Halomonas maura is a physiologically versatile bacterium of both

Antonie van Leeuwenhoek (2006)
DOI 10.1007/s10482-005-9043-9
Ó Springer 2006
Halomonas maura is a physiologically versatile bacterium of both ecological
and biotechnological interest
Inmaculada Llamas, Ana del Moral*, Fernando Martı́nez-Checa, Yolanda Arco,
Soledad Arias and Emilia Quesada
Department of Microbiology, University of Granada, Campus Universitario de Cartuja s/n, 18071, Granada,
Spain; *Author for correspondence (e-mail: [email protected]; phone: +34-958-243875; fax: +34-958246235)
Accepted in revised form 21 November 2005
Key words: Biotechnology, Ecology, Exopolysaccharide, Halomonas maura, Halophilic bacteria
Abstract
Halomonas maura is a bacterium of great metabolic versatility. We summarise in this work some of the
properties that make it a very interesting microorganism both from an ecological and biotechnological
point of view. It plays an active role in the nitrogen cycle, is capable of anaerobic respiration in the presence
of nitrate and has recently been identified as a diazotrophic bacterium. Of equal interest is mauran, the
exopolysaccharide produced by H. maura, which contributes to the formation of biofilms and thus affords
the bacterium advantages in the colonisation of its saline niches. Mauran is highly viscous, shows thixotropic and pseudoplastic behaviour, has the capacity to capture heavy metals and exerts a certain immunomodulator effect in medicine. All these attributes have prompted us to make further investigations into
its molecular characteristics. To date we have described 15 open reading frames (ORF’s) related to exopolysaccharide production, nitrogen fixation and nitrate reductase activity among others.
Halomonas maura is a moderately halophilic
bacterium
Moderately halophilic bacteria, the group to which
Halomonas species belong, include a wide array of
microorganisms that are taxonomically and physiologically distributed among many genera within
the Bacteria and Archaea domains. Their common
characteristic is that they grow best at NaCl concentrations of between 0.5 and 2.5 M (Kushner
and Kamekura 1988), although they can be found
throughout quite a diverse range of hypersaline
habitats (Oren 1999). They have numerous applications in various fields of industry and ecology
(Ventosa et al. 1998; Margesin and Schinner 2001;
Jones 2004; Quesada et al. 2004). Over the last
decade interest in Halomonas species has centred
on their ability to degrade aromatic compounds
(Garcı́a et al. 2004) and produce exoenzymes,
exopolysaccharides (EPS’s) and other commercially valuable products (Ventosa et al. 1998; Oren
2002; Quesada et al. 2004).
At present the genus Halomonas contains more
than thirty species, all belonging to the c-Proteobacteria, most of which have been isolated from
saline environments (Vreeland et al. 1980; Dobson
and Franzmann 1996; Ventosa et al. 1998; Mata
et al. 2002). Taxonomically Halomonas species
constitute a heterogeneous bacterial genus. On the
basis of 16S and 23S rRNA gene sequences and
phenotypic studies, Arahal et al. (2002) have
established various distinct phylogenetic groups
(Mata et al. 2002). Some of the Halomonas species, including Halomonas eurihalina, Halomonas
maura, Halomonas ventosae, Halomonas anticariensis and Halomonas almeriensis, which were
isolated from the rhizosphere of xerophytic plants
and characterised by our research group (Quesada
et al. 1990; Bouchotroch et al. 2001; Martı́nezCánovas et al., 2004a; 2004b; Martı́nez-Checa
et al. 2005), produce extracellular polysaccharides with potential biotechnological applications (Calvo et al. 2002; Béjar et al. 1998;
Martı́nez-Checa et al. 2002; Arias et al. 2003;
Quesada et al. 2004).
Halomonas maura is a Gram-negative rod,
occurring either singly or in pairs, or occasionally
as long filaments. The cells are capsulated, nonmotile and accumulate PHA. They do not form
endospores. They grow best in media containing
0.5 to 2.5 M NaCl, although their eurihaline
character allows them to grow within a range of
salt concentrations of between 1% and 15% w/v.
They use the following compounds as sole carbon
and energy sources: citrate, ethanol, fumarate,
D-fructose, glycerol, D-rhamnose and D-ribose; they
do not use lactose or D-trehalose (Bouchotroch
et al. 2001).
Natural habitat of Halomonas maura
Species of the genus Halomonas are to be found
growing both in hypersaline thalassal environments (containing salt compositions equal to sea
water) and in athalassal ones such as soils, salterns and brackish lakes (Ramos-Cormenzana
1993; Bouchotroch et al. 1999). In some of these
latter environments bacteria may be faced with
many factors that make survival difficult, such as
drought, strong solar radiation, high temperatures and sometimes extreme pH conditions
(Rodrı́guez-Valera 1993).
Halomonas maura was first isolated from soils
surrounding a saltern at Asilah in Morocco
(Bouchotroch et al. 2001). Since then it has been
found in various saline environments, often
adhering to the roots of halophytic plants such as
Salicornia spp. It is one of the most common
exopolysaccharide-producing species found in
saline soils (Martı́nez-Cánovas et al. 2004c) and its
strains have the ability to produce large quantities
of exopolysaccharides, some of which are of considerable biotechnological interest (Bouchotroch
et al. 2000).
It colonises root surfaces but we have no data
pointing to its being able to infect plants or
establish symbiotic relationships. Although the
exact mechanism of how it interacts with plant
roots is not yet fully understood we can affirm that
its anchoring capacity depends, among other factors, upon the production of extracellular polysaccharide, as it does with other diazotrophic and
symbiotic microorganisms such as Azospirillum
(Skvortsov and Ignatov 1998) and Sinorhizobium
meliloti (González et al. 1996).
Halomonas maura and the biogeochemical
nitrogen cycle
H. maura has a chemo-organotrophic metabolism
and possesses a complex respiratory chain that
switches and activates the different terminal oxidoreductase proteins according to the availability of
environmental oxygen, as occurs in many other
bacteria (Anraku and Gennis 1987; Bott et al.
1992). Although its metabolism is of the respiratory type with oxygen as the terminal electron
acceptor, it is capable of anaerobic respiration in
the presence of nitrate, which it uses as an alternative final electron acceptor. It has been demonstrated that respiration on nitrate in this bacterium
occurs only under anaerobic conditions and solely
via the membrane-bound nitrate-reductase
enzyme (NAR). Molecular studies have detected
the presence of the narGHJI operon (Montserrat
Argandoña, personal communication) and have
equally proved the absence of the nitrate reductase
NAP, an enzyme typically found in Gram-negative
strains capable of anaerobic nitrate respiration
(Flanagan et al. 1999).
Halomonas maura is only capable of carrying
out the first step in the denitrification process, i.e.
the reduction of NO)3 to NO)2 . When we tested for
nitrate reduction neither nitrite nor nitrogen gas
was found in the culture medium, which leads us
to suspect that nitrite is converted into ammonia
via dissimilatory nitrate reduction to ammonia
(DNRA), as it is in Escherichia coli. In DNRA,
NO)3 is used during dissimilatory NO)3 reduction
to NH+
4 and nitrogen will be conserved in a form
that is available to other organisms (Patrick et al.
1996). Although the occurrence of DNRA has
been demonstrated in different environments, such
as marine sediments (Tobias et al. 2001), the ecological significance of the process is not yet
understood (Cornwell et al. 1999).
H. maura is also able to thrive in a wide range of
oxygen concentrations by using a cbb3-type cytochrome oxidase, encoded by the gene cluster
ccoNOQP, an enzyme found in numerous nitrogenfixing microorganisms. In fact we have recently
shown, by identifying the conserved nifH gene
using molecular biological techniques and the
acetylene reduction assay, that H. maura fixes
atmospheric nitrogen under microaerobic conditions (Argandoña et al. 2005). This property,
together with its ability to colonise niches with a
wide range of saline concentrations and to grow
under different oxygen concentrations, provides it
with enormous potential interest in agriculture and
forestry. It could, for instance, be useful in the
inoculation of moderately saline soils, where it has
been shown that salt stress impedes nitrogen
fixation quite significantly, inhibiting both the
synthesis and activity of nitrogenase and/or
reducing bacterial adhesion to plant roots (Tripathi
et al. 2002).
The fact that Halomonas maura, one of the most
common bacteria found in saline soils, is a bacterial diazotroph capable of fixing nitrogen under a
wide range of saline concentrations and uses nitrate as a final electron acceptor means that it may
well play an important role in the nitrogen cycle of
these habitats.
Genetic studies of H. maura
There is still a lot of work to be done on the genetics
of moderately halophilic bacteria, although some
data, such as the presence of small and mediumsized plasmids (Fernández-Castillo et al. 1992;
Vargas et al. 1995; Llamas et al. 1997), megaplasmids (Argandoña et al. 2003), the physical size of
the chromosome (Mellado et al. 1998; Llamas et al.
2002), the genes involved in osmoregulation (Louis
and Galinski, 1997; Cánovas et al. 2000), a-amylase
production (Coronado et al. 2000), exopolysaccharide production (Llamas et al. 2003) and gene
reporter systems (Arvanitis et al. 1995; Tegos et al.
2000; Afendra et al. 2004) have been reported.
The excellent properties of Halomonas maura
due to its versatile metabolism led us to undertake
a genetic study. The strains belonging to this species have a relatively high DNA G+C content
(62.2–64.2 mol%) (Bouchotroch et al. 2001). A
study of the genome organisation of H. maura
strain S-31T reveals that it possesses a single
circular chromosome of 3500 Kb and two large
extrachromosomal DNA elements of 619 and
70.7 Kb (Argandoña et al. 2003).
The presence of megaplasmids is common to
many species of the Halomonas genus (Argandoña
et al. 2003). In other genera these types of plasmid
have been shown to play a key role in important
bacterial functions, such as nitrogen fixation
(Barloy-Hubler et al. 2000) and resistance to
antibiotics and heavy metals (Taghavi et al. 1997).
Plasmids have also been described in other diazotrophs; all Azospirillum species, for example, possess plasmids of sizes ranging from 160 to over
592 Kb. Megaplasmids in other soil bacteria are
known to carry essential information for plant
interaction (Skvortsov and Ignatov 1998). The
Agrobacterium virulence (vir) genes as well as the
Rhizobium nodulation (nod) and host-specific
nodulation (hsn) genes are encoded in megaplasmids. Although we are unsure at present of the
precise function of the plasmids and megaplasmids
in H. maura they could well contribute to its survival strategies in the saline niches that it inhabits.
The total amount of DNA sequenced from
strains S-30 and S-31T so far has revealed 15 open
reading frames (ORF’s), to which we have been
able to assign functions by studying their mutations and comparing their amino-acid sequences
(Table 1). So far we have been able to identify
within these ORF’s a narGHJI gene cluster
involved in nitrate anaerobic respiration, a
ccoNOPQ gene cluster encoding a cbb3 type
cytochrome (Montserrat Argandoña, personal
communication), a nifH gene for nitrogen fixation
(Argandoña et al. 2005) and epsABCDJ genes
involved in the assembly and polymerisation of the
EPS mauran (Arco et al. 2005). The epsABCDJ
genes form part of a gene cluster (eps) with the
same structural organisation as others involved in
the biosynthesis of group 1 capsules and some
EPS’s. Conserved genetic features were found,
such as JUMPStart and ops elements, which
characteristically precede the polysaccharide gene
clusters. We have demonstrated the possibility that
Table 1. Genes identified in Halomonas maura strains.
Gene
Size
(aa)
Function
Accession
No.
narK
narGHJI
narG
narH
narJ
narI
ccoNOQP
433
AY641547
AY641547
AY641547
AY641547
AY641547
AY641547
DQ013173
ccoN
cooO
ccoQ
ccoP
nifH
474
202
70
309
Partial
sequence
nitrate transporter
Nitrate reductase Nar
a subunit
b subunit
d subunit
c subunit
cbb3-type
cytochrome oxidase
I subunit
II subunit
Unknown
III subunit
Nitrogenase
Polymerization
and assembly of EPS
Outer-membrane porine
Phosphotyrosine
phosphatase
Autotyrosine kinase
Sodium-sulphate
symporter
Flippase
AY918062
1258
535
256
223
epsABCDJ
epsA
epsB
377
146
epsC
epsD
730
543
epsJ
446
DQ013173
DQ013173
DQ013173
DQ013173
AY827547
AY918062
AY918062
AY918062
AY918062
AY918062
mauran, just like many other polysaccharides, may
be synthesised via a Wzy-like biosynthesis system.
We have also proved by transcriptional expression
assays that the eps gene cluster reaches maximum
activity during the stationary phase in the presence
of 5% w/v marine salts (Arco et al. 2005).
Genetic studies into exopolysaccharide-producing microorganisms of industrial interest have
tended to focus upon improvements in the production or functional characteristics of their EPS’s
(Vartak et al. 1995; Martins and Sá-Correia 1993).
We have made similar studies with the mauran
producer strain S-30 using strategies such as insertional mutagenesis and have produced a mutant,
called TK26, which produces higher quantities of
exopolysaccharide than the wild-type strain does.
This strain has been registered in the Spanish collection of type cultures as CECT 5720 and patented
for its high EPS production and interesting functional properties for industry (Arias et al. 2002).
Halomonas maura produces an exopolysaccharide
suitable for biotechnological purposes
Strain S-30 of H. maura is distinguishable from the
rest of the strains of the species because it excretes
large quantities of an exopolysaccharide known as
mauran onto the outside of its cell wall. This EPS
is a high-molecular-mass (4.7 106 Da) acidic
polymer composed of repeating units of mannose,
galactose, glucose and glucuronic acid and has
a number of potential functional properties
(Table 2) (Bouchotroch et al. 2001; Arias et al.
2003), among which are its high viscosifying
capacity, similar to that of xanthan, and the
pseudoplastic and thixotropic behaviour of its
solutions. In addition, the stability of its functional
properties under a wide range of pH, saline and
freezing-thawing conditions opens up possibilities
for its use in many fields (Arias et al. 2003). In fact
mauran has shown great promise as a viscosifying
agent in trials with lactic-fermentation foodstuffs
carried out by a food company.
Mauran is also capable of emulsifying vegetable
oils, hydrocarbons and especially petroleum. It has
been shown to exert a synergic effect in association
with commercial chemical surfactants such as
Tween 20 and Polysorbate 60. It is an efficient
stabiliser and emulsifying agent that inhibits coalescence and increases emulsion viscosity, although
in itself it is not a surfactant. This property has
been positively tested by adding aqueous solutions
of mauran (1.5% w/v) to different cosmetic products during a study carried out by a cosmetics
company (Arias et al. 2003).
The anionic nature of mauran, based on its high
sulphate and uronic-acid contents, may be
responsible for the capacity of its solutions to bind
lead and other heavy metals with considerable
Table 2. Potential applications of mauran EPS.
Properties
Physical properties
Viscosifying capacity,
pseudoplastic and
thixotropic
Emulsifying agent
(emulsion stabilization)
Industrial use
Cosmetics (cream,
lotions, shampoo...)
Pharmaceuticals (Antiseptic
lotions containing H2O2)
Foodstuffs (jams, sugar syrups...)
Cosmetics (toothpastes,
hair dyes...)
Pharmaceuticals (pomades)
Foodstuffs (dressings)
Petroleum
Chemical properties
Metal-binding capacity
Bio-absorbent
Biological properties
Immunomodulator effect
Medicine
and antiproliferative activity
efficiency (Arias et al. 2003). This property would
make it a viable alternative to other more aggressive physical and chemical methods as a biosorbent in polluted water and soil environments.
Apart from this, the unusually high sulphate
content of mauran, a property that has been
related to biological activity in many microbial
polymers, has led us to assay its immunomodulator effect and its antiproliferative activity on
human cancer cells. Preliminary experiments along
these lines are showing encouraging results.
The ecological importance of EPS in Halomonas
maura: biofilm and quorum sensing
Exopolysaccharide-producing Halomonas strains
are widespread in salterns, saline soils, seawater
and marshes, where they are believed to exert a
considerable influence within their ecological niches (Quesada et al. 2004). Exopolysaccharides
benefit the bacteria by enabling them to attach
themselves to surfaces and colonise the rhizosphere
(Costernon et al. 1987; González et al. 1996;
Sutherland 2001). They also improve nutrient
acquisition (Cheng and Jaunet 1992) and provide
protection against environmental stress and host
defences (Robertson and Firestone 1992). Moreover, EPS’s also afford advantages to the plant
hosts by improving water stability, which is critical
to plant survival. Hence there is a strong selective
advantage for the production of EPS within the
rhizosphere. It is very likely that the EPS’s produced by Halomonas maura strains play similar
roles in their survival strategy. Nevertheless, the
way in which the biosynthesis of these exopolysaccharides is regulated and whether their production responds to environmental factors and/or
population-density remains to be investigated.
By carrying out adhesion assays (O‘Toole and
Kolter 1998) with wild-type and polysaccharidedeficient mutants we have recently been able to
demonstrate that H. maura strains are capable of
producing biofilms and that mauran forms an
integral part of the structural organisation of these
biofilms (Inmaculada Llamas, unpublished data).
Scanning-electron micrographs reveal small channelled protuberances in the H. maura wild-type
strain (Figure 1a), which seem then to participate
in the formation of microcolonies and the matrix
of biofilm (Figure 1b). These extracellular struc-
tures are not to be seen in an exopolysaccharidedeficient mutant of H. maura (Figure 1c) and
no comparative microcolonies are formed (Figure 1d), thus confirming that EPS is essential to
the formation of the biofims developed by the wild
strain of H. maura, just as has been described in
other bacteria.
There are many indications of biofilm-producing communities in the rhizosphere. Firstly, it is
evident that bacteria attach themselves to roots,
and various mechanisms have been described to
enable this process, involving a variety of cell
components such as outer-membrane proteins,
wall polysaccharides and cell-surface agglutinin.
Secondly, exopolysaccharide is produced by bacteria in the rhizosphere (Amellal et al. 1998). This
not only affords many advantages to bacterial cells
(as described above) but it also enhances soil
aggregation, which in turn improves water stability, a factor critical to the survival of the plant and
to the availability of nutrients and metabolic
co-operativity (Sutherland 2001; Davey and
O’Toole 2000).
The regulatory mechanisms that guide biofilm
development have recently come under scrutiny.
The cell-to-cell communication known as quorum
sensing, playing a part in the regulation of surface attachment and biofilm maturation, was first
suggested by Williams and Stewart (1994). Quorum sensing involves changes in the concentration
of a diffusible autoinducer to provide a regulatory signal in response to population density and
has been shown to be essential for the construction of a mature biofilm in P. aeruginosa (Davies
et al. 1998) Vibrio cholerae (Hammer and Bassler
2003), Aeromonas hydrophila (Lynch et al. 2002)
and Streptococcus gordonii (McNab et al. 2003).
This system has been seen to regulate other
functions such as the expression of virulence factors and exoenzymes in Pseudomonas aeruginosa
and Erwinia carotovora (de Kievit and Iglewski,
2000; Beck Von Bodman et al. 2003), conjugal
transfer in Agrobacterium tumefaciens (Farrand
1998; Fuqua et al. 1994; 2001), the production
of antibiotics in Chromobacterium violaceum
(McClean et al. 1997) and the production of exopolysaccharide in Pantoea stewartii (Beck Von
Bodman et al. 1998) and Sinorhizobium melitoti
(Marketon et al. 2003).
We have recently described a system of quorum sensing in exopolysaccharide-producing
Figure 1. Scanning electron micrographs of wild-type (a and b) and EPS-deficient mutant (c and d) strains of Halomonas grown on
coverslip surfaces for 48 h. Note the presence of exopolysaccharide films in the wild-type strain (a) and the microcolonies formed (b).
Halomonas strains, the physiological role of which
in the development of these bacteria is currently
being studied (Llamas et al. 2005). We have
also identified some of the signal molecules (AHL’s)
produced by Halomonas strains, such as N-butanoyl
homoserine lactone (C4-HL), N-hexanoyl homoserine lactone (C6-HL), N-octanoyl homoserine
lactone (C8-HL) and N-dodecanoyl homoserine
lactone (C12-HL) (Llamas et al. 2005).
In Halomonas anticariensis, another EPS-producing species, in which we detected the highest
levels of signal molecules, we have obtained transconjugants deficient in signal molecules, which will
allow us to characterise the genes involved in this
system and also the regulated cell functions.
Halomonas maura is a suitable bacterium for
use in ecology and biotechnology
All these characteristics of Halomonas maura lead
us to the conclusion that it is of an enormous
potential interest both in industry because of the
possibility of using its exopolysaccharides for a
variety of applications, and also in ecology due
to its capacity to colonise saline niches and
intervene in the nitrogen cycle. This latter
capacity could well result in its having beneficial
applications as a soil inoculate in moderately
saline, arid soils that until now have defied
agricultural use. In addition to this, it has other
specific advantages for biotechnological use such
as its lack of pathogenicity, rapid growth and
easily available nutrient requirements, which
allow it to be cultured in relatively cheap media
with a high salt content, thus reducing risks of
contamination.
Consequently, to further the possibilities of
employing Halomonas species to ecological ends
we are at present investigating the cell functions
that encode the quorum-sensing regulatory system
in various species of this genus and are also
looking into the influence of salt concentration
upon the nitrogen-fixing capacity of Halomonas
maura. On the medical front, we are continuing to
study the biological activity of the sulphate-bearing EPS mauran, produced by Halomonas maura.
Acknowledgements
This research was supported by grants from the
Dirección General de Investigación Cientı́fica y
Técnica (BOS2003–00498) and from the Plan
Andaluz de Investigación, Spain. Thanks go to our
colleague Dr. J. Trout for revising our English
text.
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