Halomonas Maura Is a Physiologically Versatile Bacterium of Both Ecological and Biotechnological Interest

Antonie van Leeuwenhoek (2006) Ó Springer 2006 DOI 10.1007/s10482-005-9043-9

Halomonas maura is a physiologically versatile bacterium of both ecological and biotechnological interest

Inmaculada Llamas, Ana del Moral*, Fernando Martı´nez-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: +34-958- 246235)

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 Jones 2004; Quesada et al. 2004). Over the last bacterium decade interest in Halomonas species has centred on their ability to degrade aromatic compounds Moderately halophilic bacteria, the group to which (Garcıá et al. 2004) and produce exoenzymes, Halomonas species belong, include a wide array of exopolysaccharides (EPS’s) and other commer- microorganisms that are taxonomically and phys- cially valuable products (Ventosa et al. 1998; Oren iologically distributed among many genera within 2002; Quesada et al. 2004). the Bacteria and Archaea domains. Their common At present the genus Halomonas contains more characteristic is that they grow best at NaCl con- than thirty species, all belonging to the c-Proteo- centrations of between 0.5 and 2.5 M (Kushner bacteria, most of which have been isolated from and Kamekura 1988), although they can be found saline environments (Vreeland et al. 1980; Dobson throughout quite a diverse range of hypersaline and Franzmann 1996; Ventosa et al. 1998; Mata habitats (Oren 1999). They have numerous appli- et al. 2002). Taxonomically Halomonas species cations in various fields of industry and ecology constitute a heterogeneous bacterial genus. On the (Ventosa et al. 1998; Margesin and Schinner 2001; basis of 16S and 23S rRNA gene sequences and phenotypic studies, Arahal et al. (2002) have strains have the ability to produce large quantities established various distinct phylogenetic groups of exopolysaccharides, some of which are of con- (Mata et al. 2002). Some of the Halomonas spe- siderable biotechnological interest (Bouchotroch cies, including Halomonas eurihalina, Halomonas et al. 2000). maura, Halomonas ventosae, Halomonas anticari- It colonises root surfaces but we have no data ensis and Halomonas almeriensis, which were pointing to its being able to infect plants or isolated from the rhizosphere of xerophytic plants establish symbiotic relationships. Although the and characterised by our research group (Quesada exact mechanism of how it interacts with plant et al. 1990; Bouchotroch et al. 2001; Martıńez- roots is not yet fully understood we can affirm that Cańovas et al., 2004a; 2004b; Martıńez-Checa its anchoring capacity depends, among other fac- et al. 2005), produce extracellular polysaccha- tors, upon the production of extracellular poly- rides with potential biotechnological applica- saccharide, as it does with other diazotrophic and tions (Calvo et al. 2002; Be´jar et al. 1998; symbiotic microorganisms such as Azospirillum Martıńez-Checa et al. 2002; Arias et al. 2003; (Skvortsov and Ignatov 1998) and Sinorhizobium Quesada et al. 2004). meliloti (Gonza´lez et al. 1996). Halomonas maura is a Gram-negative rod, occurring either singly or in pairs, or occasionally as long filaments. The cells are capsulated, non- Halomonas maura and the biogeochemical motile and accumulate PHA. They do not form nitrogen cycle endospores. They grow best in media containing 0.5 to 2.5 M NaCl, although their eurihaline H. maura has a chemo-organotrophic metabolism character allows them to grow within a range of and possesses a complex respiratory chain that salt concentrations of between 1% and 15% w/v. switches and activates the different terminal oxido- They use the following compounds as sole carbon reductase proteins according to the availability of and energy sources: citrate, ethanol, fumarate, environmental oxygen, as occurs in many other D-fructose, glycerol, D-rhamnose and D-ribose; they bacteria (Anraku and Gennis 1987; Bott et al. do not use lactose or D-trehalose (Bouchotroch 1992). Although its metabolism is of the respira- et al. 2001). tory type with oxygen as the terminal electron acceptor, it is capable of anaerobic respiration in the presence of nitrate, which it uses as an alter- Natural habitat of Halomonas maura native final electron acceptor. It has been demonstrated that respiration on nitrate in this bacterium Species of the genus Halomonas are to be found occurs only under anaerobic conditions and solely growing both in hypersaline thalassal environ- via the membrane-bound nitrate-reductase ments (containing salt compositions equal to sea enzyme (NAR). Molecular studies have detected water) and in athalassal ones such as soils, sal- the presence of the narGHJI operon (Montserrat terns and brackish lakes (Ramos-Cormenzana Argandon˜ a, personal communication) and have 1993; Bouchotroch et al. 1999). In some of these equally proved the absence of the nitrate reductase latter environments bacteria may be faced with NAP, an enzyme typically found in Gram-negative many factors that make survival difficult, such as strains capable of anaerobic nitrate respiration drought, strong solar radiation, high tempera- (Flanagan et al. 1999). tures and sometimes extreme pH conditions Halomonas maura is only capable of carrying (Rodrı´guez-Valera 1993). out the first step in the denitrification process, i.e. ) ) Halomonas maura was first isolated from soils the reduction of NO3 to NO2 . When we tested for surrounding a saltern at Asilah in Morocco nitrate reduction neither nitrite nor nitrogen gas (Bouchotroch et al. 2001). Since then it has been was found in the culture medium, which leads us found in various saline environments, often to suspect that nitrite is converted into ammonia adhering to the roots of halophytic plants such as via dissimilatory nitrate reduction to ammonia Salicornia spp. It is one of the most common (DNRA), as it is in Escherichia coli. In DNRA, ) ) exopolysaccharide-producing species found in NO3 is used during dissimilatory NO3 reduction + saline soils (Martıńez-Cańovas et al. 2004c) and its to NH4 and nitrogen will be conserved in a form that is available to other organisms (Patrick et al. The excellent properties of Halomonas maura 1996). Although the occurrence of DNRA has due to its versatile metabolism led us to undertake been demonstrated in different environments, such a genetic study. The strains belonging to this spe- as marine sediments (Tobias et al. 2001), the eco- cies have a relatively high DNA G+C content logical significance of the process is not yet (62.2–64.2 mol%) (Bouchotroch et al. 2001). A understood (Cornwell et al. 1999). study of the genome organisation of H. maura H. maura is also able to thrive in a wide range of strain S-31T reveals that it possesses a single oxygen concentrations by using a cbb3-type cyto- circular chromosome of 3500 Kb and two large chrome oxidase, encoded by the gene cluster extrachromosomal DNA elements of 619 and ccoNOQP, an enzyme found in numerous nitrogen- 70.7 Kb (Argandon˜ a et al. 2003). fixing microorganisms. In fact we have recently The presence of megaplasmids is common to shown, by identifying the conserved nifH gene many species of the Halomonas genus (Argandon˜ a using molecular biological techniques and the et al. 2003). In other genera these types of plasmid acetylene reduction assay, that H. maura fixes have been shown to play a key role in important atmospheric nitrogen under microaerobic condi- bacterial functions, such as nitrogen fixation tions (Argandon˜ a et al. 2005). This property, (Barloy-Hubler et al. 2000) and resistance to together with its ability to colonise niches with a antibiotics and heavy metals (Taghavi et al. 1997). wide range of saline concentrations and to grow Plasmids have also been described in other diazo- under different oxygen concentrations, provides it trophs; all Azospirillum species, for example, pos- with enormous potential interest in agriculture and sess plasmids of sizes ranging from 160 to over forestry. It could, for instance, be useful in the 592 Kb. Megaplasmids in other soil bacteria are inoculation of moderately saline soils, where it has known to carry essential information for plant been shown that salt stress impedes nitrogen interaction (Skvortsov and Ignatov 1998). The fixation quite significantly, inhibiting both the Agrobacterium virulence (vir) genes as well as the synthesis and activity of nitrogenase and/or Rhizobium nodulation (nod) and host-specific reducing bacterial adhesion to plant roots (Tripathi nodulation (hsn) genes are encoded in megaplas- et al. 2002). mids. Although we are unsure at present of the The fact that Halomonas maura, one of the most precise function of the plasmids and megaplasmids common bacteria found in saline soils, is a bacte- in H. maura they could well contribute to its sur- rial diazotroph capable of fixing nitrogen under a vival strategies in the saline niches that it inhabits. wide range of saline concentrations and uses ni- The total amount of DNA sequenced from trate as a final electron acceptor means that it may strains S-30 and S-31T so far has revealed 15 open well play an important role in the nitrogen cycle of reading frames (ORF’s), to which we have been these habitats. able to assign functions by studying their muta- tions and comparing their amino-acid sequences (Table 1). So far we have been able to identify Genetic studies of H. maura within these ORF’s a narGHJI gene cluster involved in nitrate anaerobic respiration, a There is still a lot of work to be done on the genetics ccoNOPQ gene cluster encoding a cbb3 type of moderately halophilic bacteria, although some cytochrome (Montserrat Argandon˜ a, personal data, such as the presence of small and medium- communication), a nifH gene for nitrogen fixation sized plasmids (Fernańdez-Castillo et al. 1992; (Argandon˜ a et al. 2005) and epsABCDJ genes Vargas et al. 1995; Llamas et al. 1997), megaplas- involved in the assembly and polymerisation of the mids (Argandon˜ a et al. 2003), the physical size of EPS mauran (Arco et al. 2005). The epsABCDJ the chromosome (Mellado et al. 1998; Llamas et al. genes form part of a gene cluster (eps)withthe 2002), the genes involved in osmoregulation (Louis same structural organisation as others involved in and Galinski, 1997; Cańovas et al. 2000), a-amylase the biosynthesis of group 1 capsules and some production (Coronado et al. 2000), exopolysac- EPS’s. Conserved genetic features were found, charide production (Llamas et al. 2003) and gene such as JUMPStart and ops elements, which reporter systems (Arvanitis et al. 1995; Tegos et al. characteristically precede the polysaccharide gene 2000; Afendra et al. 2004) have been reported. clusters. We have demonstrated the possibility that Table 1. Genes identified in Halomonas maura strains. large quantities of an exopolysaccharide known as mauran onto the outside of its cell wall. This EPS Gene Size Function Accession 6 (aa) No. is a high-molecular-mass (4.7 Â 10 Da) acidic polymer composed of repeating units of mannose, narK 433 nitrate transporter AY641547 galactose, glucose and glucuronic acid and has narGHJI Nitrate reductase Nar AY641547 narG 1258 a subunit AY641547 a number of potential functional properties narH 535 b subunit AY641547 (Table 2) (Bouchotroch et al. 2001; Arias et al. narJ 256 d subunit AY641547 2003), among which are its high viscosifying narI 223 c subunit AY641547 capacity, similar to that of xanthan, and the ccoNOQP cbb3-type DQ013173 pseudoplastic and thixotropic behaviour of its cytochrome oxidase ccoN 474 I subunit DQ013173 solutions. In addition, the stability of its functional cooO 202 II subunit DQ013173 properties under a wide range of pH, saline and ccoQ 70 Unknown DQ013173 freezing-thawing conditions opens up possibilities ccoP 309 III subunit DQ013173 for its use in many fields (Arias et al. 2003). In fact nifH Partial Nitrogenase AY827547 mauran has shown great promise as a viscosifying sequence epsABCDJ Polymerization AY918062 agent in trials with lactic-fermentation foodstuffs and assembly of EPS carried out by a food company. epsA 377 Outer-membrane porine AY918062 Mauran is also capable of emulsifying vegetable epsB 146 Phosphotyrosine AY918062 oils, hydrocarbons and especially petroleum. It has phosphatase been shown to exert a synergic effect in association epsC 730 Autotyrosine kinase AY918062 epsD 543 Sodium-sulphate AY918062 with commercial chemical surfactants such as symporter Tween 20 and Polysorbate 60. It is an efficient epsJ 446 Flippase AY918062 stabiliser and emulsifying agent that inhibits coa- lescence and increases emulsion viscosity, although mauran, just like many other polysaccharides, may in itself it is not a surfactant. This property has be synthesised via a Wzy-like biosynthesis system. been positively tested by adding aqueous solutions We have also proved by transcriptional expression of mauran (1.5% w/v) to different cosmetic prod- assays that the eps gene cluster reaches maximum ucts during a study carried out by a cosmetics activity during the stationary phase in the presence company (Arias et al. 2003). of 5% w/v marine salts (Arco et al. 2005). The anionic nature of mauran, based on its high Genetic studies into exopolysaccharide-produc- sulphate and uronic-acid contents, may be ing microorganisms of industrial interest have responsible for the capacity of its solutions to bind tended to focus upon improvements in the produc- lead and other heavy metals with considerable tion or functional characteristics of their EPS’s (Vartak et al. 1995; Martins and Sa´-Correia 1993). Table 2. Potential applications of mauran EPS. We have made similar studies with the mauran Properties Industrial use producer strain S-30 using strategies such as inser- tional mutagenesis and have produced a mutant, Physical properties Viscosifying capacity, Cosmetics (cream, called TK26, which produces higher quantities of pseudoplastic and lotions, shampoo...) exopolysaccharide than the wild-type strain does. thixotropic Pharmaceuticals (Antiseptic

This strain has been registered in the Spanish col- lotions containing H2O2) lection of type cultures as CECT 5720 and patented Foodstuffs (jams, sugar syrups...) for its high EPS production and interesting func- Emulsifying agent Cosmetics (toothpastes, (emulsion stabilization) hair dyes...) tional properties for industry (Arias et al. 2002). Pharmaceuticals (pomades) Foodstuffs (dressings) Petroleum Halomonas maura produces an exopolysaccharide Chemical properties suitable for biotechnological purposes Metal-binding capacity Bio-absorbent Biological properties Strain S-30 of H. maura is distinguishable from the Immunomodulator effect Medicine and antiproliferative activity rest of the strains of the species because it excretes efficiency (Arias et al. 2003). This property would tures are not to be seen in an exopolysaccharide- make it a viable alternative to other more aggres- deficient mutant of H. maura (Figure 1c) and sive physical and chemical methods as a biosor- no comparative microcolonies are formed (Fig- bent in polluted water and soil environments. ure 1d), thus confirming that EPS is essential to Apart from this, the unusually high sulphate the formation of the biofims developed by the wild content of mauran, a property that has been strain of H. maura, just as has been described in related to biological activity in many microbial other bacteria. polymers, has led us to assay its immunomodula- There are many indications of biofilm-produc- tor effect and its antiproliferative activity on ing communities in the rhizosphere. Firstly, it is human cancer cells. Preliminary experiments along evident that bacteria attach themselves to roots, these lines are showing encouraging results. and various mechanisms have been described to enable this process, involving a variety of cell components such as outer-membrane proteins, The ecological importance of EPS in Halomonas wall polysaccharides and cell-surface agglutinin. maura: biofilm and quorum sensing Secondly, exopolysaccharide is produced by bacteria in the rhizosphere (Amellal et al. 1998). This Exopolysaccharide-producing Halomonas strains not only affords many advantages to bacterial cells are widespread in salterns, saline soils, seawater (as described above) but it also enhances soil and marshes, where they are believed to exert a aggregation, which in turn improves water stabil- considerable influence within their ecological ni- ity, a factor critical to the survival of the plant and ches (Quesada et al. 2004). Exopolysaccharides to the availability of nutrients and metabolic benefit the bacteria by enabling them to attach co-operativity (Sutherland 2001; Davey and themselves to surfaces and colonise the rhizosphere O’Toole 2000). (Costernon et al. 1987; Gonza´lez et al. 1996; The regulatory mechanisms that guide biofilm Sutherland 2001). They also improve nutrient development have recently come under scrutiny. acquisition (Cheng and Jaunet 1992) and provide The cell-to-cell communication known as quorum protection against environmental stress and host sensing, playing a part in the regulation of sur- defences (Robertson and Firestone 1992). More- face attachment and biofilm maturation, was first over, EPS’s also afford advantages to the plant suggested by Williams and Stewart (1994). Quo- hosts by improving water stability, which is critical rum sensing involves changes in the concentration to plant survival. Hence there is a strong selective of a diffusible autoinducer to provide a regula- advantage for the production of EPS within the tory signal in response to population density and rhizosphere. It is very likely that the EPS’s pro- has been shown to be essential for the construc- duced by Halomonas maura strains play similar tion of a mature biofilm in P. aeruginosa (Davies roles in their survival strategy. Nevertheless, the et al. 1998) Vibrio cholerae (Hammer and Bassler way in which the biosynthesis of these exopoly- 2003), Aeromonas hydrophila (Lynch et al. 2002) saccharides is regulated and whether their pro- and Streptococcus gordonii (McNab et al. 2003). duction responds to environmental factors and/or This system has been seen to regulate other population-density remains to be investigated. functions such as the expression of virulence fac- By carrying out adhesion assays (O‘Toole and tors and exoenzymes in Pseudomonas aeruginosa Kolter 1998) with wild-type and polysaccharide- and Erwinia carotovora (de Kievit and Iglewski, deficient mutants we have recently been able to 2000; Beck Von Bodman et al. 2003), conjugal demonstrate that H. maura strains are capable of transfer in Agrobacterium tumefaciens (Farrand producing biofilms and that mauran forms an 1998; Fuqua et al. 1994; 2001), the production integral part of the structural organisation of these of antibiotics in Chromobacterium violaceum biofilms (Inmaculada Llamas, unpublished data). (McClean et al. 1997) and the production of exo- Scanning-electron micrographs reveal small chan- polysaccharide in Pantoea stewartii (Beck Von nelled protuberances in the H. maura wild-type Bodman et al. 1998) and Sinorhizobium melitoti strain (Figure 1a), which seem then to participate (Marketon et al. 2003). in the formation of microcolonies and the matrix We have recently described a system of quo- of biofilm (Figure 1b). These extracellular struc- rum 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 potential interest both in industry because of the in the development of these bacteria is currently possibility of using its exopolysaccharides for a being studied (Llamas et al. 2005). We have variety of applications, and also in ecology due also identified some of the signal molecules (AHL’s) to its capacity to colonise saline niches and produced by Halomonas strains, such as N-butanoyl intervene in the nitrogen cycle. This latter homoserine lactone (C4-HL), N-hexanoyl homo- capacity could well result in its having beneficial serine lactone (C6-HL), N-octanoyl homoserine applications as a soil inoculate in moderately lactone (C8-HL) and N-dodecanoyl homoserine saline, arid soils that until now have defied lactone (C12-HL) (Llamas et al. 2005). agricultural use. In addition to this, it has other In Halomonas anticariensis, another EPS-pro- specific advantages for biotechnological use such ducing species, in which we detected the highest as its lack of pathogenicity, rapid growth and levels of signal molecules, we have obtained trans- easily available nutrient requirements, which conjugants deficient in signal molecules, which will allow it to be cultured in relatively cheap media allow us to characterise the genes involved in this with a high salt content, thus reducing risks of system and also the regulated cell functions. contamination. Consequently, to further the possibilities of employing Halomonas species to ecological ends Halomonas maura is a suitable bacterium for we are at present investigating the cell functions use in ecology and biotechnology that encode the quorum-sensing regulatory system in various species of this genus and are also All these characteristics of Halomonas maura lead looking into the influence of salt concentration us to the conclusion that it is of an enormous upon the nitrogen-fixing capacity of Halomonas maura. On the medical front, we are continuing to reporter system in moderately halophilic bacteria by study the biological activity of the sulphate-bear- employing the ice nucleation gene of Pseudomonas syringae. ing EPS mauran, produced by Halomonas maura. Appl. Environ. Microbiol 61: 3821–3825. Barloy-Hubler F., Capela D., Barnett M., Kalman S., Federspie N.A., Long S.R. and Galibert F. 2000. High-res- olution physical map of the Sinorhizobium meliloti 1021 pSyma megaplasmid. J. 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