Degradation and Utilization of Alginate by Marine Pseudoalteromonas: a Review

ABSTRACT

Alginate, which is mainly produced by brown algae and decomposed by heterotrophic bacteria, is an important marine organic carbon source. The genus Pseudoalteromonas contains diverse forms of heterotrophic bacteria that are widely distributed in marine environments and are an important group in alginate degradation. In this review, the diversity of alginate-degrading Pseudoalteromonas is introduced, and the characteristics of Pseudoalteromonas alginate lyases, including their sequences, enzymatic properties, structures, and catalytic mechanisms, and the synergistic effect of Pseudoalteromonas alginate lyases on alginate degradation are introduced. The acquisition of the alginate degradation capacity and the alginate utilization pathways of Pseudoalteromonas are also introduced. This paper provides a comprehensive overview of alginate degradation by Pseudoalteromonas, which will contribute to the understanding of the degradation and recycling of marine algal polysaccharides driven by marine bacteria.

INTRODUCTION

Pseudoalteromonas is a strictly marine genus in the Gammaproteobacteria class, which was first separated from the genus Alteromonas in 1995 by Gauthier et al. (1, 2). At the time of writing, this genus contained 48 species with validly published names, representing a large genus in the class Gammaproteobacteria (https://lpsn.dsmz.de/search?word=Pseudoalteromonas). Pseudoalteromonas strains are all Gram negative, heterotrophic, aerobic, motile, and nonspore forming, require Na⁺ ions for growth, and have genomic G+C contents of 38 to 50 mol% (3, 4). Pseudoalteromonas strains have a strong capacity to produce extracellular degrading enzymes and a variety of bioactive substances that play important roles in marine biogeochemical cycles (5).

Alginate is an important marine organic carbon source. It mainly exists in the cell wall of hundreds of species of brown algae, such as kelps, bladderwrack, and gulfweed, accounting for 10 to 45% of the dry weight (6). In addition to brown algae, alginate is also produced by a small number of bacteria, mainly as the principal component of extracellular polysaccharides or biofilms (7). Alginate is a straight-chained acid polysaccharide connected by 1,4-glucoside bonds between β-d-mannuronic acid (M) and its C5 epimer α-l-guluronic acid (G), which are arranged in the homopolymeric M block (PM), the homopolymeric G block (PG), and the heteropolymeric MG (GM) block (PMG) (8). Alginate is degraded by alginate lyases, which are synthesized by marine algae (9), marine mollusks (10), and a wide range of microorganisms, including marine bacteria (11), marine fungi (12), terrestrial bacteria (13), and viruses (14).

In recent years, many studies have focused on the degradation of alginate by marine heterotrophic bacteria. Among marine heterotrophic bacteria, the Pseudoalteromonas genus was found to be an important alginate-degrading group (15). Many Pseudoalteromonas strains show alginolytic activity (15), and many Pseudoalteromonas alginate lyases, which exhibit diverse characteristics, have been identified and characterized (16, 17). In this review, our understanding of alginate degradation by Pseudoalteromonas, including the diversity of Pseudoalteromonas strains having alginate-degrading ability and the diversity and characteristics of Pseudoalteromonas alginate lyases and their synergistic degradation on alginate, are introduced. Also, the acquisition of the alginate-degradation capacity and the alginate utilization pathway of Pseudoalteromonas are discussed.

PSEUDOALTEROMONAS IS AN IMPORTANT ALGINATE-DEGRADING GROUP IN THE OCEAN

Many studies have shown that Pseudoalteromonas is an important group of alginate-degrading bacteria. In polar regions, macroalgae are abundant, contributing a significant amount of polysaccharide-rich detritus (18). Studies of alginate lyase gene sequences from sediment metagenomes from four high-latitude regions of both hemispheres showed that the alginate lyase sequences in polar environmental sediments were closely related to those of Pseudoalteromonas (19). In an ex situ microcosm experiment using Arctic bacterial communities, Jain et al. found that, compared with the unamended control microcosms, Pseudoalteromonas abundance increased significantly in the early period of alginate addition (18). Six Pseudoalteromonas strains were also identified by Dong et al., who screened 21 culturable alginate lyase-excreting strains from the Arctic brown alga Laminaria (20). Cha et al. also reported that Pseudoalteromonas strains (22/60) were dominant among the 60 culturable alginate-utilizing strains they isolated from the north and south polar regions (21). Macroalgae, including kelps, are also widespread in many temperate coastal areas. Martin et al. investigated the polysaccharide-degrading activity of the culturable bacterial subpopulation associated with the large brown alga Ascophyllum nodosum, collected from a shallow coastal area in France. They isolated 324 isolates affiliated with 36 genera, among which Pseudoalteromonas (9.1%) was one of the two most abundant groups and was consistently found in all three A. nodosum samples (22). Furthermore, 63 alginate lyase-excreting strains were identified, of which Pseudoalteromonas (22/63) was one of the dominant genera (22). These studies show that Pseudoalteromonas strains account for a large proportion of marine alginate-degrading bacteria.

Forty-eight type strains of Pseudoalteromonas have so far been identified. Their phylogenetic relationship is shown in Fig. 1, and their growth characteristics, ability to degrade alginate, and isolation sites are summarized in Table 1. The alginate-degrading ability of 28 type strains have been investigated, of which 16 type strains are positive for alginate degradation (Table 1). These strains were isolated from seawater, marine animal, and plant samples from sites around the world. Among them, 5 type strains (P. elyakovii, P. atlantica, P. carrageenovora, P. mariniglutinosa, and P. issachenkonii) were isolated from algae, all of which can degrade alginate.

FIG 1.

Phylogenetic analysis of type strains of the genus Pseudoalteromonas based on 16S rRNA gene sequences. The alginate utilization gene clusters predicted in the genomes of 8 Pseudoalteromonas strains are shown on the right of each strain. Phylogenetic trees were constructed by the neighbor-joining method with 1,000 replicons of bootstrap analysis by MEGA 7.0.26.

TABLE 1.

Characteristics of the type strains in the genus Pseudoalteromonas^a

Species	Type strain	Growth temp (°C [range])	Growth pH (range)	Growth in NaCl (% [range])	Degradation of alginate	Source	Reference(s)
Pseudoalteromonas aestuariivivens	DB-2T	15–40	5.5–/	0.5–7	—	A tidal flat sediment from the Yellow Sea of South Korea	51
Pseudoalteromonas agarivorans	KMM 255T	7–35	—	—	Yes	Sea water deep in the Pacific Ocean or from the marine ascidians	52
Pseudoalteromonas distincta	KMM 638T	—	—	—	Yes	A marine sponge	53
Pseudoalteromonas elyakovii	KMM 162T	10–37	—	—	Yes	Far Eastern mussel or the wounded fronds of Laminaria	54
Pseudoalteromonas atlantica	ATCC 19262T	5–35	5.5–8.5	—	Yes	Seaweeds collected at Inubousaki, Choshi City, Chiba, Prefecture, Japan	1, 55
Pseudoalteromonas carrageenovora	IAM 12622T	5–35	5.5–9	—	Yes	Seaweeds	1, 55
Pseudoalteromonas espejiana	ATCC 29659T	—	—	—	Yes	Seawater off the coast of northern California	1, 56
Pseudoalteromonas aliena	KMM 3562T	4–30	6–10	3–6	Yes	Seawater samples from the Pacific Ocean	57
Pseudoalteromonas amylolytica	JW1T	20–40	6–10.5	0.5–10	—	Surface seawater of the Arabian Sea	58
Pseudoalteromonas antarctica	NF3T	4–30	6–9.5	0.1–12.5	—	Antarctic coastal areas	59
Pseudoalteromonas arabiensis	k53T	6–35	5.5–9.5	0.5–10	—	Sediment from the Arabian Sea, Indian Ocean	60
Pseudoalteromonas arctica	37-1-2T	4–25	6–8	0–9	No	Seawater samples collected from Spitzbergen in the Arctic	61
Pseudoalteromonas caenipelagi	JBTF-M23T	15–40	5–/	0.5–10.0	—	Tidal flat sediment collected from the Yellow Sea	62
Pseudoalteromonas citrea	KMM216T	10–3	6–10	1–11.5	Yes	Surface water from the Mediterranean Sea near Nice, France	1, 3, 63
Pseudoalteromonas haloplanktis	ATCC 14393T	—	—	—	No	—	1, 64
Pseudoalteromonas undina	ATCC 29660T	—	—	—	No	Seawater off the coast of northern California	1, 56
Pseudoalteromonas aurantia	208T	4–30	7–10	—	No	Surface seawater off Nice, France	1, 65
Pseudoalteromonas byunsanensis	FR1199T	10–40	5–10	0.5–5	—	Tidal flat sediment in Korea	66
Pseudoalteromonas denitrificans	ATCC 43337T	4–22	—	1.5–5.5	Yes	Fjord system off the Norwegian west coast	1, 67
Pseudoalteromonas donghaensis	HJ51T	4–45	5.5–9.5	1–13	—	Seawater sample from the East Sea, near South Korea	68
Pseudoalteromonas fenneropenaei	rzy34T	20–40	6–10	1–6	—	Sediment of a pond containing farmed Fenneropenaeus chinensis, Rizhao, China	69
Pseudoalteromonas flavipulchra	NCIMS2033T	10–44	5–12	0.5–10	—	Seawater off Nice, France	70
Pseudoalteromonas maricaloris	KMM 636T	10–37	6–10	0.5–10	Yes	Australian sponge	70
Pseudoalteromonas fuliginea	KMM 216T	5–30	—	1–9	—	Swab samples of an unidentified polychaete near Canal Concepción, Chile	71
Pseudoalteromonas gelatinilytica	NH153T	15–45	5.5–9.5	0–10	—	Surface seawater of the South China Sea	72
Pseudoalteromonas issachenkonii	KMM 3549T	4–37	6–10	0.5–15	Yes	Thallus of the brown alga collected in the Pacific Ocean	73
Pseudoalteromonas prydzensis	ACAM620T	0–30	—	0.5–15	No	Antarctic sea ice	74
Pseudoalteromonas lipolytica	LMEB39T	15–37	5.5–9.5	0.5–15	—	Yangtze River estuary	75
Pseudoalteromonas luteoviolacea	ATCC 33492T	10–30	—	—	—	Surface seawater in the neritic zone near Nice, France	1, 76
Pseudoalteromonas marina	mano4T	4–37	5.3–8.8	3–12	Yes	Tidal flats of the Yellow Sea	77
Pseudoalteromonas mariniglutinosa	NCIMB1770T	5–37		1–9	Yes	Diatom collected from sea water of the Marseille Gulf	78
Pseudoalteromonas ruthenica	KMM 300T	10–35	6–10	1–9	Yes	A marine mussel	79
Pseudoalteromonas translucida	KMM 520T	4–30	6–10	1–8	Yes	Seawater	80
Pseudoalteromonas phenolica	O-BC30T	18–37	6.5–9.5	1–5	No	Seawater	81
Pseudoalteromonas spongiae	UST010723-006T	12–44	5–10	2–6	No	Sponge Mycale adhaerens in Hong Kong waters	82
Pseudoalteromonas ulvae	UL12T	4–35	5.5–12	0.1–/	No	Surface of a marine alga	83
Pseudoalteromonas paragorgicola	KMM 3548T	4–30	6–10	1–6	—	A sponge	80
Pseudoalteromonas neustonica	PAMC28425T	4–30	6–9	1–7	—	Sea surface microlayer of the Ross Sea (Antarctica)	84
Pseudoalteromonas peptidolytica	F12-50-A1T	15–37	6–10	1–10	Yes	Sea of Japan	85
Pseudoalteromonas piratica	OCN003T	14–39	5.5–10	1–6	—	Mucus of Montipora capitata	86
Pseudoalteromonas piscicida	ATCC 15057T	—	—	—	No	A killed fish in which no dinoflagellate bloom was present	1
Pseudoalteromonas profundi	TP162T	10–40	6–9	0.5–9	—	Deep-sea seamount	87
Pseudoalteromonas rhizosphaerae	RA15T	4–32	5–9	0–15	—	Rhizosphere of the halophyte plant Arthrocnemum macrostachyum growing in the Odiel marshes	4
Pseudoalteromonas rubra	ATCC 29570T	—	—	—	No	Mediterranean waters off Nice	1, 88
Pseudoalteromonas shioyasakiensis	SE3T	5–40	5.5–9.5	0.5–12	—	Pacific Ocean sediment	89
Pseudoalteromonas tetraodonis	IAM 14160T	4–35	5.5–9.5	1–10	No	Surface slime of a puffer fish	90, 91
Pseudoalteromonas tunicata	D2T	—	—	—	No	An adult tunicate collected from the western coast of Sweden	92
Pseudoalteromonas xiamenensis	Y2T	10–40	5–10	0.5–6	—	Surface seawater of Yundang Lake, Xiamen, China	93

^a—, not mentioned; /, information not provided in the source.

In addition to these type strains, many nontype Pseudoalteromonas strains have been reported to be able to utilize alginate. Pseudoalteromonas sp. strain Alg6B, isolated from the surface of the brown seaweed Laminaria japonica, is a novel seaweed-hydrolyzing strain that releases oligosaccharides from alginate (23). Pseudoalteromonas sp. strain 1400, screened from 36 marine bacterial isolates based on their alginolytic activity, had the highest alginolytic activity (24). Pseudoalteromonas sp. strain SM0524 was isolated from rotten kelp based on its high alginate lyase-producing ability, and two alginate lyases from this strain were further characterized (16, 25). P. atlantica AR06, isolated from coastal water in Japan, was able to utilize alginate as a sole source of carbon and energy and the extracellular protein fraction prepared from the cultivation media of this strain exhibited alginolytic activity and could depolymerize alginate into a dimer from a tetramer (26). Pseudoalteromonas sp. strain LJ1 is an alginate lyase-producing strain isolated from Laminaria japonica, whose extracellular alginate lyase production increased 66% via optimization of the culture conditions (27). Pseudoalteromonas agarovorans CHO-12 was isolated from seawater off the south coast of Korea for efficient saccharification from alginate (28). Pseudoalteromonas citrea KMM 3297, an associate of the holothurian Apostichopus japonicus, can produce three alginolytic enzymes that can efficiently destroy the alginate of brown algae when induced with fucoidan (29).

Together, these studies show that many Pseudoalteromonas strains from a diverse range of marine sites can secrete alginolytic enzymes that are able to efficiently degrade alginate from brown algae. Therefore, it is clear that Pseudoalteromonas is an important group involved in marine alginate degradation and recycling.

ALGINATE LYASES FROM PSEUDOALTEROMONAS

Diversity of alginate lyases from Pseudoalteromonas.

Alginate lyase is the main enzyme decomposing alginate polysaccharide, cleaving the 1,4-glycoside bonds through the β-elimination reaction. Sequences of alginate lyases are distributed in 12 polysaccharide lyase (PL) families in the Carbohydrate-Active enZYmes (CAZy) database, including families PL5, PL6, PL7, PL14, PL15, PL17, PL18, PL31, PL32, PL34, PL36, and PL39 (http://www.cazy.org/) (30). These families all contain bacterial alginate lyase sequences, with a total of 5,110 sequences. Ninety-two alginate lyase sequences from Pseudoalteromonas are present in the CAZy database; these are distributed in the PL6, PL7, PL17, and PL18 families. The proportion of Pseudoalteromonas alginate lyase sequences in each PL family varies, with 20/32 in the PL18 family, 33/616 in PL6, 23/2077 in PL7, and 16/692 in PL17. Moreover, there are 39 genomes of Pseudoalteromonas in the CAZy database. Of these, 15 contain genes encoding alginate lyases (Table 2). While Pseudoalteromonas nigrifaciens KMM 661 contains only one alginate lyase gene of the PL7 family, the other 14 strains all contain alginate lyases of the PL6, PL7, and PL17 families, among which 8 strains contain extra alginate lyases of the PL18 family. All the alginate lyase genes are located in chromosomes rather than plasmids. In the genomes of Pseudoalteromonas type strains, genes of the PL6 and PL17 families always closely locate in a gene cluster (Fig. 2), whereas the PL18 alginate lyase genes are always isolated. Thus, there is a high level of consistency in the presence of alginate lyases within this genus. Pseudoalteromonas alginate lyases are distributed in only 4 PL families, and many strains contain similar alginate lyase gene combinations, suggesting that they may have a similar mode of alginate degradation.

TABLE 2.

Alginate lyases in Pseudoalteromonas strains^a

Organism name	Protein name	Family	GenPept accession no.
Pseudoalteromonas agarivorans DSM 14585	PAGA_a0664	PL18	ATC81198.1
	PAGA_a1382	PL6	ATC81800.1
	PAGA_a1383	PL17	ATC81801.1
	PAGA_a1393	PL6	ATC81808.1
	PAGA_a3199	PL7	ATC83373.1
Pseudoalteromonas agarivorans Hao 2018	D9T18_10345	PL6	AYM88216.1
	D9T18_10380	PL17	AYM88217.1
	D9T18_10385	PL6	AYM87072.1
	D9T18_12905	PL7	AYM87517.1
Pseudoalteromonas arctica A 37-1-2	PARC_a2609	PL6	ATC87079.1
	PARC_a2610	PL17	ATC87080.1
	PARC_a3223	PL7	ATC87632.1
Pseudoalteromonas atlantica T6c	Patl_3639	PL7	ABG42141.1
	Patl_3640	PL6	ABG42142.1
	Patl_3645	PL7	ABG42147.1
	Patl_3651	PL17	ABG42153.1
	Patl_3659	PL6	ABG42161.1
Pseudoalteromonas carrageenovora IAM 12662	PCAR9_A20415	PL7	SOU39988.1
	PCAR9_A20870	PL6	SOU40429.1
	PCAR9_A20871	PL17	SOU40430.1
	PCAR9_A20878	PL6	SOU40437.1
	PCAR9_A31210	PL18	SOU42010.1
Pseudoalteromonas carrageenovora KCTC 22325	PC2016_0678	PL7	QBJ70918.1
	PC2016_1118	PL6	QBJ71345.1
	PC2016_1119	PL17	QBJ71346.1
	PC2016_1126	PL6	QBJ71353.1
	PC2016_2704	PL18	QBJ72894.1
Pseudoalteromonas espejiana ATCC 29659	PESP_a0658	PL18	ASM48884.1
	PESP_a2542	PL6	ASM50497.1
	PESP_a2549	PL17	ASM50504.1
	PESP_a2550	PL6	ASM50505.1
	PESP_a3176	PL7	ASM51031.1
Pseudoalteromonas haloplanktis TAC125	PSHAa0571	PL7	CAI85658.1
	PSHAa1748	PL17	CAI86820.1
	PSHAa1749	PL6	CAI86821.1
Pseudoalteromonas issachenkonii ECSMB14103	CPA52_10465	PL6	ATG58642.1
	CPA52_10500	PL17	ATG58648.1
	CPA52_10505	PL6	ATG59704.1
	CPA52_12905	PL7	ATG59074.1
Pseudoalteromonas nigrifaciens KMM 661	PNIG_a0689	PL7	ASM52973.1
Pseudoalteromonas sp. strain 13-15	ATS72_001490	PL6	AUL74939.1
	ATS72_001495	PL17	AUL72346.1
	ATS72_001530	PL6	AUL72353.1
	ATS72_014620	PL7	AUL74739.1
Pseudoalteromonas sp. strain 16-SW-7	FFU37_09460	PL6	QCU74676.1
	FFU37_09495	PL17	QCU74683.1
	FFU37_09500	PL6	QCU74684.1
	FFU37_12175	PL7	QCU75170.1
	FFU37_12870	PL18	QCU75291.1
Pseudoalteromonas sp. strain 1_2015MBL_MicDiv	AOR04_03175	PL7	ATG76622.1
	AOR04_06090	PL6	ATG77133.1
	AOR04_06095	PL17	ATG77134.1
	AOR04_06130	PL6	ATG77141.1
	AOR04_14635	PL18	ATG79322.1
Pseudoalteromonas sp. strain Bsw20308	D172_002815	PL18	ALQ07088.1
	D172_011625	PL6	ALQ08659.1
	D172_011660	PL17	ALQ08666.1
	D172_011665	PL6	ALQ08667.1
	D172_014650	PL7	ALQ09184.1
	D172_018980	PL18	ALQ10167.1
Pseudoalteromonas sp. strain Xi13	EJ103_02800	PL18	AZN31714.1
	EJ103_10640	PL6	AZN34301.1
	EJ103_10675	PL17	AZN34302.1
	EJ103_10680	PL6	AZN33168.1
	EJ103_13280	PL7	AZN33636.1

^aData are from the CAZy database.

FIG 2.

Synteny between the alginate utilization genes of Pseudoalteromonas strains and other strains. The sequence comparisons were performed with BLAST and drawn with the R package genoPlotR version 0.8.9. Sequence similarities are symbolized by red hues for direct comparisons and blue hues for reversed comparisons, and darker colors correspond to higher identities. Open reading frames (ORFs) were illustrated by different colors of arrows based on their functional annotations. PL families are indicated by numbers.

Enzymatic properties of alginate lyases from Pseudoalteromonas.

According to their action modes, alginate lyases are divided into either endotype or exotype modes. Endolytic alginate lyases degrade long alginate chains, releasing oligosaccharides as the main products (16, 31), whereas exolytic alginate lyases degrade small oligosaccharides into monomers and/or dimers from the ends of the chains (32–34). In addition, alginate lyases are classified into three groups based on their substrate specificities, poly(M)-specific lyases (EC 4.2.2.3), poly(G)-specific lyases (EC 4.2.2.11), and bifunctional lyases that can degrade both poly(M) and poly(G) (EC 4.2.2.-) (35). Twelve alginate lyases from Pseudoalteromonas have so far been characterized (Table 3), including one from PL6, three from PL7, four from PL18, and four with an unknown classification. No PL17 alginate lyase from Pseudoalteromonas has been characterized, although genes encoding alginate lyase of this family have been found in Pseudoalteromonas genomes. All the characterized Pseudoalteromonas alginate lyases degrade alginate by the endolytic mode, mainly producing oligomers with the degree of polymerization (DP) ranging from two to four (Table 3). Most of these alginate lyases are bifunctional enzymes, except AlyPM from Pseudoalteromonas sp. SM0524 that is poly(M) specific (Table 3) (16). All the Pseudoalteromonas strains that produce alginate lyases have been isolated from the marine environment, including seawater, sediment samples, plants, and animals. The optimal temperatures for the activity of Pseudoalteromonas alginate lyases range from 25°C to 55°C, and their optimal pHs are in the range of 7.0 to 8.5. At least one cation can increase the activity of a majority of Pseudoalteromonas alginate lyases (Table 3). These characters reflect the adaptation of Pseudoalteromonas alginate lyases to the marine environment.

TABLE 3.

Characteristics of Pseudoalteromonas alginate lyases^a

Strain	Isolation site	Protein name	Extra- or intracellular	Family	Substrate specificity	Action mode	Main products (DP)b	Optimal Tm (°C)b	Optimal pH	Ions positive for salt activationc	Reference
Pseudoalteromonas carrageenovora ASY5	Mangrove soil	Aly1281	Extra	PL7	Bifunctional	Endotype	2	50	8.0	Na+, K+	39
		Alg823	Extra	PL6	Bifunctional	Endotype	2–3	55	8.0	Na+, K+	40
Pseudoalteromonas sp. SM0524	Marine rotten kelp	AlyPM	Extra	PL7	Poly(M)	Endotype	2–3	30	8.5	Na+	16
		aly-SJ02	Extra	PL18	Bifunctional	Endotype	2–3	50	8.5	Na+, K+, Mg2+, Ca2+, Co2+, Ba2+, Ni2+, Sr2+	25
Pseudoalteromonas citrea KMM 3297	Coelomic liquid of the holothurian	AlI	Intra	—	Bifunctional	Endotype	3–5	35	—	Na+, Mg2+	29
		AlII	Intra	—	—	Endotype	>3	45	—	Na+, Mg2+	29
		AlIII	Intra	—	Bifunctional	Endotype	>3	45	—	Na+, Mg2+	29
Pseudoalteromonas sp. strain CY24	Seawater of Jiaozhou Bay of China	AlyPI	Extra	PL7	Bifunctional	Endotype	—	40	7.0	Na+, K+, Mn2+, Ca2+, Fe3+	17
Pseudoalteromonas atlantica AR06	Coastal water in Japan	AlyA	Extra	PL18	Bifunctional	Endotype	2–4	—	—	—	37
Pseudoalteromonas elyakovii IAM 14594	A decaying thallus of Laminaria -	alyPEEC	Extra	PL18	Bifunctional	Endotype	>4	30	7.0	Na+, K+, Mg2+, Ca2+, Ba2+, Mn2+	38
Pseudoalteromonas sp. 272	Sea mud in Omura bay	—	Extra	PL18	Bifunctional	Endotype	—	25	7.5–8.0	None	94
Pseudoalteromonas sp. strain 1786	Intestinal contents of an arthropod	—	Extra	—	Bifunctional	Endotype	2–4	50	7.1–7.7	Mg2+, Ca2+	95

^a—, not mentioned.

^bDP, degree of polymerization; T_m, melting temperature.

^cWe only showed the ions that activated the enzyme activity.

Structures and catalytic mechanisms of alginate lyases from Pseudoalteromonas.

Except for PL32 and PL34, the structures and catalytic mechanisms of alginate lyases from the other 10 families have been identified. Of the Pseudoalteromonas alginate lyases found in PL6, PL7, PL17, and PL18, only two structures of PL18 alginate lyases have been reported, one from Pseudoalteromonas sp. strain 272 and the other one (aly-SJ02) from Pseudoalteromonas sp. SM0524. The structures of these two enzymes are quite similar. While the alginate lyase structure of Pseudoalteromonas sp. 272 has only just been released in the PDB database, the structure and catalytic mechanism of aly-SJ02 from Pseudoalteromonas sp. SM0524 has been reported in detail (36).

The precursor of aly-SJ02 consists of a signal peptide, an N-terminal extension (NTE) predicted as a carbohydrate-binding module (CBM), a linker, and a C-terminal PL18 catalytic domain (CATD); mature aly-SJ02 contains only the CATD (36). Thus, it is not possible for NTE to function as a CBM during catalysis. This phenomenon was also reported in the PL18 alginate lyases from Pseudoalteromonas sp. AR06 and Pseudoalteromonas sp. strain IAM 14594 (37, 38). To determine the function of the NTE and the catalytic mechanism of aly-SJ02, Dong et al. described the crystal structures of the recombinant catalytic domain (r-CATD) and the catalytic domain in the recombinant precursor (P-CATD) of aly-SJ02. Two molecules (molecules A and B) were found in the asymmetric unit of r-CATD, which resulted from crystal packing. The overall structures of r-CATD and P-CATD are almost identical, adopting the β-jelly roll architecture (Fig. 3A). By comparing the conserved residues in the active centers of r-CATD, P-CATD, and the mature aly-SJ02 (M-CATD) that was modeled based on the structure of the homolog from Pseudoalteromonas sp. 272, it was found that the conformation of the two conserved residues in the active centers of r-CATD is different from those in P-CATD and M-CATD (Fig. 3B), indicating that without the NTE, the CATD of aly-SJ02 may fold incorrectly. Combined with the biochemical assays, the authors concluded that the NTE in the aly-SJ02 precursor functions as an intramolecular chaperone to help the orderly folding of the CATD. The catalytic mechanism of aly-SJ02 was further explained based on structural and biochemical data. The conserved residues at the active center of aly-SJ02, Arg219, Lys223, Gln257, His259, Tyr347, and Lys349 recognize and stabilize the carboxyl group of the substrate. Tyr353 acts as both a catalytic base and acid in catalysis (Fig. 3C). In addition, aly-SJ02 contains a Ca²⁺ ion, which is important for maintaining enzymatic activity but does not directly participate in the catalytic reaction (36).

FIG 3.

Structure and catalytic mechanism of the PL18 alginate lyase aly-SJ02 from Pseudoalteromonas sp. SM0524. (A) Overall structures of r-CATD and P-CATD of aly-SJ02. (B) Conformational comparison of the conserved amino acid residues in the active centers in different structures of aly-SJ02. The structures of M-CATD, P-CATD, molecule A, and molecule B from the r-CATD asymmetric unit are shown in green, blue, red, and yellow, respectively. Molecule A and molecule B are two molecules found in the asymmetric unit of r-CATD. (C) Schematic representation of the catalytic mechanism of aly-SJ02. Panels B and C were republished from reference 36 with permission of the publisher.

Although many Pseudoalteromonas alginate lyases have been found and characterized, studies on their structures and catalytic mechanisms are quite limited. Therefore, more studies are still needed on the structures and catalytic mechanisms of Pseudoalteromonas alginate lyases to better understand the mechanism of alginate degradation by Pseudoalteromonas.

SYNERGISTIC DEGRADATION OF ALGINATE BY PSEUDOALTEROMONAS ALGINATE LYASES

It has been found that alginate-degrading strains often secrete multiple alginate lyases to synergistically degrade alginate. Alekseeva et al. reported the synthesis of three intracellular alginate lyases of P. citrea KMM 3297 with the induction of fucoidan from the brown alga Fucus evanescens, namely, AlI, AlII, and AlIII (29). When strain KMM 3297 was grown in the presence of fucoidan, the contributions of the separate enzymes AlI, AlII, and AlIII to the total alginolytic activity were 52%, 4%, and 44%, respectively. The three alginate lyases are similar in their physical and chemical properties (optimal pH, thermal stability, and effect of bivalent metal ions) and action mode but differ in their substrate specificities (Table 3). The final degradation products from alginate catalyzed by AlI were oligosaccharides with DP from 3 to 5, and the alginate lyases AlII and AlIII catalyzed the degradation of alginate to larger fragments. A mixture of these three enzymes degraded alginate with a rate ∼1.6-fold higher than the theoretical value that was the sum of the initial reaction rates of the three enzymes, and the products from the conjoint action of these enzymes were oligosaccharides with DP from 3 to 5 (29). These results reflect a synergistic effect of the three alginate lyases on the polymeric substrate. Synthesizing alginate lyases with different substrate specificities was beneficial for P. citrea KMM 3297 to sufficiently utilize alginate in its habitat.

Pseudoalteromonas sp. strain ASY5 secretes two extracellular alginate lyases, Aly1281 and Alg823 (Table 3). The two alginate lyases have similar properties in optimal temperature and pH, salt ion activation, action mode, and main products but differ slightly in their substrate specificities. Although Aly1281 and Alg823 are both bifunctional, Aly1281 exhibits higher activity toward poly(G) than toward poly(M) (39), and Alg823 shows the highest activity toward poly(M) (40). The similar properties may enable both enzymes to exhibit maximum activity under the same environmental conditions, and the difference in substrate specificity enables a synergistic effect of the two enzymes in alginate degradation. Pseudoalteromonas sp. 0524 also produces two extracellular alginate lyases (AlyPM and aly-SJ02) with different substrate specificities (16, 25) (Table 3), which may have synergistic cooperation in alginate degradation. In addition to strains KMM 3297, ASY5, and SM0524, many other Pseudoalteromonas strains, as shown in Table 2, can secrete multiple alginate lyases, which may belong to different families with low sequence similarity and have different enzymatic properties. The synergistic effect of different alginate lyases is beneficial for the source strains to sufficiently degrade and utilize alginate. Presently, research on the synergistic degradation of alginate by Pseudoalteromonas alginate lyases is quite limited, and how they synergistically degrade is worth being further investigated.

HOW PSEUDOALTEROMONAS ACQUIRES THE ALGINATE-DEGRADING CAPACITY

The genes coding enzymes involved in polysaccharide degradation are often colocalized and coregulated in a gene cluster termed “polysaccharide utilization locus” (PUL) (41). A PUL contains adjacent genes that encode carbohydrate-active enzymes, carbohydrate transporters, and carbohydrate sensors/transcriptional regulators (42–44). PULs were first defined in the Bacteroidetes bacteria (45) and were later found to be also widely distributed in the Alpha- and Gammaproteobacteria. The alginate utilization system (AUS) was first found in Zobellia galactanivorans Dsij^T (belonging to Flavobacteriia of the Bacteroidetes phylum) (46). Many genes related to alginate utilization were found to be clustered in an alginate utilization locus (AUL) in this strain (46). In this report, the authors also investigated the evolutionary relationship of AUS between Bacteroidetes and Proteobacteria, to which Pseudoalteromonas belongs. Based on genomic analyses, they found that AUS is only present in Bacteroidetes and Proteobacteria (46). Due to limitations in evolutionary efficiency, the possibility of the existence of AUS in the common ancestor of the two bacterial phylum is very small, and it is likely that it was transmitted between different populations by horizontal gene transfers (HGTs) (46). Due to the high level of completeness and conservation of the alginolytic operon in marine Flavobacteriia, Thomas et al. believed that this AUS arose from an ancestral marine Flavobacteriia and was independently transferred to marine Proteobacteria several times (46). Marine Flavobacteriia and Proteobacteria often occur together on the surface of algae. Their phylogenetic proximity facilitates the occurrence of HGTs between them. Gobet et al. further confirmed this hypothesis that AUS of Proteobacteria was transferred from Flavobacteriia by HGTs by analyzing the genome of Pseudoalteromonas carrageenovora 9^T, a strain that can grow with alginate as the sole carbon source (47). Strain 9^T contains all necessary genes involved in alginate catabolism in a 14-gene cluster named PUL1, including 3 alginate lyase genes, 7 genes encoding other enzymes involved in alginate degradation, 3 transporter genes, and a regulation factor gene. PUL1 is very similar to the AUL identified in Z. galactanivorans Dsij^T. In addition, the gene synteny of PUL1 is conserved in 19 of the 52 Pseudoalteromonas genomes that are publicly available. The 19 strains all belong to the late diverging clade in the phylogenetic tree of Pseudoalteromonas (47). Consistent with this, among the 48 Pseudoalteromonas type strains, a majority of alginate-degrading strains belong to the late-diverging clade (Fig. 1). In addition, AULs were predicted in 8 of 40 type strains whose genomes are available, and all of these AULs are highly similar (Fig. 1). A synteny comparison of these Pseudoalteromonas AULs with those of Flavobacteriia showed that Pseudoalteromonas and Flavobacteriia have highly homologous aly, kdgF, and mfs, which encode alginate lyase, pectin degradation protein, and MFS permease, respectively (Fig. 2), indicating that Pseudoalteromonas AULs resemble those of Flavobacteriia. These findings strengthen the hypothesis that the capacity to degrade alginate is not ancestral in Pseudoalteromonas and the AUS was probably acquired by HGTs from Flavobacteriia in order to adapt to the algal niche.

THE ALGINATE UTILIZATION PATHWAY IN PSEUDOALTEROMONAS STRAINS

There are two alginate utilization pathways in marine bacteria. One pathway, which is found in Bacteroidetes and Vibrio (in the Gammaproteobacteria class), is that alginate is degraded into oligomers by extracellular alginate lyases, and the oligomers are transported into cells to be further degraded into monouronates, which are then catalyzed by several enzymes and eventually assimilated through the glycolytic pathway (46, 48). Another pathway, which was found only in Sphingomonas (belonging to Alphaproteobacteria), is that alginate is directly transported into the cells, catalyzed by enzymes such as alginate lyases, and then assimilated through the glycolytic pathway (49).

It has been reported that five key enzymes are involved in the degradation and transformation of alginate polymers to pyruvate and glyceraldehyde-3-phosphate, essential intermediates in the glycolytic pathway (48, 50). The 5 key enzymes include alginate lyase, pectin degradation protein (KdgF), 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH) reductases (DehR), 2-dehydro-3-deoxygluconokinase (KdgK), and 2-dehydro-3-deoxyphosphogluconate aldolase (Eda) (48, 50). As shown in Fig. 2, all the genomes of the 8 Pseudoalteromonas type strains contain genes encoding the 5 key enzymes in an AUL, which also contains genes encoding outer membrane transporter (TonB-dependent transporter) and permease (MFS permease) for saccharide transport (Fig. 2) Consistently, Cha et al. recently reported that the genomes of 11 nontype alginate-degrading Pseudoalteromonas strains isolated from Arctic and Antarctic marine environments all contain the 5 key enzymes in an AUL (21). Based on the analysis of the AULs of the 8 Pseudoalteromonas type strains, the alginate utilization pathway of Pseudoalteromonas can be deduced. As shown in Fig. 4, when exterior alginate is present, a Pseudoalteromonas strain secretes extracellular alginate lyases to degrade alginate, and the alginate is depolymerized into a series of alginate oligomers of different lengths. Oligomers are then transported into the periplasm through outer membrane proteins and are degraded into oligomers with lower DP by alginate lyases in the periplasm. The resultant oligomers are transported into the cytoplasm via oligoalginate transporters and are degraded to unsaturated monouronates by cytoplasmic alginate lyases. KdgF then converts unsaturated monouronates to DEH. DEH is reduced into 2-keto-3-deoxy-d-gluconate (KDG) by DehR, and KDG is further phosphorylated by KdgK to 2-keto-3-deoxy-6-phosphogluconate (KDPG). Eda in the Entner-Doudoroff pathway converts KDPG to pyruvate and glyceraldehyde-3-phosphate, which are further assimilated through the glycolytic pathway (Fig. 4).

FIG 4.

The deduced alginate utilization pathway in Pseudoalteromonas.

The alginate utilization pathway of Pseudoalteromonas is generally similar to the pathways of Bacteroidetes and Vibrio splendidus 12B01 (46, 48). However, some differences deserve attention. The outer membrane and inner membrane transporters for alginate oligomers transport in Vibro sp. strain 12B01 are porin and symporter, respectively (48) and in Bacteroidetes are TonB-dependent transporter and MFS permease, respectively (46). In addition, in Bacteroidetes, a susD-like gene encoding the oligosaccharide-binding lipoprotein always exists in pairs with the susC-like gene that encodes the outer membrane TonB-dependent transporter (46). In the AULs of the 11 Pseudoalteromonas strains, the most likely genes for oligosaccharide transport are the susC-like and mfs genes, respectively (Fig. 2A); these are the same as the genes of the alginate transporters of Bacteroidetes but different from those of Vibrio. However, no susD-like genes were found in the 11 AULs.

SUMMARY AND PROSPECTS

In conclusion, alginate, the main polysaccharide component of brown algae, is an important carbon source for marine heterotrophic bacteria. Pseudoalteromonas, widespread in the ocean, has often been found to be an important component of the alginate-degrading bacteria, and many strains have been reported to be able to decompose alginate, which emphasizes the important role it has in the degradation and recycling of marine alginate. Although nearly 100 sequences of alginate lyases from Pseudoalteromonas, which are distributed in the PL6, PL7, PL17, and PL18 families, are available in the CAZy database, only 12 Pseudoalteromonas alginate lyases from the PL6, PL7, and PL18 families have been characterized. Pseudoalteromonas strains usually secrete several alginate lyases to synergistically degrade alginate. The capacity to degrade alginate is not ancestral in the Pseudoalteromonas strains, and the AUS was probably acquired by HGTs from Bacteroides. Genome analysis suggests that the alginate utilization pathway of Pseudoalteromonas is similar to the known pathway of Vibrio splendidus 12B01.

Despite the current progress, our understanding of alginate degradation by Pseudoalteromonas is still limited, and more investigation is necessary. The following aspects, in particular, should be noted: there is a need to (i) explore more alginate-degrading Pseudoalteromonas strains; (ii) discover, identify, and characterize more novel alginate lyases from Pseudoalteromonas; (iii) investigate the effect and mechanism of synergistic degradation of alginate by multiple alginate lyases that a single strain excretes; and (iv) verify the alginate utilization pathway of Pseudoalteromonas strains proposed based on genomic analysis. More studies on these aspects would improve our understanding of the mechanism of alginate degradation by Pseudoalteromonas and the role of Pseudoalteromonas in marine organic carbon degradation and recycling.