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1 National Agricultural Research Foundation, Institute of Kalamata, Lakonikis 87, 24100 Kalamata, Greece
2 Agricultural University of Athens, Department of Agricultural Biotechnology, Electron Microscopy Laboratory, Iera Odos 75, 11855 Athens, Greece
Correspondence
Spyridon Ntougias
sntougias{at}in.gr
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7c, C16 : 0, C19 : 0 cyclo 8c, C12 : 0 3-OH and C16 : 17c/iso-C15 : 0 2-OH. Based on 16S rRNA gene sequence analysis, strain AW-7T showed almost equal phylogenetic distances from Zymobacter palmae (95.6 % similarity) and Carnimonas nigrificans (95.4 % similarity). In addition, low DNA–DNA relatedness values were found for strain AW-7T against Carnimonas nigrificans CECT 4437T (22.5–25.4 %) and Z. palmae DSM 10491T (11.9–14.4 %). The DNA G+C content of strain AW-7T is 64.4 mol%. Physiological and chemotaxonomic data further confirmed the differentiation of strain AW-7T from the genera Zymobacter and Carnimonas. Thus, strain AW-7T represents a novel bacterial genus within the family Halomonadaceae, for which the name Halotalea gen. nov. is proposed. Halotalea alkalilenta sp. nov. (type strain AW-7T=DSM 17697T=CECT 7134T) is proposed as the type species of the genus Halotalea gen. nov. A reassignment of the descriptive 16S rRNA signature characteristics of the family Halomonadaceae permitted the placement of the novel genus Halotalea into the family; in contrast, the genus Halovibrio possessed only 12 out of the 18 signature characteristics proposed, and hence it was excluded from the family Halomonadaceae.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AW-7T is DQ421388.
Figures showing the morphology of cells of strain AW-7T, as examined by negative-staining electron microscopy, and the phylogenetic position of the novel strain, calculated by the maximum-parsimony and maximum-likelihood methods, are available as supplementary figures with the online version of this paper.
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initially to accommodate the genera Deleya and Halomonas. Dobson et al. (1993) stated that the genus Halovibrio was related to the genera Halomonas and Deleya and later Dobson & Franzmann (1996) unified the genera Halomonas, Deleya and Halovibrio, together with the misclassified species Paracoccus halodenitrificans, into the genus Halomonas (Vreeland et al., 1980). Based on 16S rRNA gene sequence and DNA-DNA hybridization analyses, the genus Chromohalobacter (Ventosa et al., 1989) was proposed to be a member of the family Halomonadaceae (Kersters, 1992; Mellado et al., 1995). Dobson & Franzmann (1996) included the genus Chromohalobacter, together with the genus Zymobacter (Okamoto et al., 1993), in the emended description of the family Halomonadaceae. The genus Carnimonas was initially excluded from this family (Garriga et al., 1998), but Arahal et al. (2002b) argued that it should be classified within the family Halomonadaceae. In addition, Arahal et al. (2002a) transferred the species Halomonas marina into the genus Cobetia. The majority of the members of the family Halomonadaceae are either halotolerant or moderately halophilic bacteria that mainly originate from marine and/or saline environments (Ventosa et al., 1998). However, bacteria of the genera Carnimonas and Zymobacter are halotolerant and osmotolerant and have been isolated from processed meat products and palm sap, respectively (Okamoto et al., 1993; Garriga et al., 1998).
Olive oil mills equipped with two-phase centrifugation systems are now widespread in most olive oil-producing countries. Their operation results in the production (apart from olive oil) of a moderately acidic and viscous liquid-solid waste, termed ‘alpeorujo’ in Spain (Jones et al., 2000). To prevent the release of odours, Ca(OH)2 is added to alpeorujo, creating a moderately alkaline–saline secondary waste (Ntougias et al., 2006). A limited number of studies on bacteria originating from alpeorujo (including alkaline alpeorujo) have been performed. In a study of the osmoregulatory responses of bacteria from alpeorujo, two isolates, which tolerated high concentrations of salt and sucrose, were identified by partial 16S rRNA gene sequencing as Bacillus amyloliquefaciens and Salmonella sp. (Jones et al., 2000). In addition, several halotolerant alkaliphiles and moderate to extreme halotolerant and/or alkalitolerant bacteria have been previously identified from alkaline alpeorujo (Ntougias et al., 2006). Olivibacter sitiensis is the only non-halotolerant and non-alkalitolerant bacterium to have been isolated from this type of wastes (Ntougias et al., 2007).
In this paper, we describe the morphological, physiological, chemotaxonomic and phylogenetic characteristics of a novel halotolerant and alkalitolerant bacterium, isolated from alkaline alpeorujo. Due to its unique taxonomic position, we conclude that this isolate represents a new bacterial genus and novel species.
Strain AW-7T was isolated from alkaline alpeorujo, a waste originally produced by the two-phase centrifugal decanter system of an olive mill, which was further treated with 0.7 % w/w Ca(OH)2. Alkaline alpeorujo was disposed of in an open-air settling pond (from which our sample was obtained) next to an olive mill, located at the premises of the Toplou Monastery, Sitia (2 ° 6' 6'' N 3 ° 13' 12'' E), NE Crete, Greece. The pH and electrical conductivity (EC) of the sample (diluted in water at 1 : 1 v/v) were 8.7 and 16.2 mS cm–1, respectively.
To isolate strain AW-7T, 10 g of alkaline alpeorujo was added to 100 ml of 8.5 g l–1 NaCl solution and tenfold dilution series were prepared. Extract from alkaline alpeorujo (100 g in 1 l of distilled water, mixed for 20 min, filtered and adjusted to pH 7) solidified with agar was used as the isolation medium (Ntougias et al., 2006). After incubation at 25 °C for a week, strain AW-7T was selected and subcultured on solid medium consisting of 10 g glucose l–1, 5 g yeast extract l–1, 5 g peptone l–1, 0.1 mM MgSO4 and phosphate buffer at pH 7 (Ntougias et al., 2006). The experimental temperature for growth was 32 °C (optimum temperature). Growth of strain AW-7T was assessed on the medium described above, unless otherwise specified.
The pH range for growth (pH 4–11 in steps of one full pH unit) was investigated using the appropriate buffer solution (Ntougias & Russell, 2000). The pH was checked before inoculation and immediately after the end of the experiment. Salt tolerance was examined in presence of 0, 30, 50, 80, 100, 150 and 200 g NaCl l–1. Sugar tolerance was tested by substituting glucose in the medium described above (in the absence of agar) with 0, 100, 200, 300, 450, 500, 600 and 700 g (+)-D-glucose or maltose l–1. To investigate bacterial substrate utilization, nutrient media (pH 7, the pH was adjusted when it was necessary) containing a specific sugar, amino acid or other substrate (see species description), 0.1 mM MgSO4 and 0.05 g yeast extract l–1 were prepared (no growth was observed in the medium containing the above concentration of yeast extract as the sole carbon source). The concentration of each substrate tested was 0.05 M, unless the medium was saturated in a lower concentration and therefore a concentration up to the saturation point was used (Ntougias et al., 2007). The concentration of ethanol and glycerol was 0.5 % v/v, and 0.5 g phenol l–1 was also tested. Salts, metabolic substrates, yeast extract and the buffer were autoclaved separately and mixed aseptically. The sensitive compounds were filter-sterilized. Incubations were performed at 3, 5, 10, 15, 20, 25, 28, 32, 37, 40, 45 and 50 °C to examine the temperature range for growth. Anaerobic growth was tested using the Anaerocult A system (Merck). Antibiotic susceptibility was investigated on solid medium containing 50 µg ml–1 of the antibiotic tested (Ntougias et al., 2006). Acid production from various carbohydrates was examined in phenol red broth (18 mg phenol red l–1, pH 7.4) containing 10 g l–1 of the substrate tested (Chaturvedi & Shivaji, 2006). Reduction of nitrate, the methyl red reaction, activities of arginine dihydrolase, lysine and ornithine decarboxylases and phenylalanine deaminase, and production of H2S from L-cysteine and indole from tryptophan were tested following the methods described in Barrow & Feltham (1993). Urease, catalase and oxidase activities, spore-formation, Gram-staining, Voges–Proskauer test, and hydrolysis of gelatin, starch, Tweens 20 and 80 were examined according to Smibert & Krieg (1994). An alternative Gram test with KOH was also performed (Powers, 1995). Casein hydrolysis was tested on skim milk agar (10 % w/v skim milk). DNase test agar with toluidine blue (Sigma-Aldrich Chemie Gmbh) was used to assay DNase activity.
For transmission electron microscopy (TEM), suspensions of bacterial cells were placed on pyroxyline-coated, copper grids (AEI) and negatively stained with 1 % w/v phosphotungstic acid (pH 7.0) for 10 s. Specimens were examined and photographed with a TEM (model 9S; Zeiss).
DNA from pelleted cells was extracted as described by Wilson (1992). The nearly full-length 16S rRNA gene was amplified by using the two universal primers Afor, 5'-GGAGAGTTAGATCTTGGCTCAG-3' (sense; positions 6 to 27 according to the Escherichia coli numbering system) and Crev, 5'-AGAAAGGAGGTGATCCAGCC-3' (antisense; positions 1542 to 1525 according to E. coli numbering). A reaction mixture (50 µl) containing 1 µl (50 ng µl–1) genomic DNA, 10x PCR buffer (Finnzymes OY), 2 mM MgCl2, 200 µM each of dATP, dTTP, dCTP and dGTP, 0.5 µM each of primers Afor and Crev and 1 U DNA polymerase (Dynazyme EXT; Finnzymes OY) was prepared (Ntougias & Russell, 2001). Genomic DNA was amplified using a PTC-200 thermocycler (MJ Research Inc.) with a denaturation step of 2 min at 94 °C, followed by 35 cycles of 30 s denaturation at 94 °C, 30 s primer annealing at 55 °C and 1 min DNA chain extension at 72 °C. The PCR was completed by a 5 min DNA chain extension at 72 °C.
Three independent clones were obtained by cloning the respective amplicons in the pGEM-T Easy Vector (Promega) before insertion into DH5a-competent cells. Plasmid DNA was purified using the NucleoSpin plasmid Quick Pure kit (Macherey-Nagel). PCR sequencing using the fluorescence-labelled primers SP6 and T7 (Promega) and additional primers (5'-GGCGGCCTTCTGGACTG-3' and 5'-TCGCTGGCAAATAAGGATAGG-3') was performed at the Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Greece, via a LI-COR Long ReadIR2 4200 automated sequencer (LI-COR).
The 16S rRNA gene sequences were assembled using DNASTAR software (DNASTAR Inc.). Similarity searches were performed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and SeqMatch (Ribosomal Database Project II, http://rdp.cme.msu.edu/seqmatch/seqmatch_intro.jsp) and its closest relatives were obtained for further phylogenetic analyses. Alignment of sequences and construction of the phylogenetic tree based on the distance-matrix method were carried out using CLUSTAL W (http://www.ebi.ac.uk/clustalw/) and TREECON for Windows (version 1.3b) (Van de Peer & de Wachter, 1993), respectively. Evolutionary distances were calculated using the method of Jukes & Cantor (1969) and topology was inferred using the neighbour-joining method (Saitou & Nei, 1987) based on bootstrap analysis of 1000 trees. Phylogenetic trees were also generated by maximum-likelihood (DNAML; Cavalli-Sforza & Edwards, 1967) and maximum-parsimony (DNAPARS; Kluge & Farris, 1969) analyses using the PHYLIP version 3.6 phylogenetic package (Felsenstein, 2004), which includes the SEQBOOT program for bootstrap analysis. Phylogenetic trees calculated by maximum-parsimony and maximum-likelihood methods were drawn by TreeView software, version 1.6.6 (Page, 1996). Only almost full-length sequences were used for tree constructions. 16S rRNA secondary structures were predicted by the RNAstructure program (version 4) using the Zuker algorithm based on free energy minimization (Mathews et al., 2004).
Chemotaxonomic analyses (quinone and FAME analyses), DNA–DNA hybridization and DNA G+C content determination were carried out by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. DNA for spectroscopic DNA–DNA hybridization and G+C content determination was isolated as described by Cashion et al. (1977). DNA–DNA hybridization was performed as described by De Ley et al. (1970) and modified as suggested by Huß et al. (1983). DNA G+C content determination was carried out according to Tamaoka & Komagata (1984) and Mesbah et al. (1989). Fatty acid methyl esters were obtained as previously described (Kroppenstedt, 1985; Kämpfer & Kroppenstedt, 1996) using minor modifications of the methods of Miller (1982) and Kuykendall et al. (1988). Respiratory lipoquinone analyses were performed by Dr B. J. Tindall and the Identification Service of the DSMZ using a standard procedure (Tindall, 1990a, b).
Morphological, cultural, physiological and biochemical characteristics of strain AW-7T are given in the species description, Table 1 and in Supplementary Fig. S1 (available in IJSEM Online).
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7c and/or iso-C15 : 0 2-OH (6.2 %), C16 : 0 (28.5 %), C17 : 0 cyclo (0.4 %), C18 : 17c (35.6 %), C18 : 0 (0.4 %) and C19 : 0 cyclo 8c (12.4 %).
Strain AW-7T tolerated the greatest (+)-D-glucose and maltose concentrations (45 and 60 % w/v, respectively) ever reported for recognized members of the domain Bacteria. Members of the genera Zymomonas and Saccharibacter can tolerate up to 40 % w/v (+)-D-glucose (Swings & De Ley, 1983; Jojima et al., 2004), while strains of the genus Zymobacter can tolerate up to 20 and 50 % w/v (+)-D-glucose and maltose concentrations, respectively (Okamoto et al., 1993).
Construction of phylogenetic trees using distance-matrix, character-based (parsimony) and maximum-likelihood methods all placed strain AW-7T in a phylogenetic position related to Z. palmae and Carnimonas nigrificans (see Fig. 1 and Supplementary Figs S2 and S3 in IJSEM Online). Based on 16S rRNA gene sequence analysis, strain AW-7T was associated, but not closely related, to Z. palmae (95.6 % similarity) and Carnimonas nigrificans (95.4 % similarity).
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Bacterial strain AW-7T possessed 13 out of the 18 base signature characteristics proposed by Dobson et al. (1993) (Table 2), while it formed a 7 bp instead of 6 bp stem in its 16S rRNA secondary structure (referring to positions 76–93 in the E. coli numbering system) (Fig. 2).
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).
Bacterial strain AW-7T is an obligate aerobe which formed pale yellow colonies (not white), utilized citrate and grew at temperatures between 5 and 45 °C. In addition, cells of strain AW-7T were oxidase-positive and produced acid from (+)-D-mannose. In contrast, Z. palmae is a facultative anaerobe that grows over a relatively narrow temperature range (21–39 °C), gives positive reactions in methyl red and Voges–Proskauer tests and produces acid from D-mannitol, sorbitol and sucrose (Okamoto et al., 1993). The presence of C18 : 17c and the lack of C18 : 19 in the fatty acid profile of strain AW-7T, compared with that of Z. palmae, indicates significant chemotaxonomic differences. DNA–DNA relatedness between strain AW-7T and Z. palmae was low (11.9–14.4 %). Moreover, significant divergence between strain AW-7T and Z. palmae was detected in their respective DNA G+C contents. The G+C content of strain AW-7T was 8.2 mol% higher than that of Z. palmae (55.4–56.2 mol%) (Okamoto et al., 1993). Strain AW-7T showed 95.6 % 16S rRNA gene sequence similarity with Z. palmae.
Strain AW-7T also showed an almost equal phylogenetic distance to Carnimonas nigrificans (95.4 % 16S rRNA gene sequence similarity). Strain AW-7T and Carnimonas nigrificans showed significant differences in their DNA G+C contents. The DNA G +C content of strain AW-7T was 8.4 mol% higher than that of Carnimonas nigrificans (56.0±0.3 mol%) (Garriga et al., 1998). In addition, low DNA–DNA relatedness (22.5–25.4 %) was found between strain AW-7T and Carnimonas nigrificans. Strain AW-7T utilized D-mannitol and did not hydrolyse starch. Colonies of strain AW-7T were pale yellow and the cells were motile. The novel strain grew at 5 °C and at 37 °C (optimum temperature of 32–37 °C) and tolerated up to 15 % w/v NaCl. In contrast, Carnimonas nigrificans was a non-motile bacterium forming white colonies which failed to grow at 5 °C and 37 °C and at a salt concentration greater than 8 % w/v NaCl. The optimum growth temperature of Carnimonas nigrificans was lower (28–30 °C) than that of the novel strain. In addition, Carnimonas nigrificans did not utilize D-mannitol, but was able to hydrolyse starch (Garriga et al., 1998). Furthermore, Carnimonas nigrificans was positive for phenylalanine deaminase activity and produced acid from D-mannitol, sucrose and (+)-D-xylose. In the fatty acid profiles of the strains examined, the lack of trans isomers and the presence of significant amounts of hydroxylated fatty acids were a distinctive feature of strain AW-7T when compared to Carnimonas nigrificans.
Strains of the genus Cobetia, as compared to strain AW-7T, are oxidase-negative halophiles, that utilize myo-inositol, ethanol, L-lysine, and D-cellobiose, but not sorbitol or D-xylose. Moreover, they form cream-pigmented colonies, contain trans monounsaturated fatty acids, mainly C16 : 1t9 and to a lesser extent C18 : 1t9, as well as C18 : 19. Ubiquinone-8 is also present as a minor component in the respiratory chain of members of the genus Cobetia (Arahal et al., 2002a; Yumoto et al., 2004). In addition, members of the genus Cobetia are motile by means of polar flagella, while strain AW-7T is motile by peritrichous flagella. Strain AW-7T showed 93.0 % 16S rRNA gene sequence similarity with Cobetia marina.
In contrast to strain AW-7T, members of the genus Chromohalobacter are halophiles growing at salt concentrations of up to 30 % w/v NaCl. In addition, members of the genus Chromohalobacter produce pigments and oxidase activity is not present in all species (Arahal et al., 2001). In comparison with species of the genus Chromohalobacter, strain AW-7T contains the fatty acids C10 : 0, C12 : 0, C12 : 0 2-OH and a significant amount of C12 : 0 2-OH. Hydroxylated-C12 : 0 fatty acids have never been detected in any member of the genus Chromohalobacter so far tested (Mutnuri et al., 2005; Vargas et al., 2005). In comparison with strain AW-7T, members of the genus Chromohalobacter showed 16S rRNA gene sequence similarities of less than 91.8 %. The closest relative of strain AW-7T from the genus Chromohalobacter was Chromohalobacter israelensis (91.8 % gene sequence similarity).
In contrast to strain AW-7T, species of the genus Halomonas produce pigments and many of them have extreme upper salt concentrations for growth (Mata et al., 2002). In addition, the majority of the type strains of Halomonas species can utilize ethanol, inositol, L-isoleucine and L-lysine and they are susceptible to ampicillin and chloramphenicol (Mata et al., 2002). Due to the large number of species included in the genus Halomonas, differences in fatty acid contents depend on the specific species that is compared with strain AW-7T. As the DNA G+C content of species of the genus Halomonas ranges widely from 51.4 to 74.3 mol% (Martinez-Canovas et al., 2004; Quillaguaman et al., 2004), this characteristic cannot be used as a taxonomic tool. Concerns about the taxonomy of the genus Halomonas should be expressed as there is a significant variability in phenotypic, chemotaxonomic and molecular characteristics among species of the genus Halomonas. A study by Mata et al. (2002) showed that none of 205 phenotypic and physiological characteristics tested were common for all type strains of species of the genus Halomonas. When compared with members of the genus Halomonas, strain AW-7T showed 16S rRNA gene sequence similarities lower than 92.2 %. Its closest relative from the genus Halomonas was Halomonas pacifica (92.1 % gene sequence similarity).
Dobson et al. (1993) reported 19 characteristic signatures (18 characteristic bases and a 6 bp stem located in the 76–93 positions, E. coli numbering system) in the 16S rRNA secondary structures of members of the genera Deleya, Halomonas and Halovibrio in order to accommodate these three genera within the family Halomonadaceae. These genera were unified into a single genus by Dobson & Franzmann (1996), the genus Halomonas, and the number of signature characteristics was reduced to 15 in order to include the genus Zymobacter in the family. Arahal et al. (2002b) placed the genus Carnimonas in the family Halomonadaceae, reporting 13 elements common to all members plus six residues with two possible bases. When the presence of a 6 bp stem in positions 76–93 according to the E. coli numbering system was excluded from the analysis, we found that all species of the genera Halomonas, Chromohalobacter and Cobetia possess all the remaining characteristics (18 out of 19) proposed by Dobson et al. (1993) (Table 2), with the exception of Chromohalobacter marismortui (it possesses G instead of U in position 1124 according to E. coli numbering) and Halomonas muralis (C and U instead of U and C in positions 1297 and 1298, respectively). The above differences in the 16S rRNA secondary structure of H. muralis may due to sequence misreading (a reverse order was observed for positions 1297–1298). Taking into account the 18 characteristic base positions described by Dobson et al. (1993), Z. palmae, Carnimonas nigrificans and strain AW-7T possessed 14, 13 and 13 signature characteristics, respectively. When the bases located in the 61–106 positions (including positions 76–93) were folded, only members of the genera Carnimonas, Chromohalobacter and Cobetia formed a 6 bp stem, while members of the genus Zymobacter and strain AW-7T formed a 7 bp stem (Fig. 2). However, no formation of a 6 bp stem was observed for the type species of the genera of the family Halomonadaceae when almost complete 16S rRNA sequences were folded using the standard conditions of the RNAstructure program (Version 4.2). Based on the above data, we propose the exclusion of the 6 bp stem from the list of signature characteristics of the family Halomonadaceae. In contrast to previous results (Garriga et al., 1998; Arahal et al., 2002b), we detected a G instead of the C reported in position 1462 (E. coli numbering) in members of the genera Zymobacter and Carnimonas and a 7 bp instead of a 6 bp stem in the 16S rRNA secondary structure of the genus Zymobacter. The genus Halovibrio, consisting of the species Halovibrio denitrificans and Halovibrio variabilis (Pseudomonas halophila DSM 3050T) (Sorokin & Tindall, 2006; Sorokin et al., 2006), possessed only 8 signature characteristics (Table 2 and Fig. 2) from the 19 reported by Dobson et al. (1993). This observation, in conjunction with the distinct phylogenetic (showing 16S rRNA gene sequence similarities with the type species of the family Halomonadaceae and with strain AW-7T that are lower than 87.5 %) and chemotaxonomic position (Sorokin et al., 2006), lead us to propose the exclusion of the genus Halovibrio from the family Halomonadaceae. We also propose the reassignment of the family Halomonadaceae (see emended description), based on its 16S rRNA sequence signature characteristics, to accommodate the previous described genera Halomonas, Chromohalobacter, Zymobacter, Cobetia and Carnimonas and a new genus Halotalea gen. nov.
On the basis of phenotypic, chemotaxonomic and phylogenetic data, we conclude that strain AW-7T represents a new bacterial genus and novel species of the family Halomonadaceae, for which we propose the name Halotalea alkalilenta gen. nov., sp. nov.
Description of Halotalea gen. nov.
Halotalea (Ha.lo.ta.le'a. Gr. n. hals halos salt; L. fem. n. talea a staff, rod; N.L. fem. n. Halotalea rod-shaped cells living in saline conditions).
Cells are Gram-negative, rods, motile by peritrichous flagella and forming small, non-pigmented pale yellow colonies. Endospores are not formed. Strictly aerobic. Halotolerant and alkalitolerant. Sugar-tolerant. Oxidase- and catalase-positive. Chemo-organotrophic. Ubiquinone-9 is present in the respiratory chain. The major fatty acids are C18 : 17c, C16 : 0, C19 : 0 cyclo 8c, C12 : 0 3-OH and C16 : 17c/iso-C15 : 0 2-OH. The DNA G+C content is 64.4 mol%. The type species is Halotalea alkalilenta.
Description of Halotalea alkalilenta sp. nov.
Halotalea alkalilenta (al.ka.li.len.ta. N.L. n. alkali from Arabic al qaliy, soda ash; L. adj. lentus -a slow; N.L. n. alkalilenta slow in alkaline conditions/alkalitolerant).
Strict aerobe that tolerates up to 15 % w/v NaCl, with an optimum salt concentration of 0–3 % w/v NaCl. Tolerates up to 45 and 60 % w/v (+)-D-glucose and maltose, respectively. Grows at pH 5 to 11, with optimum pH at 7. The temperature range for growth is 5 to 45 °C, with an optimum temperature of 32–37 °C. Chemo-organotrophic. Preferentially utilizes L-glutamine and L-proline (OD600 > 0.35; growth measured at 32 °C and pH 7; OD600 values cited correspond to measurements taken after 72 h), followed by (+)-D-galactose, (+)-D-glucose, glycerol, D-mannitol, protocatechuate, L-serine, succinate, sucrose and (+)-D-xylose (respective OD600 values in the range 0.13–0.35). Weak growth on acetate, citrate, (–)-D-fructose, maltose and sorbitol (respective OD600 values in the range 0.05–0.12). Grows on gallate (106 cells ml–1 in the defined medium). (+)-D-Cellobiose, cinnamate, L-cysteine, ethanol, ferrulate, glycine, L-histidine, inositol, L-isoleucine, lactose, L-lysine, L-methionine, phenol, syringate, L-tryptophan, vanillate and o-vanillin are not utilized. Negative reactions in tests for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, phenylalanine deaminase, nitrate reduction, H2S production from L-cysteine, indole production, methyl red and Voges–Proskauer. Acid is produced from (–)-D-fructose, (+)-D-galactose, (+)-D-glucose, maltose, (+)-D-mannose and melibiose, but it is not produced from (+)-L-arabinose, (+)-D-cellobiose, inositol, lactose, D-mannitol, sorbitol, sucrose or (+)-D-xylose. Hydrolyses Tween 20, but does not hydrolyse casein, DNA, gelatin, starch, Tween 80 or urea. Susceptible to (50 µg ml–1) kanamycin, polymixin B, rifampicin, streptomycin and tetracycline, but resistant to ampicillin, bacitracin, chloramphenicol, penicillin and trimethoprim. The DNA G+C content of the type strain is 64.4 mol%.
The type strain, AW-7T (=DSM 17697T=CECT 7134T), was isolated from olive mill waste (alkaline alpeorujo) obtained from the premises of the Toplou Monastery in the region of Sitia, Crete, Greece.
Emended description of the family Halomonadaceae Franzmann et al. 1989, emend. Dobson and Franzmann 1996
The description is as given by Franzmann et al. (1988) and Dobson & Franzmann (1996), but an alteration in 16S rRNA sequence signature characteristics. The 16S rRNA sequence signature characteristics of the family Halomonadaceae are redefined as follows: position 484 (A or G), position 486 (C or U), position 640 (G), position 660 (A), position 745 (U), position 668 (A), position 738 (U), position 669 (A), position 737 (U), position 776 (U), position 1124 (U or G), position 1297 (U), position 1298 (C), position 1423 (A), position 1424 (C or U), position 1439 (U or C), position 1462 (A or G) and position 1464 (C or U). The family comprises the genera Carnimonas, Chromohalobacter, Cobetia, Halomonas, Halotalea and Zymobacter.
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