g
of pellet) for cells grown in medium containing 15% NaCl.
Glycerol was slightly consumed and degradation could only be detected in medium
with 0.01 and 0.05% (w/v) glycerol added. No significant DHA production could be
detected.
The nearly complete 16S rRNA gene presented 88% similarity with its closest relative,
S. ruber. A phylogenetic tree was built with the MEGA software as shown in Fig. 5. The
microorganism was called Salisaeta longa, type strain S4-4T, as will be further explained
in the Discussion (Vaisman & Oren, 2009), and clustered in the Bacteroidetes phylum
(Sphingobacteriales order) of the Bacteria domain.
77
99
79
100
Bacteroides fragilis DJF B083 (EU728706)
T
Cytophaga hutchinsonii ATCC 33406 (NC_008255)
T
Flavobacterium aquatile ATCC 11947 (M62797)
T
Thermonema rossianum T NR-27 (Y08956)
100 Rhodothermus marinus JCM 9785 (AF217493)
T
Rhodothermus marinus DSM 4525
(AF217494) Salinibacter ruber M31 T (AF323500)
T
Salisaeta longa S4-4 (EU426570)
100
T
Chlorobium limicola DSM 245 (CP001097)
0.02
Fig. 5 - 16S rRNA gene sequence-based phylogeny reconstructed from distance values by using the minimum-evolution
method. Bootstrap values (>50 %) are given at nodes. Chlorobium limicola DSM 245T served as the outgroup. Bar, 0.01
inferred substitutions per nucleotide position.
4.2. COMPARISON BETWEEN Salisaeta longa AND Salinibacter ruber
In order to confirm strain S4-4 as the type strain of a new species and a new genus,
Salisaeta longa, results obtained through the characterization experiments were compared
to those of Salinibacter ruber, its closest relative. Some of the different characteristics in
both species are presented in Table 1.
The polar lipids pattern of S. longa differed significantly from that of S. ruber,
especially in the glycolipid fractions (Fig. 4). The fatty acid composition was also
considerably different in both species. S. ruber has a predominance of 15:0 iso, 16:1 cis 9
and 18:1 cis 11, each representing 25-30% of the total fatty acids. 15:0 anteiso and 16:0
are also present, corresponding to 4-5% and 7-10%, respectively (Elevi Bardavid et al.,
2007b).
S. longa consumed small amounts of glycerol and did not produce significant amounts
of DHA, while S. ruber readily consumed 10 mM glycerol and produced 1.1 mM DHA
(Elevi Bardavid & Oren, 2008).
Table 1. Characteristics differentiating Salisaeta longa and Salinibacter ruber.
Characteristic
Salisaeta longa S4-4T
Salinibacter ruber M31T
Cell length
15-30 µm
0.4-2.6 µm
NaCl range
5-20%
15-30%
Indole production
-
+
Sensitivity to streptomycin
-
+
DNA G+C content (mol%)
62.9
66.5a
+ = positive; - = negative
a
The range of DNA G+C content among different strains of S. ruber was reported to be 66.3-67.7
mol%.
All the other characteristics were similar between both species, including pigment
absorption spectra and high intracellular K+ concentration.
Regarding the 16S rRNA gene sequence, S. longa presented 88% similarity with S.
ruber M31T strain (AF323500). However, the pair of primers EHB4F (Antón et al., 2002)
and EHB9R (Peña et al., 2005), designed as “specific” for Salinibacter, also amplified a
fragment of S. longa 16S rRNA gene, 91% similar to the corresponding sequence of S.
ruber M31T.
4.3. SPECIFICITY OF THE DESIGNED PAIR OF PRIMERS FOR S. longa
Primers SaetaF and SaetaR amplified a partial 16S rRNA gene sequence from S. longa
strain S4-4T. There was no amplification when DNA from Salinibacter strains and E. coli
were used as templates in the same reaction.
Four strains from experimental pond #3 (Pink 1, Pink 2, Pink 3 and Pink 4) and one
strain from experimental pond #2 (Pink 5) were isolated from S Lev plates (samples
collected in November, 2008). All isolates had long rod-shaped cells and pigments
absorption spectra typical of S. longa. The specific primer pair was able to amplify the
partial 16S rRNA gene of all five strains, and sequences were 99% similar to the
correspondent sequences of S. longa S4-4T. The almost complete 16S rRNA gene
sequences were similar as well.
4.4. DETECTION OF S. longa IN SITU
Morphological observation of pond samples revealed the presence of long rod-shaped
cells typical of S. longa (Fig. 6). DNA preparations extracted from experimental ponds
samples were used as templates in PCRs with primers SaetaF and SaetaR, in order to
evaluate if the presence of S. longa could be detected molecularly, without cultivation
methods. Amplification was obtained only from ponds #2 and #3. The amplified
fragments were also 99% similar to the corresponding sequences of S. longa S4-4T. No
amplification occurred when DNA from ponds #1, #4 and #7 were used as templates.
Fig. 6 - Phase contrast micrographs of (a) pond #2, (b) pond #3, (c) pond #4 and (d) S.
longa S4-4T strain.
4.5. CALCULATION OF THE DETECTION LIMIT OF THE PRIMERS SET
SPECIFIC FOR S. longa
Sterile experimental pond water inoculated with different numbers of S. longa cells and
109 Halobacterium salinarum cells had DNA extracted and PCR performed with the S.
longa specific pair of primers exactly as performed on the experimental pond samples.
The desired fragment was amplified from water containing a minimum of 104 S. longa
cells. No amplification was detected in water containing less than 104 S. longa cells.
4.6.
CHARACTERIZATION
OF
THE
MICROBIAL
COMMUNITY
STRUCTURE USING DGGE
The 16S rRNA genes from DNA extracted from the bacterial and the archaeal
communities present in experimental ponds containing different mixtures of Dead Sea
water - Red Sea water were amplified by universal primers for Bacteria and Archaea,
respectively. Electrophoresis was performed in polyacrylamide gels containing a
denaturing gradient from 40 to 60%, aiming to find out if the differences in the water
mixtures in the ponds influenced their microbial composition.
Results showed that the archaeal composition was significantly different between
ponds (Fig. 7). Pond #7, containing 80% Dead Sea water, presented the smallest number
of bands. The community in pond #1, containing 70% Dead Sea water, was already more
diverse, and the highest diversity was present in pond #2, containing 65% Dead Sea
water. Pond #3 (60% Dead Sea water) presented a pattern that was very similar to pond
#2, and pond #4 (55% Dead Sea water), although sharing a number of bands with ponds
#2 and #3, had a considering smaller number of bands.
1
2
3
4
5
6
7
8
9
10 11
12
13 14 15 16
Fig. 7 - DGGE of archaeal 16S rRNA gene fragments, performed in duplicates; 1, 2, 3, 9, 15
and 16 - Ladder; 4 and 10 - pond #7; 5 and 11 - pond #4; 6 and 12 - pond #3; 7 and 13 - pond
#2; 8 and 14 - pond #1.
The bacterial composition couldn’t be properly analyzed due to initial problems with the
universal PCR. Later the experiment in the experimental ponds was discontinued,
preventing the collection of new samples. Hence, only DNA extracted from ponds #1 and
#3 could be analyzed. The partial 16S rRNA gene amplified from S. longa S4-4T was also
applied to the gel. Both ponds presented only one band in the polyacrylamide gel: pond
#3 at the same position as S. longa S4-4T and pond #1 slightly below (figure not shown).
The band obtained from pond #3 band presented 99% similarity to S. longa S4-4T and the
band from pond #1 had 99% similarity with Massilia sp. and Naxibacter sp., genera not
known from hypersaline environments.
4.7.
ISOLATION
AND
CHARACTERIZATION
OF
HALOARCHAEAL
STRAINS S21 AND S22
To characterize the community present in the upper water layers of the Dead Sea in
February 2007, about 200 l of water were filtered and plated on different hypersaline
growth media. Sixteen different colonies were isolated for initial characterization. Polar
lipids were extracted and analyzed by one-dimensional TLC, and the plates were stained
for glycolipids and phospholipids. Two strains, S21 and S22, presented an archaeal
pattern different from the archaeal standards (Fig. 8, indicated by boxes).
1
2
3
4
5
6
7
1 2
3
4
5
6
7
Fig. 8- Left panel - phospholipids analysis; Right panel - glycolipids analysis. 1Halobacterium salinarum; 2- Haloferax volcanii; 3- Halorhabdus utahensis; 4S21; 5- S22; 6- Halorubrum sodomense; 7- Haloarcula marismortui
Strains S21 and S22 formed pink small colonies on plates and morphological
examination showed small pleomorphic cells when grown in regular S medium. Growth
in liquid medium was slow and cultures never achieved high densities; they only
became slightly turbid.
The complete 16S rRNA gene showed 99% similarity with Halorhabdus utahensis
(Wainø et al., 2000) and Halorhabdus tiamatea (Antunes et al., 2008). Based on the
known characteristics of those species, and notably the anaerobic or microaerophilic
nature of Hrd. tiamatea and the preferential growth of Hrd. utahensis on glucose,
strains S21 and S22 were tested for growth under anaerobic conditions and on glucose
as carbon source. Growth was similar under aerobic and anaerobic conditions, but
optical density was twice as high when both strains were grown with glucose instead of
starch. They were unable to grow in the media routinely used for both previously
described Halorhabdus species. Hence, the growth medium was changed from S to Sg,
in which starch was substituted for glucose. Hrd. utahensis was also able to grow in this
medium. Liquid cultures of S21 an S22 in Sg medium were dense and pink, and cells
became more rod-shaped.
5. DISCUSSION
The main objectives of this work were to isolate and characterize new halophilic
microorganisms from the Dead Sea and from the experimental ponds at the Dead Sea
Works, Sedom, containing different mixtures of Dead Sea and Red Sea water.
Strain S4-4, isolated from the experimental ponds, presented the typical orange-red
color of Salinibacter ruber. This red-pigmented halophilic member of the Bacteria,
found in saltern crystallizer ponds all over the world, has many physiological
characteristics similar to the Halobacteriales (Antón et al., 2002) and was indeed the
closest relative of the isolated strain.
The complete characterization of strain S4-4 showed that it was sufficiently
different from S. ruber to be classified as a new species belonging to a new genus. The
proposed name for this organism was Salisaeta longa gen. nov., sp. nov. (Sa.li.sae’ta, L.
masc. n. sal, salis salt; L. fem. n. saeta a bristle; N.L. fem. n. Salisaeta a salt bristle;
lon’ga. L. fem. adj. longa long). The type strain (strain S4-4T) was deposited in two
collection cultures: the German Collection of Microorganisms and Cell Cultures (DSM
21114T) and the Spanish Type Culture Collection (CECT 7354T).
The nearly complete 16S rRNA gene of S. longa S4-4 T (EU426570) and S. ruber
M31T (AF323500) strains presented a similarity of 88%. Part of the 16S rRNA gene of
S. longa could be amplified by primers EHB4F (Antón et al., 2002) and EHB9R (Peña
et al., 2005), designed as “specific” primers for Salinibacter. Thus, this primer pair can
no longer be considered specific for S. ruber, and therefore the discovery of new genus
related to Salinibacter requires re-evaluation of the probes/primers previously designed
for this genus.
Among the interesting characteristics of the new organism is the haloadaptation
mechanism used to enable life under hypersaline conditions. All the halophilic and
halotolerant aerobic Bacteria characterized until 2002 produce and/or accumulate
organic “compatible” solutes such as ectoine, glycine betaine, and others, to provide the
necessary osmotic balance. Synthesis and degradation of those solutes can be regulated
according to the extracellular salt concentration, enabling a considerable degree of
adaptability to changes in the salinity of the medium (Oren, 1999b; Ventosa et al.,
1998).
The aerobic halophilic Archaea, on the other hand, accumulate KCl in molar
concentrations (Lanyi, 1974; Oren, 1999b). This strategy requires far-reaching
adaptations in order for all intracellular processes to be functional at high salt
concentrations. Proteins of the Halobacteriales are typically rich in acidic amino acids,
depleted of basic amino acids, and relatively poor in hydrophobic amino acids. Such
proteins generally require the presence of high salt concentrations for stability and
activity (Lanyi, 1974). Accordingly, the microorganisms that harbor these are unable to
adapt to life below a (generally very high) minimal salt concentration. A similar strategy
of adaptation to high salt was found in the obligatory anaerobic Bacteria of the order
Halanaerobiales, phylogenetically affiliated with the low G+C branch of the Firmicutes
(Oren, 1986; Oren, 1999b).
In 2002, it was discovered that Salinibacter shares with the halophilic Archaea of
the order Halobacteriales a large excess of acidic amino acids (Oren & Mana, 2002)
and a high intracellular KCl concentration as osmotic solute (Oren et al., 2002). Thus, it
may be predicted that both groups share the same habitat and are unable to live at low
salt concentrations.
Salisaeta, in the other hand, is able to grow in a range of 5-20% NaCl, much below
the minimum 15% NaCl concentration required by Salinibacter. However, the
intracellular K+ concentration is similar to that of Salinibacter and other halophilic
Archaea and increases proportionally to the salinity of the medium. Further attempts to
detect the presence of organic osmotic solutes and determine the content of acidic
amino acids in Salisaeta’s proteins will be necessary to confirm the typical archaeal
haloadaptation in this aerobic member of the Bacteria.
Another interesting feature of the new microorganism is the presence of two
sulfonolipids in its membrane composition (Baronio et al., submitted for publication). It
is known that the modification of membrane lipid composition is an important aspect of
haloadaptation, preserving membrane integrity and function at high salt concentrations
(Russell, 1993). The membrane lipids of S. longa, like in S. ruber, are typical for the
bacterial domain, with glycerophospholipids containing ester-linked fatty acyl chains
and not ether-linked phytanyl chains. Corcelli et al. (2004) discovered a novel
sulfonolipid in S. ruber and suggested it to be used as a chemotaxonomic marker for the
detection of Salinibacter within the halophilic microbial community in hypersaline
environments. Preliminary results of the characterization of the sulfonolipds of
Salisaeta indicate that one of them is indeed an analogue of the novel Salinibacter
sulfonolipid. The second one a hydroxyl derivative of the first (Baronio et al., submitted
for publication).
The phylogenetic proximity of Salisaeta (a moderate halophilic member of the
Bacteria) to the extremely halophilic Salinibacter and the resemblance of these
organisms to the Halobacteriales, stimulate new studies about the characteristics of the
newly discovered organism. Some of the features to be analyzed in the future would be
the nature of the carotenoid pigment responsible for its red-orange color and the
presence of retinal proteins in the membrane. These proteins are common in
Salinibacter and many members of the Halobacteriales, where they function as lightdriven proton or chloride pumps. One of the retinal proteins of S. ruber, named
xanthorhodopsin, has been studied in detail and has a C40-carotenoid acyl glucoside
salinixanthin, responsible for the organism’s color, serving as a light harvesting antenna
(Lutnæs et al., 2002; Balashov et al., 2005; Balashov & Lanyi, 2007).
It is also interesting to understand the ecology of Salisaeta and the role it plays in
the microbial community of the experimental ponds. Therefore, experiments were
performed to verify the consumption of glycerol and consequent production of DHA by
this organism. Glycerol can be expected to be one of the main nutrients available in
hypersaline environments. It is produced in large quantities by the unicellular algae
Dunaliella, which is the main or only primary producer in those habitats (Oren, 1993),
including the experimental ponds. The use of glycerol by members of the
Halobacteriaceae (Oren, 1993) and Salinibacter (Sher et al., 2004) had been
demonstrated, and the overflow product formed by the latter was later identified as
DHA (Elevi Bardavid & Oren, 2008). Salisaeta, unlike Salinibacter, consumed only
small amounts of glycerol and DHA production could not be detected; therefore, no
significant importance can be attributed to Salisaeta concerning glycerol uptake in the
experimental ponds.
Salisaeta was originally isolated from a pond containing 80% Dead Sea water and
20% Red Sea water, but it was also present in ponds containing 65 and 60% Dead Sea
water (completed with Red Sea water). The presence of the new organism was
confirmed not only by the isolation of new strains, but also by PCR, from samples
containing at least 104 Salisaeta cells. It is important to notice that the methodology
developed in this work – DNA extraction from the experimental ponds and PCR
reaction with the specific primers – was designed for liquid, highly-populated
environments, similar to the experimental ponds in Sedom. It may require adaptations
for samples collected in different environments.
The detection limit of 104 Salisaeta cells per sample was satisfactory for the aims of
this work, but attempts could be made to improve it. The collection of cells by filtration
instead of centrifugation is one possibility; however, previous dilution of the samples
will be required due to their high salinity, otherwise filters may become clogged with
salt crystals. The dilution has to be carefully calculated, preventing cell lysis due to
osmolarity changes in the samples. Another possibility is using a nested or semi-nested
PCR instead of one single reaction with the specific pair of primers.
The importance of the discovery of Salisaeta longa relies on its phylogenetic
proximity with Salinibacter, both members of the Bacteroidetes but sharing similar
characteristics with halophilic Archaea. And in the future, with the implementation of
the ‘”Peace-Conduit”, it is possible that a bloom of Salisaeta will be observed at the
Dead Sea due to the water dilution, as it was observed in the experimental ponds.
DGGE experiments showed that, when the Dead Sea is diluted with 40% Red Sea water
(pond #3) and nutrients are available, Salisaeta prevails within the bacterial community
of the experimental pond. If the same scenario will become true in the lake, the more is
known about this organism, the easier it will be to monitor its presence and activities.
It is also probable that a Salisaeta bloom will occur if the Dead Sea will become
diluted with 35% Red Sea water (simulated in pond #2). Although it could not be shown
by DGGE, the presence of this organism was confirmed microscopically, molecularly
and by isolation of strains from a pond sample containing such a Dead Sea-Red Sea
water mixture. When the water mixture was composed of 70% Dead Sea water and 30%
Red Sea water (pond #1), the only bacterial 16S rRNA partial gene sequence detected in
the DGGE belonged to Massilia sp. or Naxibacter sp. This result may be due to
contamination of the sample, because some members of those genera are often present
in the air and so far, they do not include any halophilic species. However, the existence
of salt-tolerant or salt-requiring relatives of Massilia or Naxibacter cannot be excluded.
No conclusions could be obtained in this work regarding the bacterial composition of
the other ponds with different ratios of Dead Sea-Red Sea water mixtures.
From the Dead Sea samples, two strains were isolated for characterization.
Preliminary results placed the isolates in the family Halobacteriaceae, as members of
the Halorhabdus genus (Wainø et al., 2000). This genus is composed of two species,
Hrd. tiamatea and Hrd. utahensis. The type strains of both species share 99% similarity
of the 16S rRNA gene sequences, but are sufficiently different to be considered separate
species (Antunes et al., 2008). The same similarity of the 16S rRNA gene sequence was
observed in strains S21 and S22, but some of the isolates’ characteristics indicated that
they are closer to Hrd. utahensis, for instance the pink pigmentation of the cells, their
ability to grow under aerobic and anaerobic conditions, and better growth on simple
rather than complex substrates. Hrd. tiamatea is non-pigmented, grows optimally under
anaerobic condition and utilizes complex substrates for growth (Antunes et al., 2008).
The hypothesis that strains S21 and S22 represent a novel species in the
Halorhabdus genus arose due to the differences in the polar lipids patterns of Hrd.
utahensis and the Dead Sea isolates. However, the slow growth of the new strains
prevented their complete characterization before the conclusion of this work.
Nevertheless, the isolation of members of the Halorhabdus genus from the Dead Sea
was important to confirm that life still exists in this hypersaline lake, even at the current
increasingly harsh conditions. This is the first time that members of this genus have
been isolated from the Dead Sea. One reason may be the slow growth of the strains;
another hypothesis is that changes in the environment allowed the Halorhabdus
population to develop and overgrow other organisms that prevailed in the community
until now. The presence of Halorhabdus members in the Dead Sea was also confirmed
by Bodaker et al. (2009) through metagenomics of the environmental samples.
If the Peace Conduit will be implemented, changes in the archaeal community of
the Dead Sea will be expected. The DGGE experiments with 16S rRNA genes
amplified from the experimental ponds using universal primers for Archaea showed that
the community diversity will significantly change according to the amount of Red Sea
water poured into the hypersaline lake. The greatest number of bands was observed
when Red Sea water represented 35% of the water mixture (pond #2), and was very
similar to the pattern observed with 30% Red Sea water (pond #3). The sample
containing the smaller amount of bands was that containing 80% Dead Sea water and
20% Red Sea water (pond #7), probably because only a small number of species can
tolerate such a high salinity. It is important to stress that, although Dead Sea-Red Sea
mixtures at other ratios presented less bands that pond #2, it doesn't mean they were less
populated; it is just an indication of the variety of species present, not of the total
number of cells. No Halorhabdus-specific bands (as obtained with cultures of Hrd.
utahensis and of strains S21 and S22 that were also included in the DGGE experiment)
were amplified from the pond samples, indicating that Halorhabdus was not present in
the ponds or existed in a small number, below the detection limit of the PCR.
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7. ARTICLES AND ABSTRACTS BASED ON THIS THESIS
Vaisman, N. and Oren, A. 2009. Salisaeta longa gen. nov., sp. nov., a red halophilic
member of the Bacteroidetes. Int. J. Syst. Evol. Microbiol. 59, 2571-2574.
Baronio, M., Lattanzio, V. M. T., Vaisman, N., Oren, A. and Corcelli, A. The
acylhalocapnines of halophilic bacteria: structural details of unusual sulfonate
sphingoids. J. Lipid Res., submitted for publication.
Vaisman, N. and Oren, A. 2008. Salinisaeta longa gen. nov., sp. nov., a new halophilic
bacterium isolated from an experimental mesocosm at Sedom. Annual Meeting of
the Israel Society for Microbiology, Rehovot.
Vaisman, N. and Oren, A. 2008. Salinisaeta longa, gen. nov., sp. nov., a new halophilic
bacterium isolated from an experimental mesocosm with a Dead Sea water – Red
Sea water mixture. Extremophiles 2008, Cape Town.
Vaisman, N. and Oren, A. 2009. Salisaeta longa: isolation and properties of a new
halophilic bacterium from hypersaline mesocosm ponds. American Society for
Microbiology annual meeting, Philadelphia.
‫תקציר‬
‫ים המלח נמצא במגמת התייבשות‪ .‬במאה ה‪ ,20-‬מפלסו ירד ביותר מ‪ 20-‬מטרים‪ ,‬ובעשור האחרון‪ ,‬מפלס מי‬
‫הים ירד בממוצע במטר לשנה‪ .‬כיום האגם הינו רווי ב‪ NaCl-‬ובעקבות שקיעה אדירה של מלח נגרמה‬
‫ירידה בריכוזי ה‪ Na+-‬ועלייה בריכוזי ה‪ .Mg2+-‬ההרכב הכימי של האגם נעשה עם השנים עשיר יותר‬
‫בקטיונים דו‪-‬ערכיים‪ .‬בשל כך‪ ,‬התגברו תנאי הסביבה הקיצוניים לחיים המיקרוביאליים‪ .‬אף על פי התנאים‬
‫הקשים הללו‪ ,‬האגם הוכח כבית גידול המכיל מגוון מיקרואורגניזמים‪ ,‬ביניהם נציגים של קבוצת הבקטריה‪,‬‬
‫אצות חד‪-‬תאיות‪ ,‬פיטריות‪ ,‬נגיפים ובעיקר‪ ,‬ארכייה‪.‬‬
‫במטרה למנוע את ייבושו של האגם ולהביאו אל רמת המפלס הרצויה‪ ,‬הועלתה ההצעה להקים תעלת‬
‫מים התקשר בין ים המלח לים סוף )"תעלת הימים"(‪ .‬עם הקמת פרויקט "תעלת הימים" יוזרמו נפחי מים‬
‫גדולים מים סוף אל ים המלח‪ ,‬דבר שעתיד לגרום להקטנת המליחות של שכבותיו העליוניות של האגם‪ .‬על‬
‫מנת לחזות את ההשפעה העתידית של מיהול ים המלח על מאפייניו המיקרוביאליים‪ ,‬נערכים בשנים‬
‫האחרונות מחקרים בשטחם של מפעלי ים המלח בסדום‪.‬‬
‫דוגמאות נאספו מים המלח ומברכות ניסיוניות בסדום )המכילות תערובת של מי ים המלח ומי ים סוף(‬
‫לשם בידוד ואפיון של חיידקים שלא היו מוכרים טרם למדע‪ .‬בודדו שני זנים מסוג ה‪ Halorhabdus-‬מים‬
‫המלח ואופיינו באופן חלקי‪ .‬בידוד זה מתאים לתוצאות המחקר של בודקר ושות' ‪(Bodaker et al.,‬‬
‫)‪ ,2009‬שהצביעו על הימצאות רצפים השייכים לקבוצה זו בים המלח על ידי שימוש בשיטה מולקולארית‪.‬‬
‫זן שבודד מבריכה ניסיונית שהרכב המים בה ‪ %80‬מי ים המלח ו‪ %20-‬מי ים סוף אופיין כחיידק‬
‫חדש‪ ,‬הנקרא ‪ .Salisaeta longa‬החיידק הקרוב ביותר אליו הוא ‪ ,Salinibacter ruber‬בעל צורה‬
‫מאורכת‪ ,‬אדום‪ ,‬השייך לאגף ה‪ Bacteroidetes-‬של הבקטריה‪ .‬אך עם זאת מקבץ מתכונותיו הפיזיולוגיות‬
‫נמצאו דומות יותר ל‪.Halobacteriales-‬‬
‫‪ Salisaeta longa‬מאופיין בתאים ארוכים )‪ (15-30 µm‬והוא פחות הלופילי מ‪Salinibacter -‬‬
‫‪ ,ruber‬למרות עמידותו לריכוזי מגנזיום גבוהים‪ .‬גם ‪ Salisaeta‬וגם ‪ Salinibacter‬צוברים ‪ K+‬תוך‪-‬תאי‬
‫בריכוז גבוה כמנגנון של הלואדפטצייה‪.‬‬
‫צפייה מיקרוסקופית של דוגמאות מהבריכות המכילות מי ים המלח ומי ים סוף בכמויות שונות‬
‫מהבריכה שממנה נבדד ‪ S. longa‬הראו תאים ארוכים בדומה לתאי ‪ .S. longa‬אחרי זריעה של הדוגמאות‬
‫הללו על המצע ההופטימאלי לגידול ‪ ,S. longa‬בודדו חמישה זנים הנראים ‪,‬גם מבחינת המושבה וגם‬
‫מבחינת מורפולוגית התאים‪ ,‬כמו ‪ .S. longa‬בהשוות רצפי הגנים המקודדים ל‪ 16S rRNA-‬נתגלה ‪%99‬‬
‫דמיון ל‪.S. longa-‬‬
‫במטרה להגביר באופן ספציפי מקטע חלקי של הגן המקודד ל‪ 16S rRNA-‬של ‪ Salisaeta‬עוצבו זוג‬
‫פריימרים‪ .‬המקטע הרצוי הוגבר מדנ"א שהופק מ‪ ,S. longa S4-4T-‬מחמשת הזנים הנ"ל‪ ,‬ובאופן ישיר‬
‫משתי ברכות ניסיוניות‪ .‬לא הייתה הגברה כשנעשה שימוש בדנ"א שהופק מ‪,Salinibacter ruber-‬‬
‫‪ Escherichia coli‬או דוגמאות סביבתיות אחרות כדגם לריאקצית ה‪ .PCR-‬סף הדטקציה של השיטה היה‬
‫‪ 104‬תאי ‪ Salisaeta‬לדוגמה‪.‬‬
‫ניסוי ‪ DGGE‬נעשה עם מקטעים חלקיים של הגנים המקודדים ל‪ 16S rRNA-‬של ארכייה ובקטריה‬
‫שהוגברו מברכות ניסיוניות המכילות כמויות שונות של מי ים המלח ומי ים סוף )מ‪ %80-‬ל‪ %40-‬מי ים‬
‫המלח(‪ ,‬כדי לגלות האם קיימת השפעה על הקהילה המיקרוביאלית המאכלסת את הברכות‪ .‬הקהילה של‬
‫הארכייה מאוד הושפעה מההבדלים בהרכב המים‪ ,‬והראתה את השונות הגבוהה ביותר כאשר הדגימה הכילה‬
‫‪ %35‬מי ים סוף בנוסף למי ים המלח‪ .‬היה בלתי אפשרי לבדוק את הקהילה הבקטריאלית היטב בגלל בעיות‬
‫ב‪ PCR -‬הראשוני‪ .‬אף על פי זאת‪ ,‬מהדוגמאות שמהן ניתן היה לקבל הגברה )ברכות המכילות ‪ %70‬או‬
‫‪ %60‬מי ים המלח(‪ ,‬רק מקטע אחד הופיע בג'ל‪ .‬ריצוף המקטע הנלקח מברכה המכילה ‪ %60‬מי ים המלח ו‪-‬‬
‫‪ %40‬מי ים סוף הראה ‪ %99‬דמיון עם ‪.Salisaeta longa‬‬
‫עבודה זו נעשתה בהדרכת פרופ' אהרון אורן‬
‫המחלקה למדעי הצמח והסביבה‪ ,‬המכון למדעי החיים‬
‫האוניברסיטה העברית בירושלים‬
‫בידוד ואפיון של חיידקים הלופיליים חדשים מים‬
‫המלח ומברכות ניסיוניות עם תערובות מי ים המלח‬
‫ומי ים סוף‬
‫עבודת גמר‬
‫מוגשת לחוג העל‪-‬פקולטאי לביוטכנולוגיה‬
‫של האוניברסיטה העברית בירושלים‬
‫לשם קבלת תואר "מוסמך בביוטכנולוגיה"‬
‫נטלי וייסמן‬
‫‪1.12.2009‬‬