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 thixo- tropic and pseudoplastic behaviour, has the capacity to capture heavy metals and exerts a certain immu- nomodulator 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 exo- polysaccharide 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 (Garcı´a 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; Martı´nez- roots is not yet fully understood we can affirm that Ca´novas et al., 2004a; 2004b; Martı´nez-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 Martı´nez-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 demon- strated 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 (Martı´nez-Ca´novas 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.