The Pathogen of the Great Barrier Reef Sponge Rhopaloeides Odorabile Is a New Strain Of

The Pathogen of the Great Barrier Reef Sponge Rhopaloeides Odorabile Is a New Strain Of

The pathogen of the Great Barrier Reef sponge Rhopaloeides odorabile is a new strain of Pseudoalteromonas agarivorans containing abundant and diverse virulence related genes

Jayanta D Choudhury1, Arnab Pramanik1, Nicole S Webster2, Lyndon E Llewellyn2, Ratan Gachhui3,Joydeep Mukherjee1*

1 School of Environmental Studies, JadavpurUniversity, Kolkata 700 032, India

2 Australian Institute of Marine Science, PMB 3, Townsville MC, Townsville 4810, Queensland, Australia

3 Department of Life Science and Biotechnology, JadavpurUniversity, Kolkata 700 032, India

* Corresponding author

Tel. 0091 33 2414 6147

Fax 0091 33 2414 6414

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Online resource 1Description of Pseudoalteromonas agarivorans strain NW4327

Strain NW4327 was found to be a chemoorganotroph, with a respiratory metabolism. It did not form endospores or accumulate polyhydroxybutyrate as an intracellular reserve product. Strain NW4327 did not possess arginine dihydrolase activity and was oxidase- and catalase-positive. It required 1-12% NaCl for growth and good growth was observed in media containing 4-8 %NaCl. The temperature range for growth was 4 to 37 °C, with an optimum between 24-28 °C. The pH range for growth was 6.0 to 8.0. Amylase and DNase were produced but alginase and agarase were not produced. Nitrate reduction was observed. Positive reactions were obtained for hydrolysis of gelatin, casein, esculin, Tween 20 and Tween 80 while negative reactions were shown for indole, urease and H2S production. Strain NW4327 produced melanin-like pigment in basal medium with tyrosine. It aerobically utilized maltose, D-glucose, D-galactose, D-lactose, D-mannitol, citrate, L-arginine, L-histidine, glycine, L-serine and L-tyrosine as single carbon sources, but could not utilize D-sorbitol, D-xylose, L-lysine, L-threonine and D-arabinose. Acid was produced from D-galactose and maltose but not from D-glucose, D-lactose, D-xylose or sucrose. The G+C content was found to be 41.2-43.6 mol%. Ubiquinone 8 was the major respiratory quinone. The major cellular fatty acids were 18:1 ω7c, 16:1 ω7c, 16:0 and 12:0 3OH. The bacterium was susceptible to gentamicin (30 μg), polymyxin B(300 units), carbenicillin(100 μg), chloramphenicol (30 μg) and streptomycin(10 μg) and resistant to lincomycin(15 μg), tetracycline(30 μg), penicillin G (10 units).

Online Resource 2Cellular fatty acid composition of Pseudoalteromonas sp. strain NW4327, Pseudoalteromonas genus and Pseudoalteromonas agarivorans KMM 255T. Major fatty acids are shown in bold.

Fatty acid / 1 / 2 / 3
10:0 / 0.13 / NR / NR
10:0 3OH / 1.14 / NR / NR
11:0 / 0 / NR / 0
11:0 2OH / 0.19 / NR / NR
11:0 3OH / 0.38 / NR / NR
12:0 / 2.45 / 0.6-2.0 / 1.8
12:1 / 0 / 0.0-1.3 / 0.4
12:0 iso 3OH / 0.66 / NR / NR
12:0 3OH / 7.50 / NR / NR
12:1 3OH / 0.21 / NR / NR
13:0 / 0 / NR / 0.1
13:0 3OH / 0.29 / NR / NR
14:0 / 0.67 / 0.4-8.4 / 1.1
14:0 3OH / 0.11 / NR / NR
14:1 ω7c / 0 / NR / 0.4
15:0 / 0 / 0.2-8.5 / 2.7
15:0 iso / 0 / NR / 0.3
15:1 ω6c / 0 / NR / 0.2
15:1 ω8c / 0.23 / 0.5-7.3 / 1.2
16:0 / 17.70 / 14.0-33.8 / 27.3
16:0 iso / 1.05 / NR / 1.3
16:0 3OH / 0.33 / NR / NR
16:1 ω7c / 34.11 / 29.6-47.8 / 42.4
16:1 ω9c / 0.60 / NR / NR
17:0 / 2.25 / 0.5-8.7 / 3.4
17:0 iso / 0.24 / 0.0-0.9 / 0.4
17:0 anteiso / 0.79 / NR / 1.9
17:1 ω6c / 0 / 0.0-0.8 / 0.5
17:1 ω8c / 4.02 / 0.2-19.0 / 5.9
18:0 / 2.42 / NR / 0.6
18:0 iso / 0.57 / NR / 0.2
18:1 ω7c / 19.74 / 1.3-18.5 / NR
18:1 ω8c / 0 / NR / 7.1
18:1 ω9c / 0.79 / NR / 0.2
18:1 ω11c / 0 / NR / 0.3
19:0 10 methyl / 0.48 / NR / NR
19:1 ω6c / 0.35 / NR / NR
20:1 ω7c / 0.57 / NR / NR

1: NW4327; 2: genus Pseudoalteromonas (Ivanova et al. 2000); 3: P. agarivorans KMM 255T (Romanenko et al. 2003); NR: not reported.

References

Ivanova EP, Zhukova NV, Svetashev VI, Gorshkova NM, Kurilenko VV, Frolova GM, Mikhailov VV (2000) Evaluation of phospholipid and fatty acid compositions as chemotaxonomic markers of Alteromonas-like proteobacteria. Curr Microbiol 41:341-345

Romanenko LA, Zhukova NV, Rohde M, Lysenko AM, Mikhailov VV, Stackebrandt E (2003) Pseudoalteromonas agarivorans sp. nov., a novel marine agarolytic bacterium. Int J Syst Evol Microbiol 53:125-131

Online resource 3Differential phenotypic characteristics between Pseudoalteromonas sp. strain NW4327 and phylogenetically related species of genus Pseudoalteromonas. All data were obtained in this study.

Characteristic / 1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12
Melanin production / + / + / + / + / - * / - * / + / - * / + / + / + / - *
Nitrate to Nitrite / + / - * / + / + / - * / + / - * / + / - * / + / - * / - *
Esculin degradation / + / - * / + / + / + / + / + / + / + / + / + / +
Growth at/in
4°C / + / - * / + / + / + / + / + / - * / + / + / + / +
37°C / + / + / + / + / - * / + / - * / + / - * / + / - * / +
1% NaCl / + / - * / + / + / + / + / + / + / + / + / + / +
10% NaCl / + / - * / + / + / - * / + / + / + / - * / + / + / +
Production of
Amylase / + / + / + / + / + / + / - * / + / - * / + / + / - *
Alginase / - / + * / + * / + * / + * / ND / + * / + * / + * / + * / - / ND
Agarase / - / + * / - / + * / - / - / - / - / - / - / - / -
DNase / + / + / + / + / + / + / + / - * / + / + / + / +
Acid production from
Mannitol / - / - / + * / - / + * / - / - / - / - / + * / - / -
Glucose / - / - / + * / - / + * / + * / - / - / - / + * / - / + *
Lactose / - / - / + * / - / + * / + * / - / - / + * / - / - / + *
Sucrose / - / + * / + * / - / + * / + * / - / - / - / + * / - / + *
Galactose / + / - * / + / + / + / + / - * / - * / + / - * / - * / - *
Xylose / - / + * / + * / - / + * / - / - / + * / - / + * / - / -
Maltose / + / + / + / + / + / + / + / - * / + / + / + / +
Fructose / - / + * / + * / - / + * / + * / - / - / + * / + * / - / + *
Utilization of
D-Glucose / + / + / + / ND / + / + / + / + / - * / + / + / +
D-Galactose / + / + / + / ND / + / + / - * / - * / - * / + / - * / +
D-Mannitol / + / - * / + / + / + / + / + / + / - * / + / - * / +
Maltose / + / + / + / + / + / - * / + / + / + / + / + / +
D-Lactose / + / + / + / - * / - * / - * / + / + / + / + / - * / - *
D-Xylose / - / - / + * / - / + * / - / - / + * / - / + * / - / + *
D-Sorbitol / - / - / + * / - / + * / - / - / - / - / + * / - / -
L-Arginine / + / - * / + / + / + / - * / + / + / - * / + / - * / - *
L-Tyrosine / + / - * / + / + / + / - * / + / - * / - * / - * / - * / +
m-Inositol / + / + / - * / + / - * / - * / - * / + / - * / + / + / - *
D-Trehalose / + / - * / - * / + / + / - * / - * / + / - * / + / - * / +
D-Cellobiose / + / - * / - * / - * / + / - * / - * / + / - * / + / + / +
Ethylene glycol / - / + * / - / + * / + * / + * / - / - / - / + * / - / -
D-Fructose / + / - * / + / + / + / - * / + / - * / + / + / - * / +
D-Mannose / + / + / + / + / + / - * / - * / - * / - * / - * / + / +
Glycerol / + / - * / + / + / + / - * / + / - * / - * / + / - * / +
D-Arabinose / - / - / + * / - / + * / - / - / - / + * / + * / - / -
Citrate / + / - * / + / - * / + / - * / + / - * / - * / + / - * / - *
L-Alanine / - / + * / + * / - / + * / - / - / - / - / + * / + * / -
L-Asparagine / + / + / + / - * / + / - * / + / - * / - * / + / + / - *
Glycine / + / - * / - * / - * / + / - * / - * / - * / - * / - * / - * / - *
L-Histidine / + / + / + / - * / + / - * / - * / - * / + / + / - * / - *
4-hydroxy-L-proline / - / + * / - / + * / + * / - / - / - / - / + * / - / -
L-Isoleucine / - / + * / + * / - / - / - / - / - / - / + * / - / -
L-Leucine / - / + * / + * / - / + * / - / - / - / + * / + * / + * / -
L-Lysine / - / + * / + * / + * / + * / - / + * / + * / + * / + * / + * / -
L-Proline / - / + * / + * / - / + * / + * / + * / + * / + * / + * / + * / + *
L-Serine / + / - * / + / - * / + / + / - * / + / - * / + / + / +
L-Threonine / - / + * / - / - / + * / - / - / - / - / - / - / -
Susceptible/resistant to
Vancomycin / + / + / - * / + / + / + / + / + / + / - * / - * / +
Lincomycin / - / + * / - / - / - / - / - / + * / - / - / - / + *
Kanamycin / + / - * / + / + / + / + / + / - * / + / + / + / - *
Neomycin / + / - * / + / + / + / + / + / + / + / + / + / ND
Oleandomycin / + / + / - * / + / + / - * / + / + / + / + / + / - *
Polymyxin B / + / + / + / + / + / - * / + / + / + / + / + / +
Ampicillin / - / - / - / - / - / + * / - / - / - / - / - / + *
Tetracycline / - / + * / - / - / - / - / - / - / - / - / - / + *
Gentamycin / + / + / + / + / + / + / + / - * / + / + / + / ND
Carbenicillin / + / + / + / + / + / + / + / + / + / + / - / +
Chloramphenicol / + / + / - * / + / + / + / + / + / + / + / + / +

Asterisks indicate traits different from strain NW4327. ND: Not Determined. All strains studied required Na+ ions for growth, were positive for oxidase and catalase, growth at 3-6% NaCl and for hydrolysis of gelatin, casein, Tween 20 and Tween 80. Negative reactions were obtained for indole, urease and H2S production and arginine dihydrolase activity. All strains were susceptible to Streptomycin and resistant to Penicillin G. + : susceptible, - :resistant in case of antibiobic susceptibility tests. 1: NW4327, 2:P. atlantica IAM 12927T, 3:P. espejiana NCIMB 2127T,4:P. agarivorans KMM 255T, 5:P. elyakovii KMM 162T, 6:P. paragorgicola KMM 3548T, 7:P. carrageenovora IAM 12662T, 8:P. issachenkonii KMM 3549T,9:P. distincta KMM 638T, 10:P. nigrifaciens IAM 13010T, 11:P. undina NCIMB 2128T, 12:P. tetraodonis IAM 14160T.

Online Resource 4 DNA-DNA re-association values between strain NW4327 and phylogenetically closely related species. Species are highly similar in terms of 16S rRNA gene sequences (first three) and gyrB gene sequences (first, third and fourth).

Similar species / NCBI accession no.
(16S rRNA) / Pairwise similarity (%) (16S rRNA) / NCBI accession no. (gyrB) / Pairwise similarity (%) (gyrB) / DNA-DNA hybridization (%)
I / II / III
Pseudoalteromonas atlantica IAM 12927T / X82134 / 99.93 / AF007274 / 98.23 / 18.49 / 19.42 / 25.34
Pseudoalteromonas espejiana NCIMB 2127T / X82143 / 99.85 / FR668566 / 92.05 / 20.63 / 27.44 / 21.40
Pseudoalteromonas agarivorans KMM 255T / AJ417594 / 99.80 / KF793928 / 98.39 / 96.65 / 97.55 / 85.15
Pseudoalteromonas tetraodonis IAM 14160T / AF214730 / 99.18 / FR668561 / 98.14 / 17.22 / 15.94 / 19.97

Online Resource 5Putative virulence associated genes in Pseudoalteromonasagarivorans strain NW4327. GI numbers related to the proteins are provided in a separate column. Homologs of the proteins in Pseudoalteromonas agarivorans S816 was detected by BLASTP (criteria: E-value≥1e-6; identity≥30 %; coverage≥70 %). Identity (as percentage similarity) of NW4327 proteins with P. agarivorans S816 homologs are given in parentheses.

Gene Product / NCBI Protein Name / GI / Secreted Yes/No / Homolog in P. agarivorans S816
Adhesion and Colonization
Type IV pili (T4P) Formation
3-dehydroquinate synthase / 3-dehydroquinate synthase / 566570776 / No / Yes (99)
Leader peptidase (Prepilin peptidase) / N-methyltransferase (Type IV prepilin peptidase PilD) / 566572124 / No / Yes (96)
Multimodular transpeptidase-transglycosylase / Peptidase (Penicillin-binding protein 1b) / 566568439 / Yes2 / Yes (99)
Penicillin-binding protein 1a / 566570783 / No / Yes (99)
Twitching motility protein PilT / Twitching motility protein PilT / 566571887 / No / Yes (99)
Type IV fimbrial assembly protein PilC / Type II secretion system protein F / 566572123 / No / Yes (92)
Type IV fimbrial assembly, ATPase PilB / Type II secretory protein GspE / 566572125 / No / Yes (100)
Type IV fimbrial biogenesis protein FimT / Pilus biosynthesis protein / 566570953 / No / Yes (65)
Type IV pilus biogenesis protein PilF / Lytic cell-wall binding lipoprotein (Type IV pilus biogenesis protein PilF) / 566570316 / Yes2 / Yes (98)
Twitching motility protein PilG / Chemotaxis protein CheY (Twitching motility protein PilG) / 566570727 / No / Yes (99)
Type IV fimbrial biogenesis protein PilV / Pilus biosynthesis protein / 566570959 / No / Yes (99)
Type IV fimbrial biogenesis protein PilW / Pilus assembly protein PilW / 566570958 / Yes2 / Yes (98)
Type IV fimbrial biogenesis protein PilX / Hypothetical protein / 566570957 / No / Yes (100)
Type IV fimbrial biogenesis protein PilY1 / Type IV pilus assembly protein PilY1 / 566569394 / Yes1,2 / Yes (99)
Type IV pilus assembly protein PilY1 / 566570956 / Yes1,2 / Yes (88)
Type IV pilin PilA / Fimbrial protein / 566572122 / Yes2 / No
Type IV pilus biogenesis protein PilE / Fimbrial protein / 566570955 / No / Yes (90)
Type IV pilus biogenesis protein PilM / Pilus assembly protein PilM / 566570782 / No / Yes (100)
Type IV pilus biogenesis protein PilN / Pilus biosynthesis protein PilN / 566570781 / No / Yes (95)
Type IV pilus biogenesis protein PilO / Pilus assembly protein PilO / 566570780 / No / Yes (100)
Type IV pilus biogenesis protein PilP / Pilus biosynthesis protein PilP / 566570779 / Yes2 / Yes (99)
Type IV pilus biogenesis protein PilQ / Secretin / 566570778 / No / Yes (99)
Type IV fimbrial biogenesis protein FimU / Pilus biosynthesis protein / 566570954 / No / Yes (97)
Type IV pilus biogenesis protein PilZ / Pilus biosynthesis protein PilZ / 566570574 / Yes2 / Yes (100)
MSHA Pili Formation
MSHA biogenesis protein MshQ / Agglutinin biogenesis protein MshQ / 566570741 / Yes2 / No
MSHA biogenesis protein MshP / Agglutinin biogenesis protein MshP / 566570742 / Yes2 / Yes (97)
MSHA biogenesis protein MshO / Agglutinin biogenesis protein MshO / 566570743 / Yes2 / Yes (99)
MSHA pilin protein MshD / Agglutinin biogenesis protein MshD / 566570744 / Yes2 / Yes (98)
MSHA pilin protein MshC / Agglutinin biogenesis protein MshC / 566570745 / No / Yes (98)
MSHA pilin protein MshA / Agglutinin biogenesis protein MshA / 566570746 / Yes2 / Yes (74)
MSHA pilin protein MshB / Pili assembly chaperone / 566570747 / Yes2 / Yes (99)
MSHA biogenesis protein MshG / MSHA biogenesis protein MshG / 566570748 / No / Yes (99)
MSHA biogenesis protein MshE / MSHA biogenesis protein MshE / 566570749 / No / No
MSHA biogenesis protein MshN / Agglutinin biogenesis protein MshN / 566570750 / Yes2 / Yes (97)
MSHA biogenesis protein MshM / ATPase AAA (Mannose-sensitive agglutinin biogenesis protein MshM) / 566570751 / No / Yes (99)
MSHA biogenesis protein MshL / Type II secretion protein (mannose-sensitive agglutinin biogenesis protein MshL) / 566570752 / Yes2 / Yes (99)
MSHA biogenesis protein MshK / Agglutinin biogenesis protein MshK / 566570753 / No / Yes (98)
MSHA biogenesis protein MshJ / Agglutinin biogenesis protein MshJ / 566570754 / No / Yes (99)
MSHA biogenesis protein MshI / Mannose-sensitive agglutinin biogenesis protein MshI (MshI2) / 566570755 / No / Yes (98)
Mannose-sensitive agglutinin biogenesis protein MshI (MshI1) / 566570756 / No / No
MSHA biogenesis protein MshH / Diguanylate phosphodiesterase (MSHA biogenesis protein MshH) / 566570758 / No / Yes (99)
Curli Formation
CsgA / Curlin (csgA) / 566570229 / Yes1,2 / Yes (99)
CsgE / Curli production assembly protein CsgE / 566570230 / Yes1 / Yes (100)
CsgF / Curli production assembly proteinCsgF / 566570231 / Yes1 / Yes (100)
CsgG / Transporter (Curli production assembly/transport component CsgG) / 566570232 / Yes1 / Yes (100)
CsgD / LuxR family transcriptional regulator (CsgD) / 566570233 / No / Yes (99)
Tight Adherence
Type II/IV secretion system ATP hydrolase TadA/VirB11/CpaF, TadA subfamily / Type II secretion system protein E / 566569632 / No / Yes (99)
Flp pilus assembly protein TadB / Pilus assembly protein TadB / 566569633 / No / Yes (98)
Type II/IV secretion system protein TadC, associated with Flp pilus assembly / Secretion system protein / 566569634 / No / Yes (99)
Protein Secretion Systems
Type I Secretion System
Type I secretion outer membrane protein, TolC precursor / Flagellar motor protein MotB / 566569681 / Yes1,2 / No
ABC transporter, transmembrane region / ABC transporter / 566569682 / No / No
HlyD family secretion protein / Secretion protein HylD / 566569683 / No / No
Type II Secretion System
General secretion pathway protein A / ATPase AAA / 566568452 / No / Yes (99)
General secretion pathway protein B / General secretion pathway protein B / 566568451 / No / Yes (98)
General secretion pathway protein C / General secretion pathway protein C / 566570669 / Yes2 / Yes (97)
General secretion pathway protein D / General secretion pathway protein D / 566570668 / Yes1 / Yes (92)
General secretion pathway protein E / General secretion pathway protein E / 566570667 / No / Yes (100)
General secretion pathway protein F / General secretion pathway protein F / 566570666 / No / Yes (99)
General secretion pathway protein G / General secretion pathway protein G / 566570665 / Yes2 / Yes (100)
General secretion pathway protein H / General secretion pathway protein H / 566570664 / No / Yes (99)
General secretion pathway protein I / General secretion pathway protein I / 566570663 / Yes1 / Yes (99)
General secretion pathway protein J / General secretion pathway protein J / 566570662 / No / Yes (99)
General secretion pathway protein K / General secretion pathway protein K / 566570661 / No / Yes (99)
General secretion pathway protein L / General secretion pathway protein L / 566570660 / No / Yes (99)
General secretion pathway protein M / General secretion pathway protein M / 566570659 / No / Yes (99)
General secretion pathway protein N / General secretion pathway protein N / 566570658 / No / Yes (99)
Type VI Secretion System
VgrG protein / Type IV Secretion protein Rhs / 566570255 / Yes2 / No
Hcp protein / Hemolysin D / 566570256 / Yes2 / No
ClpB protein / ATPase / 566570257 / No / Yes (38)
Uncharacterized protein ImpH/VasB / Type VI Secretion Protein / 566570258 / Yes2 / No
Protein ImpG/VasA / Type VI Secretion Protein ImpG / 566570259 / No / No
Uncharacterized protein ImpF / Hypothetical protein / 566570260 / No / No
Protein ImpE / Virulence protein, SciE type / 566570261 / No / No
Uncharacterized protein ImpD / Type VI Secretion Protein / 566570262 / No / No
Uncharacterized protein ImpC / EvpB family type VI secretion protein / 566570263 / Yes2 / No
Uncharacterized protein ImpB / Type VI Secretion Protein / 566570264 / No / No
Uncharacterized protein ImpA / Type VI Secretion Protein ImpA / 566570265 / No / No
Phosphoprotein phosphatase PppA / Serine/threonine protein phosphatase / 566570266 / No / No
Protein phosphatase ImpM / Type VI secretion protein / 566570267 / Yes2 / No
IcmF-related protein / Type VI Secretion Protein VasK / 566570268 / No / No
Outer membrane protein ImpK/VasF, OmpA/MotB domain / Type VI Secretion Protein / 566570269 / No / No
Uncharacterized protein ImpJ/VasE / Type VI Secretion Protein / 566570270 / No / No
Type VI Secretion LipoProtein /VasD / Type VI Secretion LipoProtein /VasD / 566570271 / Yes1 / No
Uncharacterized protein ImpI/VasC / Signal peptide protein / 566570272 / Yes2 / No
Serine/threonine protein kinase PpkA / Serine/threonine protein kinase / 566570273 / No / No
Proteases, Toxins and Phospholipases
Cytosol aminopeptidase PepA / Multifunctional aminopeptidase A (Peptidase M17) / 566572013 / No / Yes (99)
Membrane alanine aminopeptidase N / Aminopeptidase (Peptidase M1) / 566569517 / No / Yes (99)
Aminopeptidase (Peptidase M1) / 566569959 / Yes1,2 / Yes (99)
Peptidase B / Aminopeptidase B (Peptidase M17) / 566569842 / Yes2 / Yes (99)
Cytosol aminopeptidase (Peptidase M17) / 566571533 / Yes2 / Yes (99)
Vibriolysin, extracellular zinc protease / Hemagglutinin (Peptidase M4) / 566571703 / Yes1,2 / Yes (99)
Hemagglutinin (Peptidase M4) / 566570874 / Yes1,2 / Yes (99)
Acylamino-acid-releasing enzyme / Acyl-peptide hydrolase (Peptidase S9) / 566570386 / Yes1,2 / Yes (99)
Dipeptidyl carboxypeptidase Dcp / Dipeptidyl carboxypeptidase II (Peptidase M3) / 566568355 / Yes1,2 / Yes (99)
Oligopeptidase A / Oligopeptidase A (Peptidase M3) / 566570728 / No / Yes (99)
Aminopeptidase / Peptidase M28 / 566571263 / Yes1,2 / Yes (99)
Prolyl oligopeptidase / Peptidase S9 / 566571265 / Yes1 / No
Peptidase U32 / Protease (Peptidase U32) / 566570313 / No / Yes (94)
Zn-dependent aminopeptidase / Aminopeptidase (Peptidase M1) / 566570392 / Yes1,2 / Yes (99)
Peptidyl-dipeptidase A / Peptidyl-dipeptidase A (Peptidase M2) / 566569120 / Yes1 / Yes (99)
Alkaline serine protease, subtilase family protein / Protease (Peptidase S8) / 566569203 / Yes1,2 / Yes (94)
Zn-dependent dipeptidase / Peptidase M19 / 566569007 / Yes1 / Yes (99)
Zinc-dependent carboxypeptidase / Peptidase M14 / 566568948 / Yes1,2 / Yes (99)
Peptidase M23 / Peptidase M23 / 566568342 / No / Yes (99)
Serine protease, subtilase family / Peptidase S8 (Deseasin MCP-01) / 566570228 / Yes1,2 / Yes (83)
Serine protease, subtilase family / Peptidase S8 / 566571312 / Yes1,2 / Yes (99)
Peptidase S8 / Peptidase S8 / 566570815 / Yes1,2 / No
Extracellular serine protease / Serine protease (Peptidase S8/S53) / 566571315 / Yes1,2 / Yes (99)
Protease II / Protease (Peptidase S9) / 566571311 / Yes1,2 / Yes (99)
Protease II / Protease (Peptidase S9) / 566568846 / No / Yes (99)
Leucyl aminopeptidase / Peptidase M17 / 566572049 / No / Yes (99)
Leucyl aminopeptidase / Aminopeptidase (Peptidase M28) / 566571702 / Yes1,2 / Yes (97)
Aminopeptidase N family protein / Peptidase M61 / 566571720 / Yes1 / Yes (99)
Carboxypeptidase-related protein / Carboxypeptidase (Peptidase S10) / 566568443 / Yes1,2 / Yes (99)
Iron-regulated protein A precursor / Peptidase M75 / 566568387 / Yes2 / No
Protease precursor / Tail fiber protein (Peptidase M6) / 566571727 / Yes1,2 / Yes (97)
Glutamate carboxypeptidase / Peptidase M20 / 566570300 / Yes1,2 / Yes (96)
Zn-dependent protease / Metalloprotease (Peptidase M50) / 566571363 / No / Yes (99)
Peptidase M16-like / Peptidase M16 / 566569809 / Yes1,2 / Yes (99)
Zn-dependent protease with chaperone function PA4632 / Peptidase M48 / 566569348 / Yes1 / Yes (99)
Tail-specific protease precursor / Carboxy-terminal protease (Peptidase S41) / 566569515 / Yes1 / Yes (99)
Membrane-associated zinc metalloprotease / Zinc metallopeptidase RseP (Peptidase M50A) / 566569041 / No / Yes (100)
Exported zinc metalloprotease YfgC precursor / Protease (Peptidase M48) / 566568559 / Yes1 / Yes (99)
Dipeptidyl-peptidase IV / Dipeptidyl-peptidase IV (Peptidase S9) / 566572051 / Yes1,2 / Yes (100)
Hemolysin III homolog / Hemolysin III / 566571747 / No / Yes (99)
Hemolysins and related proteins containing CBS domains / Hemolysin / 566571361 / No / Yes (99)
Hemagglutinin / Hemagglutinin / 566570714 / Yes1,2 / Yes (74)
YoeB toxin protein / Txe/YoeB family addiction module toxin / 566570297 / Yes2 / Yes (99)
YefM protein (antitoxin to YoeB) / prevent-host-death protein / 566570298 / No / Yes (100)
Phospholipase A1 precursor / Phospholipase / 566571407 / Yes1 / Yes (99)
Phospholipase / Phospholipase / 566570111 / Yes2 / Yes (97)

“1” denotes secretion predicted by SignalP 4.1 (Petersen et al. 2011) and “2” denotes secretion predicted by SecretomeP 2.0 (Bendtsen et al. 2005).

References

Bendtsen JD, Kiemer L, Fausbøll A, Brunak S (2005) Non-classical protein secretion in bacteria. BMC Microbiol 5:58. Doi:10.1186/1471-2180-5-58

Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785-786

Online Resource 6Components of Type VI Secretion System present in Pseudoalteromonasagarivorans strain NW4327. The Pfam and InterPro entries corresponding to the T6SS domains are provided. Homologous proteins of Rhizobium leguminosarum bv. Trifolii and Edwardsiella tarda strain PPD130/91 are also shown(criteria: E-value≥1e-6; identity≥24 %; coverage≥70 %).

Ser. No. / T6SS Components / Pfam Entry / InterPro Entry / NCBI Protein Name / RAST Protein Name / Homolog in R. leguminosarum biovar trifolii
(% identity/ % similarity) /

Homolog inE. tarda strain PPD130/91

(% identity/ % similarity)

*1 / Imp-A N-terminal related/ COG3515 / PF06812 / IPR010657 / Type VI Secretion Protein ImpA
(gi: 566570265) / Uncharacterized protein ImpA / ImpA
(gi:16326486)
(30/49) / EvpK
(gi:158512118)
(30/50)
*2 / IcmF-related/ DUF1215/COG3523 / PF06744 / IPR010623 / Type VI Secretion Protein VasK
(gi: 566570268) / IcmF-related protein / ImpL
(gi:16326462)
(29/48) / _
*3 / DUF879/COG3519 / PF05947 / IPR010272 / Type VI Secretion Protein ImpG
(gi: 566570259) / Protein ImpG/VasA / ImpG
(gi:16326483)
(35/51) / EvpF
(gi:158512125)
(35/53)
*4 / DUF877/COG3517 / PF05943 / IPR010269 / Type VI Secretion Protein
(gi: 566570262) / Uncharacterized protein ImpD / ImpD
(gi:16326480)
(37/59) / EvpB
(gi:40287637)
(42/60)
EvpB family type VI secretion protein
(gi: 566570263) / Uncharacterized protein ImpC / ImpC
(gi:16326479)
(64/81) / EvpB
(gi:40287637)
(52/70)
*5 / DUF876/COG3522 / PF05936 / IPR010263 / Type VI Secretion Protein
(gi: 566570270) / Uncharacterized protein ImpJ/VasE / ImpJ
(gi:16326460)
(40/60) / EvpM
(gi:158512120)
(24/44)
*6 / DUF770/COG3516 / PF05591 / IPR008312 / Type VI Secretion Protein
(gi: 566570264) / Uncharacterized protein ImpB / ImpB
(gi:16326475)
(59/79) / EvpA
(gi:40287636)
(51/65)
*7 / DUF1305/COG3520 / PF06996 / IPR010732 / Type VI Secretion Protein
(gi: 566570258) / Uncharacterized protein ImpH/VasB / ImpH
(gi:16326484)
(26/44) / EvpG
(gi:158512126)
(26/50)
*8 / ClpV/COG0542 / PF00004 / IPR003959 / ATPase
(gi: 566570257) / ClpB protein / _ / EvpH
(gi:158512127)
(49/69)
*9 / FHA domain/COG3456 / PF00498 / IPR000253 / Signal peptide protein
(gi: 566570272) / Uncharacterized protein ImpI/VasC / _ / _
*10 / COG3521 / PF12790 / IPR017734 / Type VI Secretion LipoProtein/VasD
(gi: 566570271) / Type VI Secretion LipoProtein/VasD / _ / _
*11 / DotU (IcmH)-related/COG3455 / PF09850 / IPR017732 / Type VI Secretion Protein
(gi: 566570269) / Outer membrane protein ImpK/VasF, OmpA/MotB domain / ImpK
(gi:16326461)
(29/48) / _
*12 / Pfam 04965/COG3518 / PF04965 / IPR007048 / Hypothetical protein
(gi: 566570260) / Uncharacterized protein ImpF / ImpF
(gi:16326482)
(33/49) / EvpE
(gi:45332447)
(35/54)
*13 / Hcp/DUF796/COG3157 / PF05638 / IPR008514 / Hemolysin D
(gi: 566570256) / Putative cytoplasmic protein USSDB7A / _ / EvpC
(gi:40287638)
(31/44)
*14 / VgrG/DUF586/COG3501 / PF04717 / IPR006531 / Type IV Secretion protein Rhs
(gi: 566570255) / VgrG protein / _ / EvpI
(gi:158512116)
(36/55)
15 / COG4455 / PF07024 / IPR009211 / Virulence protein, SciE type
(gi:566570261) / Protein ImpE / ImpE
(gi:16326481)
(26/49) / _
16 / COG3913 / PF09867 / IPR017748 / Type VI secretion protein
(gi:566570267) / Protein phosphatase ImpM / _ / _
17 / COG0631 / PF13672 / IPR001932 / Serine/threonine protein phosphatase
(gi: 566570266) / Phosphoprotein phosphatase PppA / _ / _
18. / COG0515 / PF00069 / IPR000719 / Serine/threonine protein kinase (gi:566570273) / Hypothetical protein / _ / _

In parentheses, GI numbers for the proteins and the identity/similarity of NW4327 T6SS components with their homologs are provided. Asterisks denote the T6SS core proteins according to Bingle et al.(2008)and Boyer et al.(2009).

References

Bingle LE, Bailey CM, Pallen MJ (2008) Type VI secretion: a beginner’s guide. Curr Opin Microbiol 11:3-8

Boyer F, Fichant G, Berthod J, Vandenbrouck Y, Attree I (2009) Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources? BMC Genomics 10:104. Doi:10.1186/1471-2164-10-104

Online Resource 7Gene organization of type VI secretion cluster present in Pseudoalteromonasagarivorans strain NW4327. Each uniquely colored box represents a particular gene. Inside the boxes, the COG (cluster of orthologous groups) names are provided. The black box without any name represents a hypothetical protein with no assigned function.

Online Resource 8List of virulence associated factors found in NW4327 with their potential homologs in terrestrial pathogens.

Category / Reported in terrestrial pathogens / References
Adhesion & Colonization
Type IV pili / Pseudomonas aeruginosa, Neisseria gonorrhoeae, Neisseria meningitidis, Vibrio cholerae / Shi and Sun 2002
MSHA pili / Vibrio cholerae / Marsh and Taylor 1999
Curli / Escherichia coli / Gophna et al. 2001
Tight adherence (tad) / Actinobacillus actinomycetemcomitans / Kachlany et al. 2000
Protein Secretion Systems
Type I Secretion System / Escherichia coli / Holland et al. 2005
Type II Secretion System / Vibrio cholerae, Aeromonas hydrophila, Pseudomonas aeruginosa, Erwinia chrysanthemi, Xantomonas campestris, Burkholderia pseudomallei / Sandkvist 2001
Type VI Secretion System / Aeromonas hydrophila, Escherichia coli, Burkholderia mallei, Edwardsiella tarda, Pseudomonas aeruginosa, Vibrio cholerae / Coulthurst 2013
Proteases, Toxins and Phospholipases
Metalloprotease family M4 / Clostridium perfringens, Enterococcus faecalis, Helicobacter pylori, Vibrio cholerae, Pseudomonas aeruginosa / Adekoya and Sylte 2009
Metalloprotease family M6 / Bacillus anthracis / Chung et al. 2006
Metalloprotease family M48 / Porphyromonas gingivalis / Walters et al. 2009
Serine protease family S8 / Stenotrophomonas maltophilia, Bacillus sp. B16 / Windhorst et al. 2002; Qiuhong et al. 2006
Serine protease family S9 / Porphyromonas gingivalis / Kumagai et al. 2000
Peptidase family U32 / Porphyromonas gingivalis, Proteus mirabilis, Helicobacter pylori,Salmonella enterica,Aeromonas veronii / Jasilionis et al. 2012
Hemolysin / Vibrio cholerae, Staphylococcus aureus / Ichinose et al. 1987; DeLeo et al. 2009
Phospholipase / Helicobacter pylori, Vibrio cholerae, Yersinia enterocolitica, Staphylococcus aureus, Campylobacter coli / Schmiel and Miller 1999
yefM-yoeB Toxin-Antitoxin System / Streptococcus pneumoniae / Nieto et al. 2007

References

Adekoya OA, Sylte I (2009) The thermolysin family (M4) of enzymes: therapeutic and biotechnological potential. Chem Biol Drug Des 73:7-16

Chung M-C, Popova TG, Millis BA, Mukherjee DV, Zhou W, Liotta LA, Petricoin EF, Chandhoke V, Bailey C, Popov SG (2006) Secreted neutral metalloproteases of Bacillus anthracis as candidate pathogenic factors. J Biol Chem 281:31408-31418

Coulthurst SJ (2013) The Type VI secretion system - a widespread and versatile cell targeting system. Res Microbiol 164:640-654

DeLeo FR, Diep BA, Otto M (2009) Host defense and pathogenesis in Staphylococcus aureus infections. Infect Dis Clin North Am 23:17-34

Gophna U, Barlev M, Seijffers R, Oelschlager TA, Hacker J, Ron EZ (2001) Curli fibers mediate internalization of Escherichia coli by eukaryotic cells. Infect Immun 69:2659-2665

Holland IB, Schmitt L, Young J (2005) Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway. Mol Membr Biol 22:29-39

Ichinose Y, Yamamoto K, Nakasone N, Tanabe MJ, Takeda T, Miwatani T, Iwanaga M (1987) Enterotoxicity of El Tor-like hemolysin of non-O1 Vibrio cholerae. Infect Immun 55:1090-1093