10 The Family Colwelliaceae John P. Bowman Food Safety Centre, Tasmanian Institute of Agriculture, University of Tasmania, Hobart, TAS, Australia

Taxonomy, Historical, and Current Short Description Taxonomy, Historical, and Current Short of the Family Colwelliaceae Ivanova Description of the Family Colwelliaceae VP et al. 2004, 1773VP ...... 179 Ivanova et al. 2004, 1773

Molecular Analyses ...... 180 Col.well.i0a.ce.ae. N.L. fem. n. Colwellia, type genus of the fam- ily; suff. -aceae, ending to denote a family; N.L. fem. pl. n. Phenotypic Properties ...... 181 Colwelliaceae, the Colwellia family. The family Colwelliaceae was first described by Ivanova and Genus Colwellia Deming et al. 1988, 328AL ...... 181 colleagues (2004) as part of an effort to create taxonomic har- mony within a large clade of almost exclusively marine bacteria Genus Thalassomonas Macia´n et al. 2001, 1283 ...... 187 located within class Gammaproteobacteria. This clade now rep- resents the order Alteromonadales (Bowman and McMeekin Enrichment, Isolation, and Maintenance Procedures . . . . . 187 2005) and consists of at least 22 genera as of late 2012. In addition to the genera Colwellia and Thalassomonas, Genome-Based and Genetic Studies ...... 191 the other members of the order include Aesturaiibacter, Agarivorans, Algicola, Aliagarivorans, Alkalimonas, Ecology ...... 192 Alishewanella, Alteromonas, Bowmanella, Catenovulum, Ferri- monas, Glaciecola, Idiomarina, Moritella, Oceanisphaera, Para- Applications ...... 193 ferrimonas, Pseudoalteromonas, Psychromonas, Rheinheimera, Salinimonas, and Shewanella. The order Alteromonadales is subdivided into several families; besides Colwelliaceae these Abstract families include Alteromonadaceae, Ferrimonadaceae, Idio- The family Colwelliaceae is part of the order Alteromonadales marinaceae, Moritellaceae, Pseudoalteromonadaceae, and within the class Gammaproteobacteria and currently comprises Shewanellaceae. The description of new genera since 2004 the type genus Colwellia and the genus Thalassomonas. prompts description of new families or perhaps more usefully, Collectively, Colwelliaceae encompasses at least 19 species. Both when more genome data becomes available, a taxonomic genera are strictly marine in terms of distribution and appear as reappraisal of the entire order. curved to straight rod-shaped cells that form primarily The genus Colwellia was first described by Deming et al. nonpigmented colonies, possess a polar or subpolar flagellum, (1988) on the basis of 5S rRNA sequence data of two very and are catalase and oxidase positive. Metabolism varies between psychrophilic strains including strain ATCC 27364T the two genera, with Colwellia species being facultatively anaer- (NRC1004) isolated from Flounder eggs collected near Trond- obic and able to grow fermentatively and also by using at least heim, Norway, and barophilic strain BNL-1T, collected from nitrate as an electron acceptor. Most Thalassomonas species are surface sediment of the Puerto Rico Trench at a depth of instead strictly aerobic; however further study is required to 7,410 m. Strain ATCC 27364T, which possessed an unusual confirm this. Both members of Colwelliaceae have attracted bright red prodigiosin-like pigment (D’’Aoust and Gerber interest in terms of extremophilic environmental research and 1974) and produced a self-toxic growth response, was initially biotechnological investigations. The genus Colwellia contains named by Da´oust and Kushner (1972)‘‘Vibrio psychroerythrus.’’ several obligately psychrophilic (cold-requiring) and piezophilic ATCC 27364T and BNL-1T were designated Colwellia (pressure-requiring) species that synthesize omega-3 polyunsat- psychrerythraea, the type species, and Colwellia hadaliensis, urated fatty acids while a number of Thalassomonas species respectively, by Deming and colleagues (1988). Subsequently possess potent agarolytic activity. The species Colwellia a further 10 species have since been added to the genus, deriving psychrerythraea represents a model for understanding how bac- from a diversity of marine environments, and include teria thrive at freezing temperatures. C. demingiae, C. hornerae, C. psychrotropica, C. rossensis

E. Rosenberg et al. (eds.), The Prokaryotes – Gammaproteobacteria, DOI 10.1007/978-3-642-38922-1_230, # Springer-Verlag Berlin Heidelberg 2014 180 10 The Family Colwelliaceae

. Fig. 10.1 16S rRNA gene sequence-based neighbor-joining tree (distances based on maximum likelihood algorithm) showing the position of members of the family Colwelliaceae (which are shown in colored type) within the order Alteromonadales. Thermotoga maritima and Coprothermobacter platensis represented out-groups. Sequences used come from the type strains of the following species (GenBank accession code): Colwellia piezophila (NR_024805), Colwellia maris (NR_024635), Colwellia rossensis (NR_025957), Colwellia psychrotropica (NR_026055), Colwellia psychrerythraea (NR_037047), Colwellia aestuarii (NR_043509), Colwellia asteriadis (EU599214), Colwellia chukchiensis (FJ889599), Colwellia hornerae (JN175346), Colwellia demingiae (U85845), Colwellia polaris (DQ007434), Thalassomonas viridans (NR_042048), Thalassomonas haliotis (NR_041662), Thalassomonas actiniarum (NR_041661), Thalassomonas agariperforans (HM237288), Thalassomonas ganghwensis (NR_025717), Thalassomonas agarivorans (NR_043649), Thalassomonas loyana (NR_043066), Agarivorans albus (NR_024788), Agarivorans gilvus (GQ200591), Aliagarivorans marinus (FJ167390), Aliagarivorans taiwanensis (FJ167391), Algicola sagamiensis (NR_027234), Alishewanella fetalis (AF144407), Alkalimonas amylolytica (AF250323), Alteromonas macleodii (Y18228), Bowmanella denitrificans (DQ343294), Catenovulum agarivorans (GQ262000), Ferrimonas balearica (CP002209), Glaciecola punicea (U85853), Idiomarina abyssalis (NR_024891), Moritella marina (AB038033), Oceanisphaera litoralis (AJ550470), Paraferrimonas sedimenticola (NR_041444), Pseudoalteromonas haloplanktis (X67024), Psychromonas antarctica (Y14697), Rheinheimera baltica (AJ441080), Salinimonas chungwhensis (AY553295), Shewanella putrefaciens (X81623)

(Bowman et al. 1998), C. maris (Yumoto et al. 1998), (Thompson et al. 2006), T. actiniarum, T. haliotis (Hosoya C. piezophila (Nogi et al. 2004), C. aestuarii (Jung et al. 2006), et al. 2009), and T. agariperforans (Park et al. 2011). C. polaris (Zhang et al. 2008), C. asteriadis (Choi et al. 2010), and C. chukchiensis (Yu et al. 2011). Thalassomonas the sister genus of Colwellia includes seven Molecular Analyses species as of late 2012. The type species, Thalassomonas viridans, was isolated from a Mediterranean sea oyster (Macia´n et al. Colwellia and Thalassomonas species cluster together and pos- 2001) and subsequent additional species also derive from sess a maximum 16S rRNA gene sequence dissimilarity of a diverse range of strictly marine sites mainly located in temper- approximately 7 %. Thalassomonas species in most 16S rRNA ate to tropical regions. These species include T. ganghwensis gene-based trees are not monophyletic but form two (Yi et al. 2004), T. agarivorans (Jean et al. 2006), T. loyana paraphyletic sub-lineages (> Fig. 10.1). One sub-lineage The Family Colwelliaceae 10 181 contains the species T. viridans, T. actiniarum, and T. haliotis evolved (Franzmann 1996). Whether the predilection towards while the second contains T. ganghwensis, T. loyana, psychrophily in the genus Colwellia reflects a bias of isolation T. agarivorans, and T. agariperforans. Colwellia is on the other from cold marine sites is unknown; however it is reasonable hand clearly monophyletic with C. chukchiensis, C. polaris, and to assume at least at this stage that Colwellia is by and large C. aestuarii forming a peripheral relatively distinct sub-lineage. a highly cold-adapted lineage of bacteria, a rather rare feature The next closest related genera to family Colwelliaceae amongst cultivated bacteria and archaea. The geographical dis- include Agarivorans (Kurahashi and Yokota 2004) and tribution of Colwellia is broad but appears to be strictly marine Aliagarivorans (Jean et al. 2009), which currently are not affili- (see > Ecology section). ated with a family level taxon. Available data however does not In comparison to the genus Thalassomonas, Colwellia species lend convincing justification that either of these genera should have lower temperature growth ranges and optima for growth be placed in Colwelliaceae, either on the basis of 16S rRNA gene- and in general are slightly larger (range 1–5 Â 0.4–1.0 mm) based phylogeny or on the basis of phenotypic data. Further data and are pronouncedly more curved in shape. Colwellia is is required and as mentioned previously a more in-depth also facultatively anaerobic and able to grow either by aerobic genome sequence data-based appraisal is required to further oxidation, fermentation of carbohydrates, and by anaerobic develop the higher level taxonomy amongst the members of respiration with nitrate and other electron acceptors such as order Alteromonadales. manganese (see > Ecology section) but likely unable to use ferric iron as electron acceptor. Acetate and a variety of other simple compounds can serve as electron donors. Most species Phenotypic Properties can form chitinases but so far none have been found that degrade agar. The species generally grow on a range of Collectively, the species of family Colwelliaceae contains Gram- carbohydrates, organic acids, amino acids, and related com- negative, rod- to curved rod-shaped cells that in almost all pounds (> Table 10.1). Overall, the data suggests Colwellia spe- cases are motile via a single polar flagella, catalase, cytochrome cies have broad ranging biochemical versatility of moderate c oxidase, and alkaline phosphatase positive. Most strains depth. require both Na+ and divalent cations (Mg2+ and/or Ca2+) for Two species of genus Colwellia, C. hadaliensis (Deming et al. growth while no strain has been isolated that grows at 1988) and C. piezophila (Nogi et al. 2004), are obligately a temperature above 42 C. Biochemical traits common amongst barophilic inhabiting hadal surface sediments of deep-sea Colwelliaceae member species include the ability to reduce trenches. Both require for growth high hydrostatic pressure nitrate to nitrite (but not further), hydrolysis of aesculin, with best growth occurring at 60–75 MPa at 10 C, equivalent Tween 80, casein, L-tyrosine, and starch. No strains so far to depths that they were isolated from (6,000–8,000 m). have been shown to form indole from L-tryptophan Growing such strains requires highly specialized methods or produce arginine dihydrolase, lysine decarboxylase, or (see ISOLATION and MAINTENANCE section) and given the ornithine decarboxylase. The primary respiratory lipoquinone technicalities little data is available on these species in general. is ubiquinone-8 while the majority of phospholipid fatty C. hadaliensis BNL-1T appears to be no longer available acids are 14–18 carbon chain (C14-18) monounsaturated for study. and unsaturated types; branched fatty acids make up The primary fatty acids vary considerably between Colwellia only a relatively small proportion of total fatty acids in most species as shown in > Table 10.2. This is unusual since normally strains. fatty acids are relatively similar between species of a given genus. To some extent the variation amongst Colwellia species reflects analysis methods but also likely reflects physiological and AL Genus Colwellia Deming et al. 1988, 328 genetic adaptations specific to species. In five species (C. aestuarii, C. asteriadis, C. chukchiensis, C. piezophila, C. polaris) Col.well0i.a. N.L. fem. dim. n. Colwellia, named in honor of the whole-cell fatty acids were analyzed that must have contained American microbiologist Professor Rita R. Colwell. a substantial level of lipopolysaccharide-derived fatty acids,

The type species of the genus Colwellia is Colwellia which tend to include shorter chain length (C10–C14) psychrerythraea (Deming et al. 1988). Given that 12 species in and largely hydroxylated types. The amounts of C14:0, genus Colwellia have been described to date, the collective meta- C15:0,C14:1,C15:1,C16:1, and C17:1 isomers, C16:0 iso, and the data (> Table 10.1) means that a good concept of the basic polyunsaturated fatty acids (PUFA) docosahexaenoic phenotypic nature of the genus can be demonstrated. (DHA, C22:6 o3c) and eicosapentaenoic acids (EPA, C20:5 o3c) All Colwellia species are able to grow at low temperature; how- vary considerably between species suggesting additional biosyn- ever the original concept that the genus being purely psychro- thetic genes are present in certain strains, especially the philic (Deming et al. 1988; Bowman et al. 1998) no longer can be more psychrophilic species, all of which can form PUFA. said to be true since one species C. aestuarii was isolated from Culture conditions likely also influence the quantitative distri- a temperate location and grows at temperatures up to 35 C. The bution of fatty acids. The ability to form PUFA is a special, albeit fact that cold-adapted species mingle with more warm species-dependent feature of genus Colwellia since the trait temperate adapted species suggests psychrophiles are recently only occurs sporadically across the order Alteromonadales 182 10

. Table 10.1 Phenotypic characteristics of Colwellia species

Phenotypic characteristica Colwellia species codeb h Family The 123 45 6789 101112 Colony pigment Vc ÀÀ ÀÀ ÀÀÀÀ À À À

Size (mm) 1.5–4.5 1.5–4.5 1.5–3.0  1.5–3.0 1.5–3.0 0.7–1.0 1.8–3.1 0.9–4.0 2.0–4.0 3.0–5.0  0.8 2.0–4.0  1.1–4.5  Colwelliaceae  0.4–0.6  0.4–0.6 0.4–0.8  0.4–0.8  0.4–0.8  0.4–0.5  0.4–0.5  0.6–0.9  0.8–1.0 0.6–1.0 0.5–1.0 Gas vesicles ÀÀ+ ÀÀ ÀÀÀÀ À À À Isolation site Fish Sea ice Sea ice/ Sea ice Meromictic Starfish Tidal flat Sea ice Deep-sea Deep-sea Seawater Seawater eggs, sea seawater saline lake skin sediment sediment sediment (Puerto (off (Arctic ice interface (Japan Trench) Rico Trench) Hokkaido) Ocean) Temperature growth range <0–15 <0–15 <0–15 <0–20 <0–25 0–25 4–35 <0–25 <0–10 <0–10 <0–25 <4–30 Optimum temperature for 10 10 10 10–15 15–20 15–20 25–30 20 5–10 5–10 20 25 growth (C) Requires sea salts or divalent +++ ++ ÀÀÀ++ À + cations Seawater salinity tolerance 20–60 20–60 20–50 20–60 10–100 0–100 0–60 10–60 20–60 10–80 range (psu) Optimum seawater salinity 30 30–40 30 30–40 30–40 20–30 20–30 20–30 30–40 30–40 30–40 30–40 for growth (psu) High hydrostatic pressure ÀÀÀ ÀÀ ÀÀÀ++ ÀÀ needed for growth Optimum pressure for 60 75 growth (MPa) Produce Arginine dihydrolase, indole ÀÀÀ ÀÀ À À from L-tryptophan Lysine decarboxylase, ÀÀÀ ÀÀ ÀÀ À À ornithine decarboxylase Urease V À + À + ÀÀ +

H2S from L-cysteine ÀÀ b-galactosidase (ONPG test)* ÀÀÀ ÀÀ ÀÀÀ À API ZYM kit enzymatic activity Alkaline phosphatase + + + + + + + + + Acid phosphatase + + ÀÀ Esterase (C4), esterase (C8) + + À + Esterase (C14) ÀÀÀ À Naphthol-AS-BI- ++À + phosphohydrolase Trypsin, a-chymotrypsin ÀÀÀ À a-galactosidase, ÀÀÀ À a-mannosidase, a-fucosidase, b-glucuronidase b-glucosidase À + ÀÀ N-acetyl-b-glucosaminidase + ÀÀ À Hydrolysis of Aesculin + + À + ÀÀÀ++ Tween 20, Tween 40, ++ Tween 60 Tween 80 + ÀÀ ++ À ++ + + Lecithin À Xanthine, hypoxanthine À Urate ÀÀÀ À+ DNA ÀÀÀ ÀÀ +

L-tyrosine V ÀÀ À+ À Gelatin V ÀÀ ÀÀ ÀÀ++ + + Casein + + À ++ À ++ÀÀ+ Starch + + + + À + À + ÀÀÀ Chitin + + + À ++ÀÀÀ + ÀÀ Agar ÀÀÀ ÀÀ ÀÀÀÀ À À Assimilates or metabolizes

L-arabinose ÀÀ+ ÀÀ À++ÀÀÀ

D-fructose ÀÀ+ ÀÀ + À + ÀÀÀ

D-galactose ÀÀ+ ÀÀ À++À +

D À ÀÀ À -glucose + + ++ + + Family The

D-mannose ÀÀÀ ÀÀ À+ ÀÀÀ

L-rhamnose ÀÀÀ + À + ÀÀ

DL-xylose ÀÀÀ ÀÀ + À + ÀÀ Colwelliaceae Cellobiose V ÀÀ ÀÀ À++ÀÀ

D-lactose ÀÀÀ ÀÀ À+ ÀÀ À À

D-maltose + ÀÀ ÀÀ À+++ ÀÀ

D-melibiose ÀÀÀ ÀÀ À++ ÀÀ 10 Sucrose ÀÀÀ ÀÀ À++ÀÀÀ

D-trehalose ÀÀÀ ÀÀ À+ ÀÀ 183 184 10 h Family The Colwelliaceae

. Table 10.1 (continued)

Phenotypic characteristica Colwellia species codeb

D-raffinose ÀÀÀ ÀÀ À À À À Glycogen + V + ÀÀ À Adonitol ÀÀÀ ÀÀ À DL-arabitol ÀÀÀ ÀÀ À Myoinositol ÀÀÀ ÀÀ À À À

D-mannitol ÀÀÀ ÀÀ À+ ÀÀ

D-sorbitol ÀÀÀ ÀÀ À+ ÀÀ

D-gluconate ÀÀ+ ÀÀ À

N-acetyl-D-glucosamine + + + À + À Glycerol ÀÀ++À + ÀÀÀ DL-lactate V + À ++

L-aconitate ÀÀÀ ÀÀ Oxaloacetate + + À ++ Citrate À ++ +À ÀÀÀ + Fumarate + + + + + DL-b-hydroxybutyrate + À + À + DL-malate ÀÀ+ À + À + À 2-oxoglutarate ÀÀÀ À+ Pyruvate + + + + + À + Succinate + + + + + À Acetate + + + + + + À + Propionate V + À + À Butyrate + + + + + Isobutyrate À V ÀÀ+ Valerate, caproate À ++ ++ Isovalerate, nonanoate, ÀÀÀ ÀÀ adipate, pimelate Heptanoate, azelate À V À + À Caprate À V ÀÀÀ Malonate À V+ ÀÀ Glutarate À + À + À Caprylate À V ÀÀÀ

L-alanine À V+ À +

L-asparagine À ++ À +

L-aspartate À V+ À +

L-glutamate, L-proline + + + + + À

L-phenylalanine À V ÀÀÀ

L-serine À + ÀÀÀ

L-threonine, putrescine ÀÀÀ ÀÀ

L-tyrosine V ÀÀ À+ Gamma-aminobutyrate À ++ ++

Hydroxy-L-proline À V À + À DNA base G+C composition 35–38 37 38 39 42 40 39 39 39 45–46 39 41 (mol%) aAll Colwellia species are Gram-negative; appear as straight to curved rodlike cells; are motile via a single polar flagellum, catalase, and cytochrome c oxidase positive; able to reduce of nitrate to nitrite; and have a facultatively anaerobic metabolism and can grow anaerobically via either fermentation of carbohydrates and by using nitrate as an electron acceptor. The optimum pH for growth is approximately that of seawater (pH 7–8) bColwellia species code: 1, C. psychrerythraea;2,C. demingiae;3,C. rossensis;4,C. hornerae;5,C. psychrotropica;6,C. asteriadis;7,C. aestuarii;8,C. polaris;9,C. piezophila; 10, C. hadaliensis; 11, C. maris, and 12, C. chukchiensis cAbbreviations: + test positive, À test negative, V test result data varies between different strains of the species. A blank cell indicates no data is available h Family The Colwelliaceae 10 185 186 10 The Family Colwelliaceae

. Table 10.2 Fatty acid profiles of the species of the genus Colwellia

Colwellia species codea 1234567891011 Fatty acidsb % fatty acid composition

C10:0 1222

C10:0 3-OH TR 3

C11:0 TR 1 TR

C11:0 3-OH 451

C12:0 1–2 2 3 TR TR TR

C12:0 3-OH 3TR3 3 2

C12:1 3-OH c C12:0 3-OH iso TR 563

C13:0 TR d C13:0 3-OH/C15:1 iso TR

C13:0 iso TR TR TR TR TR e C14:0 3-OH/C16:1 iso TR

C14:0 5–8 7–8 5 3 TR 9 2 2 2 TR

C14:1 o7c 5–7 9–10 3 3 2 2 2

C14:1 o5c TR TR TR TR TR

C14:0 iso TR TR TR TR TR

C15:1 o8c 0–2 2–3 4 20 4 3 6 3 11 18 9

C15:1 o6c 0-TR 1.1 TR TR

C15:0 2–11 TR-1 3 14 3 3 4 4 5 6

C16:0 27–33 22–24 27 13 22 31–33 25 27 10 8 13

C16:0 3-OH TR

C16:0 iso 0-TR 10 TR 10 6 7

C16:1 o9c 6–9 9–12 2 2 6 4 6 4 5 f C16:1 o7c/C15:0 2-OH iso 31–36 37–38 43 15 57 48–50 45 37 24 23 28

C17:0 0–1 TR TR 2 2 3 2 3 2 4

C17:0 iso TR TR TR TR TR

C17:1 o8c 0–1 TR TR 6 5 4 12 14 13

C17:1 o7c TR TR TR TR TR

C17:1 o6c TR 2 TR

C18:0 TR-2 TR TR 2 TR TR TR TR

C18:1 o9c 0–2 TR TR 1 TR TR TR TR TR

C18:1 o7c TR-2 1–2 4 2 2 1 3

C20:5 o3c 0–2 TR TR

C22:6 o3c 5–8 3–4 2 1 1 aColwellia species code: 1, C. psychrerythraea;2,C. demingiae;3,C. rossensis;4,C. hornerae;5,C. psychrotropica;6,C. piezophila;7,C. maris;8,C. asteriadis; 9, C. aestuarii; 10, C. polaris; and 11, C. chukchiensis. No data is available for C. hadaliensis b Fatty acid nomenclature: Cn, carbon chain length; 2-OH or 3-OH, a-andb-hydroxy fatty acids; iso, iso-branched fatty acids; :n, number of double bonds present; onc, cis-isomer monounsaturated fatty acid with the bond located at the indicated number of carbon units from the methyl end of the molecule. In the case of polyunsaturated fatty acids, the first double bond is located at the third carbon unit from the methyl end cTR, trace fatty acid making up <1 % of total analyzed fatty acids dSummed feature 1 – the indicated fatty acids cannot be separated in the MIDI fatty acid analysis system eSummed feature 2 of the MIDI system fSummed feature 3 of the MIDI system. Based on more sophisticated separation and mass spectrometric confirmatory methods, the correctly identified fatty acid

of this summed feature is C16:1 o7c. C15:0 2-OH iso was not detected in any strain even at trace levels. C15:0 2-OH iso is in general absent in all members of the order Alteromonadales The Family Colwelliaceae 10 187

(e.g., also found in some Shewanella, Psychromonas, Moritella Enrichment, Isolation, and Maintenance species) and in the related order Vibrionales (Photobacterium). Procedures PUFA synthesis also occurs in some marine members of the phylum Bacteroidetes (e.g., the genera Psychroflexus, Isolation of Colwellia and Thalassomonas species is generally Aureospirillum). straightforward except for barophiles, which is a challenging, dangerous, and expensive enterprise. Fortunately, most species can be isolated from readily accessible marine locations and Genus Thalassomonas Macia´n et al. 2001, primary isolation and subsequent routine cultivation utilizes 1283 standard easily prepared marine media. Colwellia species are common in sea ice; however distribu- Tha.lass’.o.mo.nas. Gr. n. thalassa, the sea; Gr. n. monas, a unit; tion is extremely patchy (Bowman 2013). To improve one’s N.L. fem. n. Thalassomonas, a monad from the sea. chances of obtaining Colwellia spp., sea ice should ideally possess The type species of genus Thalassomonas is Thalassomonas visible algal assemblages, appearing as olive to brown bands in viridans (Macia´n et al. 2001). Thalassomonas and its 7 species the ice layer, especially at the base of the layer. Sea-ice cores are represent the warm climate relatives of genus Colwellia with obtained using an ice auger or ‘‘jiffy’’ drill. The core is then sliced most strains able to grow well at 30 C but either grow poorly using an electric saw and ice pieces are melted in sterile seawater or not grow at 4 C(> Table 10.3). As such there is no evidence at 2–4 C. Once melted the samples can either be directly plated of true psychrophily (or barophily) within this genus onto marine agar or preincubated at 2–4 C for 1–2 days in based on the fairly random process of species descriptions. marine broth at a ratio of 1:10 to 1:100. Marine broth consists of The species of Thalassomonas to date have been isolated 0.5 % w/v bacteriological peptone and 0.2 % w/v yeast extract in from temperate to tropical ecosystems from a wide range of either 1,000 ml seawater or instead of seawater 35 g sea salts marine sources, including fauna and flora. The temperature (purchased from aquarium supply company) in 1,000 ml dis- dichotomy between Colwellia and Thalassomonas suggests that tilled water. Raw seawater can be filtered and/or aged in the dark each genus diverged from an ancestor and subsequently (and then autoclaved) as desired but has little any effect on expanded to populate essentially climactically different oceanic cultivation success. Commercial sources of the medium include, regions. Phenotypically, Thalassomonas is rather similar to for example, marine 2216 from Difco Laboratories. To create Colwellia species in a broad sense as described in the marine agar usually 1.5 % w/v agar is added to marine broth introduction. prior to autoclaving (15 min at 121 C); however alternative The phenotypic traits that consistently separate solidifying agents could be used as desired (i.e., gellan gum). Thalassomonas from Colwellia are relatively few given the inher- Sea-ice isolates are typically isolated, purified, and maintained ent variation possible between individual species. Overall, on marine agar at 2–10 C. Colwellia spp. from sea ice have Thalassomonas spp. seem more consistently saccharolytic than relatively patchy growth and often slowly create crystalline pre- Colwellia species with most species able to degrade a number cipitates in the medium. of polysaccharides (> Table 10.3); however they are Colwellia and Thalassomonas species have been successfully non-fermentative. With the exception of T. agariperforans, isolated from marine-sourced samples by simple direct plating most reports claim Thalassomonas species are strictly and subsequent purification using on marine agar. This includes aerobic. T. agariperforans was found like Colwellia spp. to be marine fauna (anemone, starfish, oyster, coral), with tissue or able to anaerobically respire by using nitrate as electron acceptor. coral skeleton homogenized or ground up as appropriate before The testing of anaerobic growth in the genus has been cursory dilution in marine broth or buffer (e.g., 3 % NaCl in 10 mM Tris and further work is clearly needed to confirm if Thalassomonas buffer at pH 8.0) and spread plating. Other media have been like Colwellia is able to engage in anaerobic respiration. used including 1:5 to 1:10 diluted marine agar; PY broth/agar Other differential traits that can broadly separate Thalassomonas (0.3 % w/v polypeptone, 0.1 % yeast extract, 2.5 % w/v NaCl, from Colwellia include the ability by some species to attack agar 0.5 % w/v MgCl2.6H2O, pH 7.8; 1.5 % w/v agar); PYSE broth/ and alginate while most species also degrade extracellular DNA agar (0.8 % w/v peptone, 0.3 % yeast extract, 3 % w/v NaCl, but not chitin. 0.07 % w/v KCl, 0.53 % w/v MgSO4.7H2O, 0.13 % w/v The lipoquinones have been examined in most species via CaSO4.2H2O, 0.11 % w/v MgCl2.6H2O, pH 7.9; 1.5 % w/v liquid chromatography and ubiquinone-8 (Q-8) predominates agar); A1 broth/agar medium (1 % w/v soluble starch, with low levels of Q-7 and Q-9 also present (> Table 10.4). 0.4 % w/v peptone, 0.2 % yeast extract, seawater, pH 8.1; 1.8 % Similar to Colwellia species, fatty acid composition variation agar); and MR2A, a marine version of R2A using seawater or occurs between Thalassomonas species; however PUFA is absent. water containing sea salts instead of distilled water. In the case of

Shorter chain components (C9 to C13) are most likely associated MR2A, it is best to make up this medium from individual with LPS given several are hydroxylated. In other respects fatty components (0.05 % w/v proteose peptone, 0.05 % w/v acid profiles are very analogous to that of Colwellia species casamino acids, 0.05 % w/v D-glucose, 0.05 % w/v soluble starch, > ( Table 10.2). Clearly, fatty acid analysis as a genus level 0.03 % w/v sodium pyruvate, 0.005 % w/v MgSO4.7H2O, in diagnostic tool is not very useful given the species-level seawater or 3.5 % w/v/sea salts solution, approx. pH 8; 1.5 % w/v variation. agar) and adding, if desired, the K2HPO4 (0.03 % w/v final 188 10 The Family Colwelliaceae

. Table 10.3 Phenotypic characteristics of Thalassomonas species

Phenotypic characteristics T. viridans T. haliotis T. ganghwensis T. loyana T. agarivorans T. agariperforans Colony pigment Green-blue Brown Yellow Cream Off-white Yellow-white (diffusible) (diffusible) Size (mm) 1.5–2.0 1.0–2.0 1.5–2.3 1.0–2.0 1.4–2.2 1.5–3.0 Â 0.8–1.0 Â 0.8–1.0 Â 0.5–0.7 Â 0.5–0.8 Â 0.5–0.8 Â 0.4–0.7 Gas vesicle formation ÀÀÀ ÀÀÀ Isolation site Oyster Abalone Tidal mud Diseased Seawater Marine sand coral Temperature growth range 15–37 15–30 15–40 18–35 15–35 4–37 Optimum temperature for growth (C) 30 25 30–35 30 25–30 30 Requires sea salts or divalent cations + + + ÀÀ + Seawater salinity tolerance range (psu) 20–40 20–40 10–80 10–80 10–40 0–60 Optimum seawater salinity for growth (psu) 30 30 30–40 30–40 30 20 Cytochrome c oxidase + + + À ++ Catalase + + + + + + Strictly aerobic + + + À Anaerobic growth (via nitrate respiration) ÀÀ + Nitrate reduction to nitrite À ++ ++ + Produces

Arginine dihydrolase, indole from L-tryptophan ÀÀÀ ÀÀÀ Lysine decarboxylase, ornithine decarboxylase ÀÀÀÀÀ Urease ÀÀ ÀÀ À

H2S from L-cysteine ÀÀÀÀ API ZYM enzyme activities Alkaline phosphatase, naphthol-AS-BI- ++ ++ + phosphohydrolase Acid phosphatase + + ÀÆ + Esterase (C4) + + + + À Esterase (C8) + À ++ À Esterase (C14) ÀÀ ÀÀ À Trypsin, a-chymotrypsin ÀÀÀÀ a-galactosidase, b-glucosidase, a-mannosidase, ÀÀ ÀÀ À a-fucosidase, b-glucuronidase b-galactosidase ÆÀÆ ÆÆÀ a-glucosidase ÀÀ ÀÆ À N-acetyl-b-glucosaminidase ÀÀ À+ À Hydrolysis of Aesculin + + + + + Tween 20, Tween 40, Tween 60 + + + + + Tween 80 Æ ++ +Æ + Lecithin + ÀÀ ÀÀ Xanthine, hypoxanthine ÀÀÀÀ DNA + + + + + +

L-tyrosine, gelatin, casein + + + + + À Starch + + À ++ + Chitin ÀÀ Agar ÀÀÀ++ Alginate ÀÀÀ ++ The Family Colwelliaceae 10 189

. Table 10.3 (continued)

Phenotypic characteristics T. viridans T. haliotis T. ganghwensis T. loyana T. agarivorans T. agariperforans Cellulose, xylan À + Assimilates or metabolizes

L-arabinose ÀÀÀ ÀÀÀ

D-fructose + ÀÀ À

D-galactose ÀÀ++++

D-glucose, D-maltose + + + + + +

D-mannose, L-rhamnose, DL-xylose ÀÀÀ À

D-ribose V + ÀÀÀ Cellobiose V + À + À

D-lactose + ÀÀ À+

D-melibiose À + ÀÀÀ Sucrose + + À

D-trehalose + ÀÀÀÀ

D-raffinose ÀÀ À Glycogen À + ÀÀÀ Inulin ÀÀ + Amygdalin + + ÀÀÀ Arbutin + ÀÀ ÀÀ Salicin + ÀÀ ÀÀ i-erythritol ÀÀÀ ÀÀ

Myo-inositol, D-mannitol, D-sorbitol ÀÀÀ À

D-xylitol À + ÀÀÀ

D-gluconate ÀÀ À 2-ketogluconate + + + + À 5-ketogluconate À ++ ÀÀ

N-acetyl-D-glucosamine À ++ Glycerol À + ÀÀÀ Citrate ÀÀÀ ÀÀÀ DL-malate ÀÀÀ ÀÀÀ Succinate V + ÀÀ À Tartrate À + À Formate ÀÀÀÀ Acetate À ++ Caprate + ÀÀ ÀÀ Benzoate ÀÀÀÀ

L-arginine V À +

L-asparagine, glycine À + À

L-glutamate À + ÀÀ

L-tyrosine + + À

L-ornithine V ÀÀ DNA G+C composition (mol%) 48–49 50 42 39 41–43 44 aAll Thalassomonas species are Gram-negative and appear as straight to curved rodlike cells; are motile via a single polar (or subpolar) flagellum, catalase, and cytochrome c oxidase positive; and can reduce nitrate to nitrite. Fermentation of carbohydrates has not been observed for any species of the genus so far. The optimum pH for growth is approximately that of seawater (pH 7–8) bAbbreviations: + test positive, À test negative, V test result data varies between different strains of the species, Æ test has conflicting data between reports. A blank cell indicates no data is available 190 10 The Family Colwelliaceae

. Table 10.4 Fatty acid and lipoquinone composition of Thalassomonas species

Lipid components T. viridans T. actiniarum Thaliotis T. ganghwensis T. loyana T. agarivorans Tnn agariperforans Fatty acida b C9:0 0-TR

C10:0 TR 3–5 1–2 TR

C10:0 3-OH TR 2 4

C11:0 1–2 TR TR 1–2

C11:0 3-OH 3–4 2 2 TR-1.4

C11:0 3-OH iso TR

C12:0 1–2 2 2 TR 3 5–7 2

C12:0 iso 0-TR

C12:0 3-OH 5–6 2 2 3 7 2 TR

C12:0 3-OH iso TR

C12:1 3-OH 5

C13:0 TR TR 5–7 c C13:0 3-OH/C15:1 iso 1–2 TR 2–3

C13:0 iso 0–1

C14:0 2–3 2 3 TR 9–13 4–5 2

C14:0 iso TR TR 1–2 d C14:0 3-OH/C16:1 iso 1 0–2 3

C15:1 o8c 4–6 TR 2 2 2 2–3 TR

C15:1 o6c TR-1

C15:0 6–11 2 4 1 3 0–6 TR

C15:0 iso TR

C16:0 11–14 32 31 19–22 9 17–20 31

C16:0 iso TR 3–7 TR

C16:1 o9c 4–5 5 2–4 e C16:1 o7c/C15:0 2-OH iso 21–28 45 39 20–23 27–31 8–13 39

C17:0 3–5 2 3 1 TR 6–11 TR

C17:0 iso TR

C17:1 o8c 14–20 5 6 4–7 10–12 9–13

C17:1o6c TR

C18:0 TR 1 TR 1 TR 1–2 TR

C18:1 o9c TR 2 3 2–3

C18:1 o7c 3–6 6 3 11–19 9 4–5 14

C20:1 o7c 0-TR Lipoquinone type: Ubiquinone-7 1 6 1 0 2 2 Ubiquinone-8 98 81 98 96 97 97 100 Ubiquinone-9 1 13 1 4 1 1

a Fatty acid nomenclature: Cn, carbon chain length; 2-OH or 3-OH, a-andb-hydroxy fatty acids; iso, iso-branched fatty acids; :n, number of double bonds present; onc, cis-isomer monounsaturated fatty acid with the bond located at the indicated number of carbon units from the methyl end of the molecule. In the case of polyunsaturated fatty acids, the first double bond is located at the third carbon unit from the methyl end bTR, trace fatty acid making up <1 % of total analyzed fatty acids cSummed feature 1 – the indicated fatty acids cannot be separated in the MIDI fatty acid analysis system dSummed feature 2 of the MIDI system eSummed feature 3 of the MIDI system The Family Colwelliaceae 10 191

MR2A medium concentration) separately after autoclaving to psychrophile. As a result the 34H genome has proven very useful avoid precipitation occurring. It is likely these media can be used for understanding the types of traits that allow for life at interchangeably for most Colwelliaceae species. The water tem- freezing temperature. This includes detailed information on perature from where the sample was obtained dictates the incu- the amino acid composition of the 34H proteome that has lead bation temperature subsequently used with psychrophiles to a flurry of publications on protein thermostability in growing well at 10–15 C. recent years. For long-term preservation most strains can be kept either One important aspect of the psychrophilic lifestyle is mem- (1) on marine agar slants at 4 C to room temperature brane fluidity. At low temperature membrane lipids become (depending on temperature preference of the strain), increasingly rigid, and without alteration to the chemical (2) cryopreserved in marine broth (or broth versions of the makeup to maintain an optimal viscosity, the membrane can other medium described above) containing 15–20 % v/v glycerol deform leading to its rupture, subsequent cell leakage, and at À80 C or on cryopreservation beads, (3) as a suspension in death. Amongst various psychrophilic bacteria, one aspect of sterile seawater at 10–20 C. Long-term preservation such as this homeoviscosity adaptation is the synthesis of PUFA (Russell lyophilization and freezing in liquid N2 is possible (detailed and Nichols 2000). C. psychrerythraea strains can form both protocols are described by DiFernando and Vreeland 2006). DHA and EPA (> Table 10.2). Omega-3 PUFA is synthesized Some strains, such as the type strain of C. psycherythraea via polyketide synthases (Metz et al. 2001) and the pfaABCD ATCC 27374T, form autotoxic compounds or proteins. To main- gene cluster coding these enzymes in 34H is similar in conserved tain these strains, the seawater suspension option must be domain structure to those found in other gamma- performed since these cultures completely inactivate within proteobacteria (Shulse and Allen 2011). a few days on agar media. Accumulating compatible solutes is extremely important for For barophilic Colwellia species deep-sea samples, usually maintenance of protein stability since proteins constantly inter- superficial benthic mud, obviously need to be collected by rela- act with water within the cell and are subject to denaturation tively sophisticated means using manned (such as the famous processes from a number of sources (Wiggins 2008). Compatible DSV Alvin or the Shinkai 6500) or remote-controlled submers- solute accumulation is a fundamental basis for adaptation to low ibles (such as the Mir). Samples are held at constantly low temperature whether the organism is cold adapted or not temperature (0–2 C) to offset pressure change. Samples are (e.g., Hoffmann and Bremer 2011), since low temperature enriched or isolated in bags in pressure vessels made of stainless directly compromises protein stability (Privalov 1990). On the steel (i.e., SAE grade 304). The pressure bag isolation system, basis of genome data, strain 34H has the ability to synthesize the which employs low melting point agar (Kato et al. 1995; Kato major osmoprotectants betaine and glycine betaine either de 2006, 2011) has been used to successfully isolate barophiles of novo or from imported choline. The 34H genome also includes the order Alteromonadales including Colwellia piezophila (Nogi sarcosine oxidase, which suggests betaine and glycine betaine et al. 2004). To maintain oxygen ‘‘at high’’ pressure, the hydro- also may serve as sources of carbon and energy, and likely fluorocarbon fluorinert FC-72 (3 M Inc.) saturated with oxygen these compounds are constantly turned over in the cell thus can be added at 20 % (v/v). Barophiles when grown at higher balancing cellular metabolic requirements under constant sub- temperature must have higher pressure applied; thus typically zero temperatures. they are grown and maintained at low temperature (<4 C). The 34H genome also revealed the ability to synthesize Because of the technical demands and safety issues in working polyhydroxyalkanoate granules and to also be able to synthesize with barophiles, few are available in culture except those that can and degrade cyanophycin (L-arginyl-poly-L-aspartic acid)-like grow at low temperature at atmospheric pressure. polyamides. Cyanophycin is a natural nitrogen storage polymer often formed by cyanobacteria that has engendered biotechno- logical interest in recent years (Mooibroek et al. 2007). Genome-Based and Genetic Studies It was originally claimed that genes were present in 34H that suggested the potential to metabolize aromatic hydrocar- Only relatively limited genome studies and no genetic knockout bons; however further examination suggests these enzymes are or manipulation have been performed on members of the family related to catabolism of aromatic amino acids such as L-tyrosine. Colwelliaceae so far. Most research has focussed at the protein In general, based on accumulated taxonomic reports, members level and cell product level (e.g., PUFA, exopolysaccharides). As of the Colwelliaceae cannot degrade aromatic hydro- of late 2012, only one representative of family Colwelliaceae sea- carbons; however the ability to use straight chain ice bacterium Colwellia psychrerythraea 34H has had its genome alkane hydrocarbons and grow on fatty acids is evident sequence determined (Methe´ et al. 2005). The genome of strain (see > Ecology section). 34H is complete and has a size of 5,373,180 bp with a G+C of The cold-active enzyme capacity of strain 34H has been 37.9 mol% and contains 4,937 predicted protein coding genes, extensively studied in order to understand protein thermosta- 85 tRNAs, 9 rrna (16S/23S/5S rRNA) operons, and 1 structural bility and function at low temperature. Several enzymes of strain RNA gene. Strain 34H has attracted scientific interest since it 34H involved in housekeeping processes, intermediary metabo- dwells at constantly sub-zero temperatures in its native sea-ice lism, and nutrient acquisition have been characterized by habitat and the genome was the first obtained from an obligate various approaches such as X-ray crystallography, including 192 10 The Family Colwelliaceae

phenylalanine hydroxylase (Leiros et al. 2007), a secreted highly Colwellia species thrive at sub-zero temperatures within the cold-active aminopeptidase structurally similar to human brine channels of sea ice (see review on sea-ice microbiology for bifunctional leukotriene A4 hydrolase (Huston et al. 2004; more information Bowman 2013) where they are motile and Bauvois et al. 2008), isocitrate dehydrogenases (Maki et al. divide at temperatures of À10 C or less (Junge et al. 2003, 2006), cold shock (RNA chaperone) proteins (Moon et al. 2006). In these environments Colwellia are infected by 2009), DEAD box RNA-dependent RNA helicases involved in equally cold-adapted bacteriophages and thus are part of ribosome assembly and stabilization (Cartier et al. 2010), DNA a psychrophilic microbial loop specially adapted to the highly gyrase subunit A (Jung et al. 2010), and the Hsp60 molecular dynamic and extreme sea-ice environment (Borriss et al. 2003; chaperone GroEL/GroES (Yamauchi et al. 2012). Similar studies Wells and Deming 2006a, b). Metazoa within these loops may have also been performed using a strain that is now the benefit from PUFA synthesized by Colwellia species since it is type strain of Colwellia maris (Watanabe and Takada 2004; required for higher life-form neurological development. Yoneta et al. 2004) as well as other uncharacterized Colwellia Metazoa are unable to synthesize EPA or DHA de novo and strains (Wang et al. 2005, 2006; Olivera et al. 2007). Few if any must acquire them in their diet (Nichols 2003). Highly cold- studies have tackled transcriptional regulation in Colwellia spe- adapted enzymes still surprisingly active at 0 C no doubt allow cies; studies on heat shock however have revealed that RNA these bacteria to be successful in what would be considered polymerase sigma subunits are involved in stress adaptation a hostile environment (Huston et al. 2000; Yu et al. 2009). In responses in Colwellia (Yamauchi et al. 2003, 2006). sea ice exopolysaccharides secreted by Colwellia strains as well as other sea-ice dwelling species appear to be capable of influencing ice crystal formation. Accumulated evidence suggests polysac- Ecology charide formation is also crucial in controlling nutrient access and acting as a cryoprotectant during freezing in this ice brine In terms of ecosystem function, Colwelliaceae are classic marine environment (Marx et al. 2009; Ewert and Deming 2011; Krembs secondary producers with a broad-based marine distribution. et al. 2011). Colwellia spp. also likely produce ice-binding pro- Based on observational and functional studies, Colwelliaceae are teins that allow them to actively colonize sea ice as it forms collectively ubiquitous and are involved in decomposing organic (Raymond et al. 2007). Dispersal of Colwellia species into the material of intermediate complexity (hydrocarbons, lipids, pro- atmosphere by the wind was speculated as potentially impacting teins, polysaccharides) within pelagic zone particulates, sea ice, atmospheric ice formation and cloud formation via nucleation and within algal, faunal, and floral associations and engaged in reactions due to these ice-affecting properties (Junge and anaerobic respiration and/or fermenting simple compounds Swanson 2008). within superficial marine sediments. Studies using stable isotope probing showed unexpectedly Colwellia and Thalassomonas species have been detected in that Colwellia species and other related bacteria within the order numerous studies. The low-temperature association of Colwellia Alteromonadales, the family Oceanospirillaceae, and species results in its ubiquitous presence in cold and polar marine within the genus Arcobacter of the class Epsilonproteobacteria ecosystems including the pelagic zone, sea ice, relic marine are capable of manganese reduction while using acetate as an salinity meromictic lakes, epi-shelf lakes forming on ice shelves, electron donor (Vandieken et al. 2012). The results suggest and effectively the benthos of the entire ocean regardless of Colwellia (and perhaps also Thalassomonas) is involved in anaer- depth (Deming et al. 1988; Bowman et al. 1997; DeLong et al. obic respiration processes in surface sediments using nitrate and 1997; Junge et al. 2002; Bowman and McCuaig 2003; Nogi et al. manganese as electron acceptors to oxidize simple organic com- 2004; Zeng et al. 2005; Yu et al. 2006; Prabagaran et al. 2007; pounds. This is consistent with Colwelliaceae being relatively Collins et al. 2010; Veillette et al. 2011). Besides low-temperature abundant in cold surficial sediments. ecosystems, Colwelliaceae have been found in many marine Evidence suggests that Colwellia species can assimilate bicar- contexts including brines to preserve or process food (Abriouel bonate directly in the dark via anaplerotic fixation reactions in et al. 2011), endocytic or epiphytic relations with dinoflagellates which tricarboxylic acid cycle intermediates are created via car- (Seibold et al. 2001; Wichels et al. 2004), marine biofilms (Gillan boxylases including malic enzyme, pyruvate carboxylase, and et al. 1998; Finnegan et al. 2011), faunal associations (Du et al. pyruvate carboxykinase (Alonso-Sa´ez et al. 2010). It is uncertain 2010), and aquaculture systems (McIntosh et al. 2008). The how significant bicarbonate take-up is in terms of the bigger

species T. loyana appear to be associated with coral disease picture of oceanic CO2 adsorption or what controls the uptake (Thompson et al. 2006) and the use of bacteriophage specific or indeed the physiological benefits. Metatranscriptomic studies against this species has been suggested as a control agent to suggest Colwelliaceae are highly responsive to bacterioplankton reduce disease (Efrony et al. 2009). Algae-associated Colwellia activity (Eilers et al. 2000; Stewart et al. 2012), which suggests and other epiphytic bacteria may produce enzymes that combat Colwelliaceae and other heterotrophic bacteria may have influ- photooxidative stress for the algae (Hu¨nken et al. 2008). The ence in carbon budgets in marine ecosystems, especially loca- genome of C. psychrerythraea 34H was noted to contain several tions that are highly productive. catalases and two classes of superoxide dismutase suggesting an Several studies indicate Colwellia species are able to degrade active capacity to deal with reactive oxygen species (Methe´ et al. hydrocarbons, including straight chain alkanes, indicating 2005). strains may contain alkane monooxygenases and other similar The Family Colwelliaceae 10 193 enzymes. This is consistent with Colwellia and Thalassomonas References species being able to utilize various monocarboxylic and dicar- boxylic compounds such as caprate and azelate (> Tables 10.1 Abriouel H, Benomar N, Lucas R, Ga´lvez A (2011) Culture-independent study of and > 10.3). Oil contaminated sites or culture enrichments with the diversity of microbial populations in brines during fermentation of moderate length alkanes as carbon sources have been shown to naturally-fermented Aloren˜a green table olives. Int J Food Microbiol 144:487–496 become enriched in members of the family Colwelliaceae (Powell Alonso-Sa´ez L, Galand PE, Casamayor EO, Pedro´s-Alio´ C, Bertilsson S (2010) et al. 2004; Brakstad et al. 2008; Schwermer et al. 2008; High bicarbonate assimilation in the dark by Arctic bacteria. ISME Korenblum et al. 2010; Baelum et al. 2012); with suggestions J 4:1581–1590 that degradation may also occur in anaerobic sites (Gittel et al. Baelum J, Borglin S, Chakraborty R, Fortney JL, Lamendella R, Mason OU, Auer 2012). M, Zemla M, Bill M, Conrad ME, Malfatti SA, Tringe SG, Holman HY, Hazen TC, Jansson JK (2012) Deep-sea bacteria enriched by oil and dispersant from the Deepwater Horizon spill. Environ Microbiol 14:2405–2416 Bauvois C, Jacquamet L, Huston AL, Borel F, Feller G, Ferrer JL (2008) Crystal Applications structure of the cold-active aminopeptidase from Colwellia psychrerythraea, a close structural homologue of the human bifunctional leukotriene Owing to the characteristic psychrophily of Colwellia species, A4 hydrolase. J Biol Chem 283:23315–23325 Borriss M, Helmke E, Hanschke R, Schweder T (2003) Isolation and characteri- their psychoactive enzymes have attracted interest ranging zation of marine psychrophilic phage-host systems from Arctic sea ice. beyond the study of protein thermostability to application- Extremophiles 7:377–384 oriented research (Wang et al. 2008; Kuddus and Ramteke Bowman JP (2007) Bioactive compound synthetic capacity and ecological 2012). An expression system that allows recombinant protein significance of marine bacterial genus Pseudoalteromonas. Mar Drugs production in yeast could aid in studying specific protein prod- 5:220–241 Bowman JP (2013) Sea-ice microbiology. The prokaryotes: prokaryote commu- ucts directly in assay screening (Seok et al. 2012). nities, In: DeLong EF, Lory S, Stackebrandt E, Thompson F (eds) Ecophys- Potent agarolytic enzymes produced by Thalassomonas strains iology. Springer, New York (in press) (Ohta et al. 2005) have also been studied in the context of Bowman JP, McCammon SA, Brown MV, Nichols DS, McMeekin TA biotechnological applicability. An interesting application of the (1997) Diversity and association of psychrophilic bacteria in Antarctic sea latter is the utilization of Thalassomonas agarases to improve the ice. Appl Environ Microbiol 63:3068–3078 Bowman JP, McMeekin TA (2005) Order X. Alteromonadales ord. nov. In: medicinal potency of seaweed-derived polysaccharides (Hatada Brenner DJ, Krieg NR, Staley JT, Garrity GM (eds) Bergey’s manual of et al. 2006). These polysaccharides are used as a food (e.g., nori, systematic bacteriology, 2nd, Vol. 2 (The Proteobacteria), Part B (The laver) but also in Chinese traditional medicine and have been Gammaproteobacteria). Springer, New York, p 443 shown to have antioxidant properties, acting as scavengers of Bowman JP, Gosink JJ, McCammon SA, Lewis TE, Nichols DS, Nichols PD, reactive oxygen species (Rocha de Souza et al. 2007). Agarolytic Skerratt JH, Staley JT, McMeekin TA (1998) Colwellia demingiae sp. nov., Colwellia hornerae sp. nov., Colwellia rossensis sp. nov. and Colwellia activity has been proposed to release sulfated galactan antioxi- psychrotropica sp. nov.: psychrophilic Antarctic species with the ability to dants from porphyran and other similar highly sulfated, com- synthesize docosahexaenoic acid (22:6o3). Int J Syst Bacteriol 48:1171–1180 plex polysaccharides from brown algae. Colwellia species Bowman JP, McCuaig RD (2003) Biodiversity, community structural shifts, and capacity to produce PUFA has attracted interest. One goal is to biogeography of prokaryotes within Antarctic continental shelf sediment. reduce the reliance of fish oil in farmed fish production Appl Environ Microbiol 69:2463–2483 Brakstad OG, Nonstad I, Faksness LG, Brandvik PJ (2008) Responses of microbial (e.g., salmon mariculture). Fish oil supplies contain needed communities in Arctic sea ice after contamination by crude petroleum oil. EPA and DHA required for proper fish development and Microb Ecol 55:540–552 growth. Rotifers fed DHA/EPA-producing Colwellia Cartier G, Lorieux F, Allemand F, Dreyfus M, Bizebard T (2010) Cold adaptation psychrerythraea ACAM 605 and EPA-producing Shewanella in DEAD-box proteins. Biochemistry 49:2636–2646 gelidimarina ACAM 456T accumulated these PUFA and thus Choi EJ, Kwon HC, Koh HY, Kim YS, Yang HO (2010) Colwellia asteriadis sp. nov., a marine bacterium isolated from the starfish Asterias amurensis. Int could transfer nutriceutical lipids to fish larvae (Lewis et al. J Syst Evol Microbiol 60:1952–1957 1998). Bacterial sources of PUFA are however likely to be too Collins RE, Rocap G, Deming JW (2010) Persistence of bacterial and archaeal low yielding and attention has turned to other sources of PUFA. communities in sea ice through an Arctic winter. Environ Microbiol However, cloning of PUFA synthesis genes into other organisms, 12:1828–1841 such as plants, remains a potential. Other applications involving D’Aoust JY, Gerber NN (1974) Isolation and purification of prodigiosin from Vibrio psychroerythrus. J Bacteriol 118:756–757 members of the Colwelliaceae include an interesting adaptation D’Aoust JY, Kushner DJ (1972) Vibrio psychroerythrus sp. n.: class- of Colwellia psychrerythraea 34H-derived DNA ligase gene ligA ification of the psychrophilic marine bacterium, NRC 1004. J Bacteriol to develop temperature-sensitive versions of pathogens such as 111:340–342 Francisella tularensis and Salmonella enteritidis that allow for Delong EF, Franks DG, Yayanos AA (1997) Evolutionary relationships of culti- larger delivery of live cellular vaccine that would be effective in vated psychrophilic and barophilic deep-sea bacteria. 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