In: Proteobacteria: Phylogeny, Metabolic Diversity and Ecological EffectsISBN: 978-1-61761-810-9
Editors: Maria L. Sezenna
©2010 Nova Science Publishers, Inc.
Chapter 5
A HARSH LIFE TO INDIGENOUS
PROTEOBACTERIA AT THE ANDEAN MOUNTAINS:
MICROBIAL DIVERSITY AND RESISTANCE
MECHANISMS TOWARDS EXTREME CONDITIONS
Virginia Helena Albarracín 1,2, Julián Rafael Dib1,3,
Omar Federico Ordoñez1, María Eugenia Farías*1
1
Laboratorio de Investigaciones Microbiológicas de Lagunas Andinas (LIMLA),
Planta Piloto de Procesos Industriales Microbiológicos (PROIMI),
CCT, CONICET, Tucumán, Argentina
2
Facultad de Ciencias Naturales e Instituto Miguel Lillo,
Universidad Nacional de Tucumán, Tucumán, Argentina
3
Facultad de Bioquímica, Química y Farmacia,
Universidad Nacional de Tucumán, Tucumán, Argentina
SUMMARY
High-altitude Andean lake (HAAL) ecosystems of the South American Andes are
almost unexplored systems of shallow lakes formed during the Tertiary geological period,
distributed in the geographical area called the Puna at altitudes from 3,000 to 6,000 m
above sea level, and isolated from direct human activity. They present a broad range of
extreme conditions which makes the indigenous microbial communities exceptionally
interesting to study physiological mechanisms of adaptation to chemical and physical
stresses such as hypersalinity and high levels of UV radiation. Previous work have
revealed the outstanding diversity of these environments, being Proteobacteria the most
extended and best represented microbial taxa within the extremophilic communities. The
aim of this work is to review the microbial diversity of Proteobacteria present at the
HAAL and to describe their multiple resistance properties towards the extreme factors
that these microbial communities thrived in their natural environments. A special
* Corresponding author: María Eugenia Farias, LIMLA-PROIMI-CCT, Av. Belgrano y Pasaje Caseros. 4000
Tucumán, Argentina. Tel: +54-381-4344888 Int. 24. Fax: +54-381-4344887 www.limla.com.ar
2
Virginia Helena Albarracín, Julián Rafael Dib, Omar Federico Ordoñez, et al.
reference to the representatives of the genus Acinetobacter found at the HAAL is also
presented.
Due to the isolation program held at LIMLA (www.limla.com.ar) during the past
four years a one-of-a-kind collection of extremophilic strains from the HAAL was
assembled. HAAL microbial diversity was investigated by sampling bacterioplankton,
benthonic microorganisms, microbial-mat associated microbes as well as gastrointestinal
symbiotic organisms from flamingoes living at the lakes. Representatives of
Proteobacteria has been profusely isolated from these samples, more exactly from Lakes:
Azul, Verde, Negra, Vilama, Aparejos, Chaxas, Salina Grande, Socompa, Dead Man
Salar, Tolar Grande, Brava, Diamante, Huaca-Huasi, all of them located above 4000 m,
at the Northwest of Argentina. In addition, a more extended coverage of Proteobacteria
was detected by non-culture dependent techniques (mainly DGGE), suggesting that much
more efforts will be needed to isolate most novel Proteobacteria present at the HAAL.
Within Proteobacteria, all the four main groups were represented in our culture
collection being the Gammaproteobacteria the class with better coverage. The
gammaproteobacteria strains were classified as belonging mainly to Pseudomonas
Acinetobacter, Halomonas, Stenotrophomonas, Moraxella, Enterobacter, Serratia,
Salinivibrio, Pseudoalteromonas, Aeromonas and Marinobacter. 16S rDNA gene
sequence comparison of some isolates with the ones presented at the database indicated
an identity lower that 94%, which should point out that these extremophilic communities
harbour yet unraveled species.
The extreme conditions suffered by these microorganisms at the HAAL made them
resistant to factors present as well as not present in their natural environments. Exposure
to UV-B radiation during 24 h revealed that most isolates were highly resistant: 33.3% of
betaproteobacteria, 44.4% of gammaproteobacteria, 40% of alphaproteobacteria were
able to survive through the whole exposition time. In addition, resistance to hipersalinity
in most isolates was also observed.
Interestingly, antibiotic resistance was also observed in spite of the pristinely and
isolation of these lakes. In light of the great adaptability strength of the strains to
changing conditions in their original environment, antibiotic resistance may be
considered as a consequence of a high frequency of mutational events, which also, may
be enhanced by the intense solar irradiation present at the HAAL (UV index in summer:
16- 18).
A special reference can be made to the representatives of the genus Acinetobacter
isolated from the HAAL. Most of these strains appeared to have multiple resistance
profiles to hipersalinity, UV-B irradiation, antibiotics and even arsenic. These
“superbugs” can be subjected to further studies as they can be clues to discover new ways
of surviving at extreme conditions, a matter that has applications in astrobiology. On the
other hand, it will be very interesting to further research on these strains biotechnological
potential because as extremophiles they can be source of novel bioactive compounds.
Key words: extremophiles, High-Altitude Lakes, Proteobacteria
PUNE-ANDINE HIGH-ALTITUDE LAKES: A DIVERSE SOURCE OF
POLI-EXTREMOPHILIC MICROORGANISMS
Extreme environments are defined as habitats that experience steady or fluctuating
exposure to one or more environmental factors, such as salinity, osmolarity, desiccation, UV
radiation, barometric pressure, pH, and temperature. Under these physical conditions human
life can not be possible. Nevertheless, some microorganisms can colonize these extreme
A Harsh Life to Indigenous Proteobacteria at the Andean Mountains: …
3
environments and they are called extremophiles; this group includes representatives of all
three domains (Bacteria, Archaea, and Eukarya); they are categorized into subgroups
according to the specific environmental characteristics of their habitats, i.e. psycrophilic,
thermophilic, halophilic, alkalophilic, acidophilic (Seufferheld et al., 2008).
Extreme environments have been subject to intensive studies focusing attention on the
diversity of organisms and molecular and regulatory mechanisms involved. The products
obtainable from extremophiles such as proteins, enzymes (extremozymes) and compatible
solutes are of great interest to biotechnology. Examples include biochemicals used for
detergent formulations, leather and paper processing, biofuels, bioremediation, UV-blocking,
and new antibiotics (Sanchez et al., 2009; Bowers et al., 2009). Nevertheless, potentially
beneficial biomolecules still remain to be discovered from unexplored extreme environments.
This field of research has also attracted attention because of its impact on the possible
existence of life on other planets (Rothschild and Mancinelli, 2001, Cavicchioli, 2002).
Figure 1. Six extreme lakes were proteobacteria diversity was mainly studied.
Typical examples of extreme environments are the High-Altitude Andean Lakes
(between 3,000 and 6,000 m asl) at the northwest of Argentina in the Puna and Andean
regions (Fig. 1). Most of these wetlands are completely isolated, experience a wide daily
range in temperatures (35 ºC), are slight saline to hypersaline, and are subject to low
phosphate availability and to high intensity of solar UV-B radiation (Table 1). Microbial
communities living in such aquatic ecosystems are tolerant to large fluctuations in
environmental factors in addition to steady-state extreme conditions (Fernandez Zenoff et al.,
4
Virginia Helena Albarracín, Julián Rafael Dib, Omar Federico Ordoñez, et al.
2006; Zenoff et al., 2006; Dib et al., 2008). These ecosystems have demonstrated to be a great
source of microbial diversity and interesting strategies that allow microorganisms to survive
under severe conditions.
Table 1. Characteristics of High altitude Andean lake (HAAL) in Argentina: L.
Aparejos, L. Negra, L. Verde, L. Azul, L. Vilama, Salina Grande and L. Socompa.
Wetland
Geographic position
Global position
L. Aparejos L. Negra L. Verde L. Azul L. Vilama Salina Grande L. Socompa
Catamarca Catamarca Catamarca Catamarca Jujuy
Jujuy
Salta
27º 34´S 27º40´S 27º 38'S 27º 38´S 22º 35´S
23º 36´S
24º 28´ S
68º 23´W 68º 23´W 68º 32' W 68º 32´W 66º 55´W 66º 55´W
68º 17´ W
10
20
20
100
20
ND
30
4,200
4,400
4,400
4,400
4,600
3,400
4,000
6.5
6.8
6.7
7.5
7.1
ND
8.5
2.5
3
0.8
0.8
11.8
ND
33.81
ND**
<0.05
<0.012
<0.012
ND**
ND
34.96
0.4
32
5
5
117
113
85
6.05
0.63
1.04
0.2
12.8
ND
ND
Depth (cm)
Altitude (masl)
pH
Arsenic (mg L-1)
Phosphorus (mg L-1)
Salinity (ppm)
Chlorophyll (µg L-1)
Max UV-B registered in
situ (W m-2; 280-312
nm)
9.8
10.8
10.78
ND Non determined; **Below detection limits.
8.94
ND
ND
10.78
Due to the isolation program held at LIMLA (www.limla.com.ar) during the past four
years a one-of-a-kind collection of extremophilic strains from the HAAL was assembled
(Fernandez Zenoff et al., 2006, Zenoff et al., 2006, Dib et al., 2008; 2009; Ordoñez et al.,
2009; Farías et al., 2009; Flores et al., 2009). HAAL microbial diversity was investigated by
sampling bacterioplankton, benthonic microorganisms, microbial-mat associated microbes
(including modern stromatolites) as well as gastrointestinal symbiotic organisms from
flamingos living at the lakes (Ordoñez et al., 2009; Farías et al., 2009; Flores et al., 2009;
Belluscio, 2009; 2010). Most isolates belonged to Eubacteria, Firmicutes, Actinobacteria,
Proteobacteria (gamma, alpha and beta) and Archaea; they displayed resistance to multiple
environmental stress such as UV-B radiation, arsenic, hipersalinity, alkalinity, and antibiotics
(Ordoñez et al., 2009; Dib et al., 2009; Flores et al., 2009) and, in this sense, they can be
considered poly-extremophiles (Bowers et al., 2009).
Representatives of proteobacteria has been profussally isolated from these samples, more
exactly from Lakes: Azul, Verde, Negra, Vilama, Aparejos, Chaxas, Salina Grande,
Socompa, Dead Man Salar, Tolar Grande, Brava, Diamante, Huaca-Huasi, most of them
located above 4,000 m, at the Nortwest of Argentina.
The aim of this chapter is to review the microbial diversity of Proteobacteria present at
the HAAL and to describe their multiple resistance properties towards the extreme factors
that these microbial communities thrived in their natural environments. A special reference to
the representatives of the genus Acinetobacter found at the HAAL is also presented.
A Harsh Life to Indigenous Proteobacteria at the Andean Mountains: …
5
ENVIRONMENTAL DESCRIPTION OF THE GEOGRAPHICAL
AREA AT THE HAAL
The HAAL are located in the Eco-region of the Puna and High-Andes, herein a brief
description of the area taking in account previous works (Reboratti, 1994; Braun Wilke and
Guzmán, 2003; Chebez, 2005; Reboratti, 2010).
The Puna (P) is a plateau area with an extension of 12,500 ha, located above 3,000 m in
the Northwest of Argentina, including the provinces of Salta, Jujuy, Catamarca, La Rioja and
San Juan (Fig. 1). The Puna also extended itself across political limits through Bolivia and
Chile. By the east border, series of valleys and “quebradas” (Humahuaca, del Toro,
Calchaquíes) are present and constitute biological connection areas as well as important
communication roads between cities.
The Puna has a clear plane relief, with occasional mountains crossing the area that allow
to demarcate close basins, very characteristic of this environment. In fact, most Puna region
represent (except from the North Section) a big arreic basin, fragmented in a system of minor
basins interrelated among each other. At the bottom of those basins, big lakes are developed
(Guayatayoc, Vilama), with variable limits due to the alternance of inter-annual irregular
precipitations and also because of the sporadic presentation of the meteorological
phenomenon called “El Niño”, which favour the dryness in the Northwest. When the lakes get
dry, they give birth to large salterns (Olaroz, Hombre Muerto), due to the high concentration
of minerals in the water.
Precipitations in the area are scarce, less than 350 mm per year and due to the highaltitude, the temperatures are low with an average of 10 ºC. In winter, minimal temperatures
can reached -15 ºC. But the dryness of the weather, favour a daily wide temperature range,
differences between day and night’s temperature can reached 35 ºC. Soils are generally sandy
or formed by stones, with scarce organic matter. The biome is typically an “estepa” colonize
by small bushes.
The High Andes (HA; 12,000,000 ha) are all the mountains located above 3,000 m at the
West of Argentina. These environments displayed long areas, isolated among each other.
High Andes are fused in some areas with the Puna Plateau and Prepuna also. The relief is
covered by mountains, quebradas, deep valleys, shaped by glacial activities and abundant
“morrenas”. Due to the high altitudes, the temperatures are low, even in summer with a wide
thermal range. Precipitations are scarce and most of them are in form of snow. The High
Andes are important reservoirs of frozen water as glaciers and “eternal snows”.
Within the P-HA landscape, almost all lakes harbouring extreme microbial communities
have been profusely sampled and studied by our research group since 2002 (Ferrero et al.,
2004; Zenoff et al., 2006; Fernandez Zenoff et al., 2006; Dib et al., 2008; Ordoñez et al.,
2009; Farías et al., 2009; Flores et al., 2009; Dib et al., 2010a; 2010b). These lakes are
distributed at the provinces of Salta, Jujuy, Catamarca, La Rioja and Tucumán, Northwest
Argentina and they are called as Lagunas (L) Vilama, Pozuelos, Azul, Verde, Negra, Brava,
Diamante, Aparejos, Chaxas, Salina Grande, Huaca-Huasi, Dead Man Salar, Laguna
Socompa, Sea Eyes of Tolar Grande (Fig. 1). Difficult to explore, these aquatic ecosystems
present several interesting properties to study extreme biological systems: (i) they are pristine
and isolated with no access roads; (ii) they are distant from each other (more than 500–700
km); (iii) they are located at high altitudes (between 3,000 and 6,000 m above sea level: m
6
Virginia Helena Albarracín, Julián Rafael Dib, Omar Federico Ordoñez, et al.
asl) and surrounded by desert, implying that few clouds shade the UV irradiation. (iv) they
are the habitat of enormous populations of three flamingos species, that migrate among these
wetlands and act as microbial dispersers; (v) they are oligotrophic, resulting in deep UV
penetration in the water column; (vi) they are subject to daily large temperature fluctuations
(up to 35 ºC of difference within day and night); (vii) they displayed high salinity and high
arsenic content (of geochemical origin) (Flores et al., 2009; Farías et al., 2009; Dib et al.,
2009). For instance, L. Azul is an oligotrophic lake located at 4,560 m asl. It is part of the
Salar de la Laguna Verde in the Andean region of Catamarca province, Argentina (27º 34’S,
65º 32’W). The location is a very isolated site, with no access roads. Rainfall is scarce so the
lakes are shallow and present a high metal content. Measured arsenic content was 0.014 mg
L-1, and salinity was 5 mg L-1. In the sampling day at noon, in the austral summer, the
maximal UV-B irradiance reached 10.8 W m-2 for the 300 to 325 nm range.
In turn, L. Vilama is located in the plateau of Jujuy Province at 4,600 m asl (22º 30’S, 66º
50’W). Climatic and geographical conditions are similar to those at L. Azul. The arsenic
concentration found in this lake was 3.1 mg L-1, and the salinity, 117 mg L-1, during the dry
season. It is included in the List of Wetlands of International Importance (RAMSAR). More
information about these and the other HAAL is given in Table 1.
Thus, the HAAL are natural laboratories for exploring and monitoring in situ interactions
between the geophysical environment and the dynamics of biodiversity. UV irradiation is
without doubt the one factor with most pressure on the ecology of the microbial communities
thriving on these shallow lakes (Fernandez Zenoff et al., 2006; Zenoff et al., 2006; Ordoñez et
al., 2009).
PROTEOBACTERIA: WORLDWIDE DIVERSITY AND
ECOLOGICAL FUNCTIONS
The Proteobacteria class was first proposed as the name for a new higher taxon to
circumscribe the α, β, γ, δ and ε groups that were once included among the phylogenetic
relatives of the purple photosynthetic bacteria (Trust et al. 1994). This group has evolved
relatively rapidly to generate a number of branches, including organisms of great biological
significance but startlingly different physiological attributes.
Within the alphaproteobacteria, a diverse class of organisms with many important
biological roles can be found (Williams et al., 2007). They frequently adopt an intracellular
lifestyle as plant mutualists or plant or animal pathogens (Batut et al., 2004). This has led to
independent paths of genome reduction in several alphaproteobacterial lineages, but lineagespecific genome expansions are also apparent, with some genomes divided among multiple
replicons that can include linear chromosomes (Boussau et al., 2004).
Special interest attaches to the alphaproteobacteria as the ancestral group for
mitochondria (Williams et al., 2007). The Rickettsiales are most often cited as the
alphaproteobacterial subgroup from which mitochondria arose, but there has been
disagreement on this point (Esser et al., 2004).
The alphaproteobacteria include the most abundant of marine cellular organisms
(Giovannoni et al., 2005). A variety of metabolic strategies are found in the class, including
photosynthesis, nitrogen fixation, ammonia oxidation, and methylotrophy. Stalked, stellate,
A Harsh Life to Indigenous Proteobacteria at the Andean Mountains: …
7
and spiral morphologies are found. Developmental programs occur that switch between cell
types, controlled by a web of regulatory systems (Viollier and Shapiro, 2004).
Alphaproteobacteria have been also described as important microorganisms belonging to
the bacteria community from sediments, plankton or neuston of diverse lakes (Hervas and
Casamayor, 2009; Ordoñez et al., 2009; Hutalle-Schmelzer et al., 2010; Shi et al., 2010).
Not less diverse and widespread are the Beta, Delta and Gammaproteobacteria divisions;
they have been described as part of microbial population of soils, marine and freshwater
sediments, bacterioplankton, among other many biotopes, depicting important ecological
functions on most of them. For instance, in Jiaozhou Bay, China, sediment ammoniaoxidizing betaproteobacteria have been used as bioindicators of environmental gradients and
coastal eutrophication (Dang et al., 2010). Betaproteobacteria also play key roles in nutrient
cycling and plant growth promotion in the rhizosphere environment (Inceoglu et al., 2010).
Betaproteobacteria were described as important constituents in freshwater epilithic biofilms
of Lake Constance, Germany which were dominated by the diatom Cymbella microcephala
(Bruckner et al., 2008). The special contribution of the proteobacteria group to the biofilm
formation seems to rely in their ability for inducing the polysaccharide secretion by the algae
(Bruckner et al., 2008). In addition, gammaproteobacteria and betaproteobacteria were the
most abundant microorganisms found associated with cyanobacterial blooms (Berg et al.,
2009). Indigenous marine bacteria occurring in most marine sediments belong mainly to
different subclasses of Proteobacteria and are actively involved in geobiochemical cycles
(Teske et al., 2000; 2002). Ettoumi et al., (2010) found by denaturing gradient gel
electrophoresis DGGE a high proportion of proteobacteria (20 sequences representing
74.07% of the eluted bands) with the gamma subclass being predominant, representing 55%
(n = 15) and recovered from all stations and depths. On the basis of DGGE results and taking
into account the prevalence of gammaproteobacteria as the major active marine community,
strain isolation was performed. The results confirmed that the most abundant group on the
collection was the gammaproteobacteria (n=31) representing 77% of the isolates.
In turn, the Epsilonproteobacteria division or rRNA superfamily VI include
physiologically and phylogenetically diverse members from a variety of habitats (for a
review, see On, 2001) such as the gastrointestinal tracts of animals (Engberg et al., 2000),
sulfurous springs (Angert et al., 1998; Rudolph et al., 2001), activated sludge (Snaider et al.,
1997), oil fields (Gevertz et al., 2000), Antarctic Ocean water (Bano and Hollibaugh, 2002),
and deep-sea cold seep sediments (Li et al., 1998; Inagaki et al., 2002). Deep-sea
hydrothermal systems, however, may host the largest biomass and diversity of
epsilonproteobacteria on earth (Haddad et al., 1995; Takai et al., 2003; Nakagawa et al.,
2005).
PROTEOBACTERIAL DIVERSITY AT THE PUNE-ANDINE HIGHALTITUDE LAKES
At a global level, the biodiversity of the Earth’s aquatic systems can be approached by
sampling different ecosystems, each with a different diversity. In this respect, both low and
high salinity systems have received considerable attention. At the seawater end, many studies
have been performed in the last 15 years (Giovannoni et al., 1990; Giovannoni and Rappé,
8
Virginia Helena Albarracín, Julián Rafael Dib, Omar Federico Ordoñez, et al.
2000; Pommier et al., 2007). At the other end, crystallizer ponds from solar salterns have also
been studied extensively (Benlloch et al., 1996; 2001; 2002; Rodríguez-Valera et al., 1999;
Casamayor et al., 2000; 2002; Oren, 2002; Estrada et al., 2004; Pedro´s-Alió, 2005;
Maturrano et al., 2006). Aquatic systems with intermediate salinities (such as the HAAL),
however, have not received much attention. In the case of solar salterns, ponds with
intermediate salinities show relatively high levels of heterotrophic activities (Gasol et al.,
2004).
HAAL microbial diversity was investigated by sampling bacterioplankton, benthonic
microorganisms, microbial-mat associated microbes as well as gastrointestinal symbiotic
organisms from flamingos living at the lakes; this biodiversity has been studied by cultured
and cultured independent methods (Fernandez Zenoff et al., 2006; Zenoff et al., 2006; Dib et
al., 2008; Ordoñez et al., 2009; Flores et al., 2009). Metagenomic determinations have been
performed by DGGE in order to have an overall view of the bacterial community. A total of
182 good quality sequences were obtained from the excised DGGE bands from gels. Most of
the DGGE bands shared similarities with uncultured sequences from GenBank and belonged
to gammaproteobacteria (42%), Cytophaga/Flavobacterium/Bacteroides (CFB) (18%),
firmicutes (11%), alphaproteobacteria (11%), betaproteobacteria (6%), HGC (2%) and
deltaproteobacteria (1%). Gammaproteobacteria and bacteroidetes were the most abundant
groups in all the studied environments (Ordoñez et al., 2009).
On the other hand, an strain collection of extremophilic isolates have been assembled.
These bacteria were isolated by their intrinsic resistances to UV, salinity, arsenic and
antibiotics (Zenoff et al., 2006; Dib et al., 2008; Ordoñez et al., 2009; Flores et al., 2009).
Until this moment, the collection summarizes 160 strains distributed in diverse taxonomical
groups: gamma, beta, alphaproteobacteria, firmicutes, high G-C bacteria, CFB and archaea.
These strains were phenotypic and genotypically characterized by diverse methods; mainly,
16S rDNA sequencing of all bacteria was carried out to achieve identification and their
sequences were deposited in NCBI GenBank. This strains collection is permanently updated
since more bacteria species are periodically added to it after each sampling campaign.
Socompa
10%
VERDE
11%
Alphaproteobacteria
APAREJOS
7%
Betaproteobacteria
Gammaproteobacteria
L. Socompa
L. Azul
NEGRA
15%
S.G.
L. Aparejos
S.G.
21%
AZUL
3%
L. Vilama
L. Negra
L. Verde
VILAMA
33%
A
0%
25%
50%
75%
100%
B
Figure 2. Diversity of Proteobacteria class at the HAAL. A Distribution of proteobacteria within the
extremophile culture collection. B Porcentages of different subclases of Proteobacteria at the studied
lakes.
A Harsh Life to Indigenous Proteobacteria at the Andean Mountains: …
9
Table 2. Phylogenetic affiliation of the Andean Proteobacteria isolated
Closest related
% 16S rDNA Accesion
similarity
number
Sphingomonas sp. Ap5
Sphingomonas sp. N3
Sphingomonas sp. N9
99
99
99
AM711590
AM711581
AM711585
Caulobacter sp. N13
Chelatococcus asaccharovorans
Rhodopseudomonas sp.
Sphingomonas paucimobilis
Agrobacterium tumefaciens LC1
Burkholderia cepacia Ap8
Variovorax paradoxus
Janthinobacterium sp.
82
97
98
97
99
99
98
98
AM712181
AM882692
AM882698
AM882688
In process
AM711592
AM882682
AM882691
Water and
faeces
Water
Water
Water and
Faeces
Faeces
Water
Faeces
Water
Water
Faeces
Faeces
Curvibacter lanceolatus V14
97
Beta proteobacterium Aquaspi A
V15
97
AM765997
Water
Pseudomonas plecoglossicida Ap1 99
AM711587
Pseudomonas plecoglossicida Ap3 99
Pseudomonas plecoglossicida Ap17 99
AM711588
AM711596
Stenotrophomonas maltophilia N7
Burkholderia cepacia N12
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Holomonas sp. SNE
Idiomarina loihiensis SND
Acinetobacter sp. LCE1
Acinetobacter sp. LCE2
Stenotrophomonas sp. LAP1
Pseudomonas sp. LAM2
Aeromonas salmonicida RP1
Aeromonas sp RP3
Stenotrophomonas maltophilia RP5
Halomonas sp. SV 2.18
Marinobacter sp. SV 10.18
Alkalilimnicola sp. SV 12.18
Alkalispirillum sp. SV 5.18
Halomonas sp. SV 9.25
Halomonas sp. SV 125.18
99
99
97
97
98
99
99
99
87
99
99
99
77
96
96
96
94
97
96
Halomonas sp. AVb
Halomonas sp. AVf .18
AM765998
Isolated from
Resistant
Profile
Taxonomy
ATBr
ATBr
ATBr
Alfa proteobacteria
Alfa proteobacteria
Alfa proteobacteria
ATBr
ATBr
ATBr
ATBr
ClNa 10%
ATBr
ATBr
ATBr
Alfa proteobacteria
Alfa proteobacteria
Alfa proteobacteria
Alfa proteobacteria
Alfa proteobacteria
Beta proteobacteria
Beta proteobacteria
Beta proteobacteria
UVRr
Beta proteobacteria
r
Beta proteobacteria
UVR
AM711584
AM711586
AM882694
AM882689
In process
In process
FM865881
FM865882
FM865888
FM865887
In process
FM865884
FM865885
FN996001
FN996002
FN996003
FN996005
FN996006
FN996007
Water
Water and
faeces
Water and
faeces
Water
Water and
Faeces
Water
Faeces
Faeces
Sediment
Sediment
Water
Water
Water
Water
Water
Water
Water
Sediment
Sediment
Sediment
Sediment
Sediment
Sediment
97
FN996008
Water
98
FN996009
Water
Marinobacter sp. AVdch .18
98
FN996004
Halomonas sp. AVdgde .18
Acinetobacter sp. N40
Pseudomonas sp. N23
Pseudoalteromonas sp. N32
Acinetobacter sp. Ver5
Acinetobacter sp. Ver3
Acinetobacter junii Ver7
Stenotrophomonas sp. Ver4
Pseudomonas sp. Ver6
Stenotrophomonas sp. Ver8
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Acinetobacter johnsonii A2
Pseudomonas sp. V1
Enterobacter V4
98
99
100
98
99
98
98
97
98
96
98
99
99
98
98
FN996010
AM778696
AM778697
AM778701
AM778688
AM778686
AM778690
AM778687
AM778689
AM778691
AM903332
AM903334
AY963294
AM403128
AM403125
ATBr
Gamma proteobacteria
ATBr
ATBr
Gamma proteobacteria
Gamma proteobacteria
ATBr
ATBr
ATBr
ATBr
ClNa 10%
ClNa 10%
ClNa 10%
ClNa 10%
ClNa 10%
ClNa 10%
ClNa 10%
ClNa 10%
ClNa 10%
ClNa 18%
ClNa 18%
ClNa 18%
ClNa 18%
ClNa 18%
ClNa 18%
ClNa 18%
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
ClNa 18%
Gamma proteobacteria
Water
ClNa 18%
Gamma proteobacteria
Water
Water
Water
Water
Water
Water
Water
Water
Water
Water
Water
Water
Water
Water
Water
ClNa 18%
UVRr
UVRr
UVRr
UVRr
UVRr
UVRr
UVRr
UVRr
UVRr
UVRr
UVRr
UVRr
UVRr
UVRr
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
10
Virginia Helena Albarracín, Julián Rafael Dib, Omar Federico Ordoñez, et al.
Table 2. (continued)
Closest related
Serratia marcescens V10
Stenotrophomonas maltophilia V11
Salinivibrio costicola V16
Marinobacter sp. LCA6
Halomonas sp. LCA7
Stenotrophomonas maltophilia LDc
Pseudomonas aeruginosa LDd
Pseudomonas plecoglossicida LDe
Halomonas sp. LD2
% 16S rDNAAccesion
similarity
number
95
AM765993
98
AM765994
94
AM765999
99
FM865892
99
FM865893
99
FM865895
98
FM865896
98
FM865897
97
FM865899
Isolated from
Water
Water
Water
Water
Water
Water
Water
Water
Water
Select to
UVRr
UVRr
UVRr
UVRr
UVRr
UVRr
UVRr
UVRr
UVRr
Taxonomy
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Halomonas arcis LD3
Pseudomonas sp. S20
Halomonas sp. S32
Salinivibrio costicola S34
98
99
99
93
FM865883
FN994189
FN994190
FN994183
Water
Sediments
Sediments
Sediments
UVRr
ND
ND
ND
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Shewanella sp. S6
99
FN994184
Microbial mats ND
Gamma proteobacteria
Shewanella putrefaciens S7
Salinivibrio costicola S10B
Salinivibrio costicola S14
99
97
94
FN994185
FR668583
FN994182
Microbial mats ND
Microbial mats ND
Microbial mats ND
Gamma proteobacteria
Gamma proteobacteria
Gamma proteobacteria
Interestingly, proteobacteria is the most predominant taxonomic group within our
collection, covering 71 out of a total of 160 isolates. Thirty-three percent of them were
isolated from L. Vilama, 21% from Salina Grande, 15% from L. Negra, 11% from L. Verde,
10% from L. Socompa and 7% and 3% from L. Aparejos and L. Azul respectively (Fig. 1A).
Within the proteobacteria, it was observed a prevalence of gammaproteobacteria at all lakes
studied; for instance, all isolates from L. Socompa and L. Verde belonged to this group while
93% of the proteobacteria from Salina Grande also correspond to the gamma division. Minor
percentages were found for the other lakes: L. Vilama (73%), L. Negra (72%), L. Aparejos
(60%) and L. Azul (50%) (Fig. 1B).
Further molecular studies let us classified the obtained gammaproteobacteria strains as
belonging to Pseudomonas, Acinetobacter, Stenotrophomonas, Enterobacter, Serratia,
Salinivibrio, Pseudoalteromonas, Aeromonas and Marinobacter genera (Table 2). 16s rDNA
gene sequence comparison of some isolates with the ones presented at the database indicated
a identity lower that 96%, which should point out that these extremophilic communities
harbour yet unravelled species. Most isolates (11) belonged to the genera Stenotrophomonas
sp. and Halomonas sp. Stenotrophomonas spp. were isolated from both, bacterioplankton and
flamingo faeces and as stated by Dib et al. (2010a), it was evident the widespread distribution
of Stenotrophomonas maltophilia at the HAAL. In turn, Halomonas spp. were obtained from
sediments and bacterioplankton only. Another well represented genus within the HAAL was
Pseudomonas sp. (10) whose strains were isolated from bacterioplankton, faeces and
sediments.
HAAL microbial diversity can be compared with those from similar environments; Lake
Tebenquiche is one of the largest saline water bodies in the Salar de Atacama at 2,500 m asl
in northeastern Chile. The analysis of metagenomic data from this lake showed that the
community was clearly dominated by bacteroidetes and gammaproteobacteria (Demergaso et
al., 2008). These results support the idea of the preponderance of the proteobacteria group in
this type of environment. Others studies also show the wide diversity of the group of
proteobacteria, especially gammaproteobacteria in the Lake Chaka, a hypersaline lake on the
A Harsh Life to Indigenous Proteobacteria at the Andean Mountains: …
11
Northeastern Tibetan Plateau (Jiang et al., 2007). The bacterial isolates were distributed into
three major groups: gammaproteobacteria, actinobacteria and firmicutes. In the
gammaproteobacteria group, all isolates belonged to three genera: Halomonas, Pseudomonas
and Shewanella.
RESISTANCE PROFILES OF PROTEOBACTERIA FROM HAAL
EXTREMOPHILIC BACTERIA COLLECTION
The extreme conditions suffered by these microorganims at the HAAL made them
resistant to factors present as well as not present in their natural environments, i.e., UV
radiation, arsenic, hipersalinity and antibiotics.
Ultraviolet Radiation Resistance as the Rule for HAAL´s Proteobacteria
Due to the high altitude and the geographical and physicochemical characteristics of
these lakes, UV-B radiation is one of the most limiting abiotic factors for bacterioplankton
communities (Wilson et al., 2004; Agogué et al., 2005; Alonso-Saez et al., 2006; Fernandez
Zenoff et al., 2006; Hernandez et al., 2007). Solar irradiance at the HAAL can be 165%
higher than at sea level with instantaneous UV-B flux reaching 17 W m−2 (Ordoñez et al.,
2009).
According to biological responses, UV can be divided into three bands: UV-A, UV-B,
and UV-C, and high UV doses are particularly related to cell damage (Coohill et al., 1996).
UV-B (280–320 nm) is detrimental to life because of the strong absorption of wavelengths
below 320 nm by DNA molecules. UV-A (320–400 nm) causes only indirect damage to
DNA, proteins, and lipids through reactive oxygen intermediates (Smith and Walker, 1998;
George et al., 2002).
Exposure to UV radiation is considered to be especially harmful to microorganisms
because they have haploid genomes with little or no functional redundancy, because they are
small, and because they lack thick, protective cell walls (García Pichel, 1994; Martin et al.,
2000; Ponder et al., 2005). Conversely, damage caused by UV in bacterial systems from
aquatic environments eventually affects the whole community, having an impact on
photosynthesis, biomass production, and the community composition (Gascón et al., 1995;
Winter et al., 2001; Alonso and Pernthaler, 2006). The effects of UV on different aquatic
systems have been thoroughly studied, especially in marine environments (Joux et al., 1999;
Alonso-Saez et al., 2006; Alonso and Pernthaler, 2006; Häder et al., 2007). Studies on the
impact of UVR on bacterioplankton have also been carried out in other aquatic systems, such
as alpine lakes, measuring the solar UVR incidence on plankton (Williamson Craige and
Role, 1995; Halac et al., 1997; Winter et al., 2001; Häder et al., 2007). Other authors have
done research on biodiversity in the Himalayas (Liu et al., 2006; Jiang et al., 2007). Previous
studies at our laboratory have demonstrated that bacteria isolated from different Andean
wetlands presented high UV-B resistance profiles. Exposure to UV-B radiation during 24 h
revealed that most isolates were highly resistant: 33.3% of betaproteobacteria, 44.4% of
gammaproteobacteria, 40% of alphaproteobacteria were able to survive through the whole
12
Virginia Helena Albarracín, Julián Rafael Dib, Omar Federico Ordoñez, et al.
exposition time (Fernández-Zenoff et al., 2006; Zenoff et al., 2006; Dib et al., 2008, Ordoñez
et al., 2009). Among them, some gammaproteobacteria showed remarkable resistance i.e.:
Acinetobacter johnsonii A2 from L. Azul (4,400 m), Pseudomonas sp. V1 from L. Vilama
(4,600 m), and Pseudomonas sp. MF10 from L. Pozuelos (3,600 m). No relationship was
found between UV-B resistance of the isolates and the biotope from which they were isolated,
i.e. a hypersaline (L. Vilama or SG) or oligosaline (L. Aparejos or L. Verde) lake (Ordoñez et
al., 2009).
In addition, the impact of solar radiation on bacterioplankton in a hypersaline Andean
lake (L. Vilama, 4,600 m) was measured in situ, demonstrating that bacterioplankton was
well-adapted to high solar irradiance due to the relatively low impact on bacterioplankton
diversity (Farías et al., 2009). Also Agogue et al. (2005) investigated UV resistance from 90
marine isolates of the northwest Mediterranean Sea and they determined that
gammaproteobacteria and bacteroidetes were the most abundant and resistant UV strains.
Similar results by Alonso-Saez et al. (2006) when studying the effects of natural sunlight on
heterotrophic marine bacterioplankton from the Northwestern Mediterranean in short-term
experiments. Members of the gammaproteobacteria and bacteroidetes groups appeared to be
highly resistant to solar radiation, with small changes in membrane integrity and viability
(Alonso-Saez et al., 2006).
Arsenic Resistance as a Natural Phenomenon at the HAAL
Arsenic is a toxic metalloid naturally found as inorganic oxyanion arsenate As(V) and
arsenite As(III) species. Arsenate is the predominant species in oxygenated aqueous
environment, whereas arsenite species predominate under anoxic or reduced conditions, being
100 times more toxic than As(V) (Neff, 1997; Mukhopadhyay et al., 2007; Taerakul et al.,
2007). Arsenic compounds are derived from both natural geothermal and anthropogenic
sources, are widely distributed in the environment (Smedley and Kinniburgh, 2002). Due to
the natural abundance of arsenic in the environment, representatives from various bacterial
genera have developed different resistance mechanisms for arsenic compounds
(Mukhopadhyay et al., 2002; Rosen, 2002). Metal-accumulating bacteria are often found
among metal-resistant bacteria (Pümpel et al., 1995; Srinath et al., 2002; Hussein et al.,
2005). Some bacteria are resistant to arsenic either due to the presence of a strictly phosphatespecific transport system, which prevents the uptake of arsenate which is analogous to
phosphate (Willsky and Malamy, 1980), or due to an efflux system mediated by the plasmidor chromosomally-encoded ars operon (Cervantes et al., 1994; Diorio et al., 1995; Cai et al.,
1998).
Bacterial populations associated with arsenic transformations have been characterized
from diverse environments such as in oxic environments (Macur et al. 2004) and in anoxic
sediments of lakes and rivers naturally contaminated with arsenic (Cummings et al., 1999;
Oremland et al., 2005).
Although pristine environments, the HAAL displayed a high concentration of arsenite in
the water due to a natural geochemical phenomena. For instance, arsenite concentration in L.
Aparejos is 2.5 mg L-1 while L. Vilama present even a higher concentration: 11.8 mg L-1
(Ordonez et al., 2009). Dib et al. (2008) have described in most of the isolated bacteria from
the HAAL arsenic resistance phenotypes, especially to arsenite. Among isolated
A Harsh Life to Indigenous Proteobacteria at the Andean Mountains: …
13
proteobacteria, Pseudomonas sp. V1 and Enterobacter sp. V4 were sensible to arsenite,
whereas A. jhonsoni A2 was the most resistant bacteria (up to 10 mM As(III)). Otherwise,
arsenic resistant bacteria from these environments could thus be candidates for
bioremediation, a methodology considered as low cost environmentally friendly technology
on the bioremediation of metals (Clausen, 2000; Srinath et al., 2002; Tsuruta, 2004).
Halophilic Proteobacteria from the HAAL
A variety of saline and hypersaline ecosystems are present on Earth. The salt
concentration in these environments can vary from 3.5% (w/v) of total dissolved salts, as in
seawater, to concentrations close to saturation (35%). Hypersaline environments are those
containing salt concentrations in excess of seawater. All these environments are ecological
niches of halophilic microorganisms (Oren, 2002a). Moderately halophilic bacteria are a
group of halophilic microorganisms able to grow optimally in media containing 3–15% NaCl
(Ventosa et al., 1998) while extremely halophilic bacteria grow optimally at salt
concentrations from above 20% (w/v) to saturation (Ventosa et al., 2006).
These microorganisms are subject to basic studies in relation to the origin of life in our
planet and the molecular mechanisms of adaptation and strategies to maintain cell structure
and function under saline and hypersaline conditions (DasSarma and Aora, 2002). Apart from
their evolutionary and ecological significance, halophiles have promising biotechnological
applications including food industry pigments, organic osmotic stabilizers, surfactants,
enzymes able to function at low water activities, bacteriorhodopsin applications including
holography, optical computers and optical memory, production of renewable energy and
biodegradation of organic pollutants (Margesin and Schinner, 2001b; Oren, 2002a; 2002).
Most of the HAAL that we are currently studying can be considered hypersaline lakes
exposed to extreme conditions, with salinity ranges at or near saturation (Table 1); yet, they
often maintain remarkably high microbial cell densities and are biologically very productive
ecosystems (Ordoñez et al., 2009; Flores et al., 2009). These features have been observed
before in other hypersaline environments (Rodriguez-Valera 1988; Ramos-Comenzana, 1993;
Ventosa et al., 2006).
Moderately halophilic proteobacteria, including gammaproteobacteria (Pseudomonas,
Enterobacter, Stenotrophomonas, Serratia, Salinivibrio, Acinetobacter, Halomonas,
Aeromonas,
Marinobacter),
betaproteobacteria
(Curvibacter,
Burkolderia)
alphaproteobacteria (Sphingomonas, Caulobacter, Agrobacterium) have been isolated from
the HAAL (Ordoñez et al., 2009; Flores et al., 2009). DGGE analysis in all saline lakes
showed a predominance of gammaproteobacteria and additionally proteobacteria were the
most abundant among of isolated bacteria. Comparable biodiversity for such environment
have been described also in Howz Soltan Lake, in Iran (Rohban et al., 2009), the solar saltern
of Sfax, in Tunisia (Baati et al. 2010), and saline environments in Alexandria Egypt (Ghozlan
et al., 2006).
Flores et al. (2009) tested salinity tolerance and the relation with UV-B resistance of
bacteria isolated from HAAL. Regarding proteobacteria, Pseudomonas sp. N23 and
Acinetobacter sp. Ver5 were grouped among high salinity tolerant since they presented low
difference in specific growth rate of (less than 10%) between 1 and 5 % of NaCl. Medium
salinity tolerance was observed in Acinetobacter sp. Ver3. Only Pseudomonas sp. N23 and
14
Virginia Helena Albarracín, Julián Rafael Dib, Omar Federico Ordoñez, et al.
Pseudoalteromonas sp. N32, both isolated from L. Negra (32 mg L-1 salinity), were able to
grow in media amended with 10% NaCl.
An Unexpected Outcome: Widespread Antibiotic Resistance in the
HAAL´S Proteobacteria
Interestingly, antibiotic resistance was also observed in spite of the pristinely and
isolation of these lakes. Antimicrobial resistance has recently been recognized as a worldwide
ecological problem (Levy, 1997; Levy, 2001; Summers, 2002; Singer et al., 2006). A high
frequency of bacterial resistance to various antimicrobials is well documented in most clinical
isolates (Kadavy et al., 2000; Cole, et al. 2005). Moreover, this phenomenon has also been
reported in wild animal populations and natural water samples (Gilliver et al. 1999; Levy,
1997; Ash et al., 2002; Nascimento et al., 2003; Levy, 2005; Pontes et al., 2007). The
selection pressure applied by the antibiotics that are used in clinical and agricultural settings
has promoted the evolution and spread of genes that confer resistance, regardless of their
origins.
Many of the known antibiotic resistance genes are found on transposons, integrons or
plasmids, which can be mobilized and transferred to other bacteria of the same or different
species (Witte, 1998).
Argentinean high-altitude lakes have showed to be a natural rich reservoir for antibiotic
resistant bacteria as was established previously. (Dib et al., 2008, 2009, 2010a; 2010b).
Antibiotic resistance bacteria were found in the four pristine high-altitude environments
studied: L. Negra, L. Azul, L. Aparejos, and L. Vilama. Fifty-six bacteria were isolated from
water and flamingos feces and identified by 16S rDNA sequencing; most of them belonged to
the proteobacteria taxa,. Antibiotic resistance profiles of isolated bacteria were determined for
22 different antibiotics. All identified bacteria were able to growth in multiple antibiotics.
Colistin, ceftazidime, ampicillin/sulbactam, cefotaxime, cefepime, cefalotin, ampicillin, and
erythromycin were the most distributed resistances among tested bacteria (Dib et al., 2009;
2010c). These results support the preliminary ideas presented in a previous publication by our
group, which postulated that exists a correlation between antibiotic resistance and UV-B
radiation in extreme environments as the HAAL (Dib et al., 2008). Under extreme UV stress
conditions, bacteria are known to increase mutational events as a last resistance mechanism,
called error prone repair (Smith et al., 1998). In many cases, spontaneous resistance to
antibiotics is known to emerge under such mutagenic conditions, as a consequence of
mutagenesis modified potential target genes.
Other authors established a possible connection between oxidative stress resistance and
resistance to antibiotics (Ariza et al., 1995). It is known that UV produces high oxidative
stress and consequently, a highly irradiated environment is expected to select oxidative stress
resistant bacteria. There could also be a relationship with antibiotic resistance found in other
irradiated environments. The fact that antibiotic resistance is more common in irradiated
environments than in non irradiated ones could support this idea.
The ubiquity of multiple antibiotic resistant bacteria in the assayed environments
supports the idea that pathogenic bacteria resistant to multiple antibiotics are not a
phenomenon that is restricted to human-modified environments. Besides, it shows that
pristine environments could be considered important reservoirs of multiple antibiotic
A Harsh Life to Indigenous Proteobacteria at the Andean Mountains: …
15
resistances in bacteria like Staphylococcus sp., Aeromonas sp., Stenotrophomonas maltophila
and a large group of enteric bacteria. Actually, these resistant bacteria could transfer the
resistance genes to human pathogens (Rhodes et al., 2000).
As flamingoes are the main birds inhabiting these lagoons and because they migrate
among these lakes, their antibiotic resistant symbionts could be spread to other places as they
migratory patterns are not well established yet. This may become even more important in the
context of global warming that could modify the behavior and migratory patterns of birds and
bring them and their antibiotic resistant microbiota in close proximity to human communities.
Indeed, there are alarming statistics that probe in the past 2 decades approximately 75% of all
types of emerging human diseases came from wildlife (Bengis et al., 2004). Thus, from an
epidemiological point of view, pristine, UV irradiated environments should receive more
attention as reservoirs of multiple antibiotic resistances (Dib et al., 2009).
ACINETOBACTER: A GENUS WELL REPRESENTED AT THE HAAL BY
POLYEXTREMOPHILIC MICROBES.
The genus Acinetobacter has a long and convoluted taxonomic history dating back to
1911 when Beijerinck isolated and described the first example of an organism that would now
be recognized as Acinetobacter (Beijerinck, 1911). Today, the use of molecular methods has
established the identity of at least 33 different species belonging to the genus Acinetobacter,
of which 18 have now been assigned formal species names (Towner, 2009). A further 28
unnamed groups have been identified that contain multiple strains, and there are also at least
21 ungrouped single strains.
Members of the genus Acinetobacter first began to be recognized as significant
nosocomial pathogens during the early 1970s. In early in vitro studies, most clinical isolates
were susceptible to commonly used antimicrobial agents, such as ampicillin (60-70% of
isolates susceptible), gentamicin (92.5%), chloramphenicol (57%) and nalidixic acid (97.8%),
so that infections caused by these organisms could be treated relatively easily (BergogneBerezin and Towner, 1996). However, multidrug-resistant (MDR) clinical isolates of
Acinetobacter spp. have been reported increasingly during the last two decades, almost
certainly as a consequence of extensive use of potent broad-spectrum antimicrobial agents in
hospitals throughout the world (Towner, 2009).
Likewise, almost the majority of previous works concerning the genus Acinetobacter are
related to the importance of this group in causing nosocomial diseases and because of their
multi-resistance patterns (Smith et al., 2007; Lee et al., 2008; Cevahir et al., 2008).
Nevertheless, less attention has been paid to environmental Acinetobacter strains (Zenoff et
al., 2006; Fernandez Zenoff et al., 2006). In fact, the natural habitats of most species
belonging to the genus Acinetobacter are still poorly defined (Towner, 2009).
Acinetobacter spp. are widespread in nature, and can be obtained from water, soil, living
organisms and even from human skins (Abdel-El-Haleem, 2003). They are oxidase-negative,
non-motile, strictly aerobic and appear as gram-negative coccobacilli in pairs under the
microscope. They can use various carbon sources for growth, and can be cultured on
relatively simple media, including nutrient agar or trypticase soya agar. Also, most members
16
Virginia Helena Albarracín, Julián Rafael Dib, Omar Federico Ordoñez, et al.
of Acinetobacter show good growth on MacConkey agar with the exception of some A.
lwoffii strains (Bergogne-Bérézin, 1996; Towner, 1996).
Natural competence as well as high metabolic capacities has been reported in several
Acinetobacter species (de Vries et al., 2001) making those species very attractive for
environmental and biotechnological use (Abdel-El-Haleem, 2003). For example,
Acinetobacter baylyi ADP1 is highly competent and may grow on a large variety of
compounds (Barbe et al., 2004).
Acinetobacter spp. are known to be involved in biodegradation of a number of different
pollutants such as biphenyl and chlorinated biphenyl, amino acids (analine), phenol, benzoate,
crude oil, acetonitrile, and in the removal of phosphate or heavy metals. Acinetobacter strains
are also well represented among fermentable bacteria for the production of a number of extra
and intracellular economic products such as lipases, proteases, cyanophycine, bioemulsifiers
and several kinds of biopolymers (Abdel-El-Haleem, 2003).
An special reference can be made to the representatives of the genus Acinetobacter that
were profusely isolated from the HAAL in previous screening programs (Fernandez Zenoff et
al., 2006; Zenoff et al., 2006; Ordoñez et al., 2009). These are the unique cases where
Acinetobacter spp. strains were isolated from highly UV-B irradiated bacterioplankton (Mean
value of UV-B irradiance reached 10.8 W m-2 for the 300- to 325-nm range). These works
intends to highlight the importance of this genus on freshwater environments which so far has
not properly studied.
Acinetobacter johnsonii A2 was isolated from an oligotrophic lake located at 4,560 m asl
which is part of the Salar de la Laguna Verde in the Andean region of Catamarca Province
(Zenoff et al., 2006). This strain was isolated by irradiating the water samples with increasing
UV-B doses (up to 106 KJ m-2). This resistance performance was similar to that found in
most co-isolated Gram-positive pigmented bacteria in the same work belonging to the
Actinobacteria group (Zenoff et al., 2006). Only some previous reports have shown the effect
of solar UV radiation on viability of an Acinetobacter sp. strain isolated from Antartic
environments (Helbling et al., 1995). Nevertheless, this high resistance to extreme conditions
seems to be a genus feature since Acinetobacter sp. strain was also the unique Gram-negative
bacteria found in spacecraft assembly. It exhibited resistance to desiccation, H2O2 exposure,
and gamma radiation (La Duc et al., 2003). In a hospital environment, Acinetobacter strains
may survive several days under severely dry conditions (Wendt et al., 1997). In addition,
Vidal et al. (1996) reported the ability of the nosocomial pathogen A. baumannii biotype 9
ACAB715 to form a biofilm on a glass surface, suggesting that this phenomenon plays a key
role for the survival of this bacterium under unfavorable environmental conditions. Recently,
the availability of three genomes of three different Acinetobacter strains, have given more
light on the physiological mechanisms involved in their pathogenicity and also in their
resistance towards severe environmental conditions (Barbe et al., 2004; Fournier et al., 2006;
Vallenet et al., 2008).
The UV-B resistance pattern of A. johnsonii A2 was more deeply studied by assessing the
repairing ability of DNA photoproducts (Fernandez Zenoff et al., 2006). Experiments to study
the percentage of survival population after 20 min of UV-B exposure were conducted and, at
the same time, the authors measured the number of cyclobutane-pirimidine-dymers (CPD)
accumulated on the DNA of the exposed population. The planktonic A. johnsonii A2 from
Laguna Azul showed the highest survival while a reference strain, Acinetobacter johnsonii
ATCC 17909 showed the lowest survival values (in the order of 15 to 20%). Conversely,
A Harsh Life to Indigenous Proteobacteria at the Andean Mountains: …
17
Acinetobacter johnsonii ATCC 17909 accumulated 160 CPD/Mb and Acinetobacter johnsonii
A2, only 75 CPD/Mb. After the UV-B exposure, dark and photoreactivation asssays were
done. Both A. johnsonii strains had very effective photorepair mechanisms, recovering their
initial CFU values after 60 min. However, while strain A2 required 120 min to reduce the
number of CPD/Mb significantly, in strain ATCC 17909, the number of CPD/Mb was
completely reduced during the first 60 min. Under dark conditions, A. johnsonii A2, achieved
full recovery of CFU despite its failure to reduce the number of CPD/Mb. In contrast, A.
johnsonii ATCC 17909 did not recover initial CFU values, and neither decreased the number
of CPD/Mb in the same conditions (Fernandez Zenoff et al., 2006).
The UV-B resistance profiles of other Acinetobacter strains isolated from different lakes
were similarly studied; Ordoñez et al. (2009) described six more strains with taxonomical
identity to A. jhonsonnii, A. lwoffii and A. junii (97-99%) isolated from L. Azul, L. Verde, L.
Negra and Salina Grande. All of them were able to resist up to 12 h UV-B irradiation while
Acinetobacter sp. A2, N40, Ver5 and Ver7 were the ones considered extremely resistant as
they were able to survive after 24 h of UV-B exposure. This outstanding resistance phenotype
may be related to the presence of highly efficient DNA-damage repairing systems, most
probably both, photo and dark repair mechanisms. At the moment, we are trying to
overexpress photolyase genes that were already targeted in two of these strains, Acinetobacter
sp. Ver3 and Acinetobacter sp. Ver7 (Albarracín et al., unpublished data) that did prove to
have very efficient photorepairing actitivities.
All of these strains as well as A. jhonsonni A2 appeared to have multiple resistance
profiles to salinity, antibiotics and even arsenic (Dib et al., 2008; Ordoñez et al., 2009; Flores
et al., 2009). A. jhonsonni A2 was able to grow in a synthetic media amended with up to 10
mM As(III) while it also depicted a multiple resistance profile to diverse antibiotics (Dib et
al., 2008). This later characteristic is not surprising for the genus as clinical strains of
Acinetobacter baumannii or even A. jhonsonnii have been studied by their MDR patterns
(Towner, 2009). But it is unexpected to find high ATB resistance levels in pristine
environments as the HAAL ecosystems where it is not supposed to be any selection pressure
towards antibiotics (Dib et al., 2008; Dib et al., 2010a; 2010b).
On the other hand, the UV-B resistant Acinetobacter sp. N40, Ver3 and Ver5 isolated
from L. Negra (salinity: 32 mg/L) and L. Verde (salinity: 5 mg/L), respectively were also
considered as moderately halophilic by Flores et al. (2009). Conversely, halophilic
Acinetobacter spp. have been isolated from solar salterns and hypersaline soils in Europe (del
Moral et al., 1987; Nieto et al., 1989) and from marine sources in Japan (Van Qua, et al.,
1981).
A BREAK-THROUGH: PROTEOBACTERIA FROM STROMATOLITES
OVER 4,000 M ABOVE SEA LEVEL.
Among microbial mats, microbialites are organosedimentary structures accreted by
sediment trapping, binding and in situ precipitation due to the growth and metabolic activities
of microorganisms. Stromatolites and thrombolites are morphological types of microbialites
classified by their internal mesostructure: layered and clotted, respectively. Microbialites first
appeared in the geological record, 3.5 billion years ago, and for more than 2 billion years they
18
Virginia Helena Albarracín, Julián Rafael Dib, Omar Federico Ordoñez, et al.
were the main evidence of life on Earth (Desneus et al., 2008). In turn, modern stromatolites
have been so far recorded in four locations: i) an hypersaline region of Hamelin Pool, Shark
Bay in Western Australia (Goh et al., 2009), ii) shallow subtidal regions at the margin of
Exuma Sound in the Bahamas (Foster et al., 2009), iii) fresh-water areas at the Cuatro
Ciénagas basin in Mexico (Desnues et al., 2008); and iv) Yellowstone Hot Spring (Lau et al.,
2005). All of these locations are situated at the sea level and mainly in warm environments
where microorganisms cope with little or no stress conditions. In the dessertic region of
Northwestern Argentina, we have found characteristic stromatolite-like ecosystems laying
and developing in shallow hypersaline lakes located above 4,000 meters, under the pressure
of harsh conditions (Table 1), very similar to the ones present in the Early´s Earth atmosphere
(Belluscio, 2009; 2010; Farías et al., 2010).
The microbial diversity from these one-of-a-kind ecosystems thriving at Laguna
Socompa, Tolar Grande and Laguna Diamante (Northwest Argentina) under harsh conditions
was preliminary studied (Belluscio, 2009; 2010; Farías et al., 2010) and we have detected that
Proteobacteria is also very well represented in these microbial communities surviving above
4,000 m. For example, Salinivibrio costicola strains have been isolated from these
environments (unpublished data) and we could address that they presented low similarity to
previous described ones (Table 2). In addition, in preliminary metagenomic studies of
stromatolites from Laguna Socompa, we detected the presence of proteobacteria associated
with the main metabolic groups previously described for microbialites (Desnues et al., 2008):
i.e. anoxygenic photoautotrophs, aerobic heterotrophs, heterotrophic bacteria, sulfur reducers,
sulfur oxidizers and fermenting proteobacteria. Most of them presented low similarities with
previous published bacteria at the gene database and these may point out that microbialites at
the HAAL could be a source of novel proteobacteria (unpublished data).
CONCLUDING REMARKS AND FUTURE PROSPECTIVE
Bacterial diversity is a reservoir of potentially interesting genes for biotechnology and
medicine (Baldauf 2003; Pedros-Alió 2007). In these sense, proteobacterial diversity
discovered at the HAAL may be of biotechnological potential because as extremophiles they
can be source of novel bioactive compounds (Sanchez et al., 2009). Present work is being
conducted towards this direction, especially for the bioprospection of highly efficient
photoreceptors (flavoproteins) and also, citotoxic molecules that can be used as antitumorals,
antibiotics, etc.
On the other hand, the large seed-bank of bacterial taxa hidden in the HAAL microbial
communities should be of interest to better delineate both the taxonomy and the evolutionary
relationships among the group of proteobacteria. It is known that the HAAL resemble more
than any other place on Earth, the conditions of Early Earth´s atmosphere (Belluscio, 2009;
Belluscio 2010). Taking this into account, it would be really interesting to perform more
thoughtfully phylogenetic comparison among the HAAL´s strains and the ones available in
the database.
As it was evident, proteobacteria inhabiting the HAAL have to deal with extreme changes
in salinity, temperature and UV dose (i.e., high environmental dissimilarity in the physical
landscape). Under these pressures, extensive genotypic and physiological diversification at
A Harsh Life to Indigenous Proteobacteria at the Andean Mountains: …
19
the species level may be expected, however, the question that still remains is if this
microdiversity arises as an adaptive strategy or as genomic modification due to the UVinduced direct mutations or stress oxidative damage (as a consequence of salinity,
desiccation, and UV damage). The widespread antibiotic resistance displayed by these
microbes may be a consequence of these two mechanisms. Research studies are starting to be
conducted on the molecular nature of the resistance mechanisms in order to reveal metabolic
pathways and special molecules that can be used by the proteobacteria to endure the drastic
environmental conditions.
Acinetobacter strains isolated from the HAAL were specially highlighted to give more
importance on the environmental representatives within this genus, and specially on
freshwater environments which so far has not properly studied. Particularly, A. jhonsonni A2
isolated from L. Azul showed multiple resistance profiles to hipersalinity, UV-B irradiation,
antibiotics and even arsenic. This “superbug” can be subjected to further studies as it can give
clues to discover new ways of surviving at extreme conditions, a matter that has applications
in astrobiology.
Finally, it is important to emphasize that these pristine environments at the P-HA
ecoregion are extremely fragile because low human disturbances can produce big destruction
on them. At the moment, they are under big risk due mainly to mining activities local or
regional (e.g. Chile) that prompted the exploitation of minerals (lithium, copper, gold among
others) and/or water from these pristine areas. The aim of our research project is not only of
scientific interest; by performing studies on the microbial communities of the P-HA we will
bring more attention to the biological uniqueness and fragile nature of these environment, and
in this sense, we will help to support an intensive environmental protection program.
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