Christiangramia qingdaonensis sp. nov., a novel polysaccharide-degrading Bacteroidota bacterium, isolated from intertidal sediment

doi:10.21203/rs.3.rs-8043186/v1

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Christiangramia qingdaonensis sp. nov., a novel polysaccharide-degrading Bacteroidota bacterium, isolated from intertidal sediment

https://doi.org/10.21203/rs.3.rs-8043186/v1

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A novel polysaccharide-degrading bacterial strain, designated ASW11-125^T, was isolated from intertidal sediments in Aoshan Bay, Qingdao, China. The strain was strictly aerobic, Gram-stain-negative, catalase-positive but oxidase-negative, short rod-shaped, and exhibited gliding motility without flagella. Growth occurred at 4–35°C (optimum 28°C), pH 6.0–8.0 (optimum pH 7.0), and in 0.5–16.0% NaCl (optimum 2.5–3.0%). The predominant polar lipid was phosphatidylethanolamine. Major fatty acids were iso-C_15:0 and iso-C_17:0 3-OH, and the primary respiratory quinone was menaquinone-6 (MK-6). Based on the phylogenetic analyses of 16S rRNA gene sequences and 1542 single copy orthologous clusters, strain ASW11-125^T affiliated with the genus Christiangramia and was closely related to Christiangramia portivictoriae MCCC 1A00585^T (98.8%), Christiangramia aquimixticola KCTC 42706^T (98.8%) and Christiangramia marina KCTC 12366^T (98.6%). Comparative genomic analysis revealed that the average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between strain ASW11-125ᵀ and its closely related species (74.6–91.5% and 18.6–44.3%, respectively) were clearly lower than the proposed species cutoff values. Based on a polyphasic characterization integrating phenotypic, phylogenetic, and chemotaxonomic evidence, strain ASW11-125ᵀ represents a novel species of the genus Christiangramia, for which the name Christiangramia qingdaonensis sp. nov. is proposed. The draft genome of strain ASW11-125ᵀ is 3.2 Mb in size with a G + C content of 38.3%. Notably, genomic analysis revealed an abundance of genes encoding putative carbohydrate-active enzymes (CAZymes), particularly those associated with starch, laminarin, and fructan utilization, suggesting its potential role in the marine carbon cycle. The type strain is ASW11-125ᵀ (= KCTC 102340ᵀ = MCCC 1K09555ᵀ).

Christiangramia qingdaonensis

intertidal sediment

polyphasic taxonomy

polysaccharide utilization loci

Marine phytoplankton fuel approximately 50% of Earth’s net primary production and exude a sizeable fraction as dissolved organic carbon (DOC) dominated by high-molecular-weight polysaccharides (Tang et al. 2017). Degradation of this reduced-carbon reservoir is disproportionately mediated by Bacteroidota, a lineage specialised in the enzymatic breakdown of structurally diverse marine polysaccharides (Lapébie et al. 2019; Zhu et al. 2023). From epipelagic waters to hadal trenches, marine Bacteroidota oscillate between three lifestyles: free-living, attached to algae cells (Mann et al. 2013), or embedded within sponges (Yoon and Oh 2012), corals (Sweet et al. 2011) and echinoderms (Romanenko et al. 2007; Gavriilidou et al. 2020). Gliding motility and rapid biofilm formation let them colonise different habitats (Mann et al. 2013). Once settled, polysaccharide-utilization loci (PULs) arm them with bespoke carbohydrate-active enzymes to unlock whatever polysaccharide the new niche offers. Each PUL forms a catabolic module of glycoside hydrolases, carbohydrate esterases, polysaccharide lyases and glycosyl-transferases coupled to TonB-dependent SusC transporters and SusD substrate-binding proteins, that synchronizes sensing, scission and import of oligosaccharides (Cantarel et al. 2009; Mann et al. 2013). Because the system is co-transcribed, cellular SusC/D abundance acts as an in-situ sentinel for which polysaccharide is being consumed at any given moment(Kappelmann et al. 2019). Even with the same PUL blueprint, sister Bacteroidota divide the glycan landscape at sub-genus resolution: free-living Hel1_33–49 keeps four PULs and a protease-heavy toolkit, while facultatively particle-attached Hel1_85 expands to eight PULs, swaps proteases for CAZymes, and rides living algae to mine a broader polysaccharide spectrum. This split in PUL repertoire epitomizes metabolic plasticity of Bacteroidota, reflecting their ability to adapt to diverse ecological niches (Xing et al. 2015).

The genus Christiangramia, a member of the family Flavobacteriaceae, is a recognised taxon within the phylum Bacteroidota. Initially proposed by Nedashkovskaya et al. with Christiangramia echinicola as the type species (Nedashkovskaya et al. 2005), the genus currently comprises twenty-two validly described species, isolated from diverse marine habitats including tidal flats (Jeong et al. 2013; Park et al. 2015a), seawater (Liu et al. 2014; Shahina et al. 2014; Shin et al. 2018), marine sediments (Hameed et al. 2014; Yoon et al. 2015; Li et al. 2018; Yang et al. 2023), solar salterns (Joung et al. 2011), and marine organisms (Nedashkovskaya et al. 2005; Nedashkovskaya et al. 2010; Liu et al. 2020). Most members are Gram-stain-negative, rod-shaped, strictly aerobic or aerobic, exhibit gliding motility, and contain menaquinone-6 (MK-6) as the major respiratory quinone (Yang et al. 2023; Wang et al. 2025). The genomic DNA G + C content ranges from 36.6 to 48.0% (Park et al. 2015b; Panschin et al. 2017). Functionally, Christiangramia species arm each niche with bespoke CAZyme batteries. C. forsetii KT0803 packs 164 CAZymes into starch-, laminarin- and alginate-specific PULs (Kabisch et al. 2014), whereas C. flava JLT2011 swaps alginate for two xylan PULs and a pectin PUL, parking half of its GH genes inside these loci (Tang et al. 2017). Multi-omics show that such differences in PUL composition directly influence substrate utilization patterns. Yet we still lack a genus-wide map of which loci are universal, which are niche-exclusive, and how this mosaic quantitatively gates Christiangramia’s share of marine carbon flux.

During an investigation of bacterial diversity in the Qingdao intertidal zone, a novel strain, ASW11-125ᵀ, was isolated from the sediment and assigned to the genus Christiangramia. To determine its precise taxonomic status and explore its functional potential, a polyphasic taxonomic characterization was conducted, including physiological, chemotaxonomic, and phylogenetic analyses, as well as genomic analyses. The results confirmed that strain ASW11-125ᵀ represents a novel species equipped with a private suite of starch-, laminarin- and fructan-specific PULs. Within the genus, laminarin and starch are universal metabolic pillars; fructan utilization is an accessory module, switched on only by those lineages that encounter it.

Sample collection and bacterial isolation

A sediment sample was collected from the tidal flat of Aoshan Bay (36°22′1″N, 120°41′51″E) in Qingdao, China. Approximately 1 g of sediment was serially diluted in 3% sterile artificial seawater (Sigma, USA), and appropriate dilutions were spread onto 1/3-strength marine agar 2216 (MA). After incubation at 20°C for 7 days, a single yellow colony was picked and purified through repeated subculturing on fresh plates, designated ASW11-125ᵀ. For long-term storage, the strain was cultured in marine broth 2216 (MB) at 28°C and preserved at − 80°C in MB supplemented with 20% (v/v) glycerol. For further comparative taxonomic analysis, the reference strains C. portivictoriae MCCC 1A00585^T, C. aquimixticola KCTC 42706^T and C. marina KCTC 12366^T were included in all subsequent experiments under the same conditions.

Phylogenetic analysis

The 16S rRNA gene of strain ASW11-125^T was amplified via PCR with the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGYTACCTTGTTACGACTT-3′). The resulting nearly full-length sequence (1491 bp) was compared with related strains using the EzBioCloud database and the BLAST algorithm. Multiple sequence alignments were performed with the CLUSTAL W algorithm (Thompson et al. 1994) in MEGA 11 (Tamura et al. 2021). Phylogenetic trees were constructed using the maximum-likelihood (Felsenstein 1981), neighbor-joining (Saitou and Nei 1987), and minimum-evolution methods (Pardi et al. 2010) in MEGA 11, with evolutionary distances calculated under the Kimura two-parameter model (Kimura 1980). Bootstrap analyses were performed based on 1000 replicates to evaluate the robustness of tree topologies (Felsenstein 1985). For the genome-based phylogenetic analysis, genomic sequences of closely related species were obtained from GenBank. The 1542 single-copy orthologous clusters (OCs) from 25 bacterial genomes were selected with Proteinortho v5.16 (Lechner et al. 2011) and aligned with MUSCLE v3.8.31 (Edgar 2004). Phylogenetic trees were subsequently inferred from the concatenated alignment of these clusters using MEGA 11.

Physiological and biochemical analysis

Colony morphology of strain ASW11-125^T was observed on MA after 48 h of incubation at 28°C. For detailed morphology features, including cell size, shape and flagella, transmission electron microscopy was used with cells negatively stained with 3% uranyl acetate for 1 min. Gram staining was performed using a Gram-stain kit (Solarbio, China) following the manufacturer’s instructions. Gliding motility was examined as described by Bowman (2000) and flexirubin-type pigment production was evaluated according to Bernardet and Bowman (2006). Growth under different pH conditions was assessed in marine broth (MB) adjusted to pH 4.0–10.0 (in increments of 0.5 units) and pH 10.0–12.0 (in increments of 1.0 unit) using the following buffers (50 mM each): MES (pH 4.0–6.0), MOPS (pH 6.5–7.0), Tris (pH 7.5–8.5), CHES (pH 9.0–10.0), and CAPS (pH 11.0–12.0). NaCl tolerance was assessed in NaCl-free basal MB supplemented with 0–20% NaCl (0–4% at 0.5% intervals; 4–20% at 1% intervals). The temperature range for growth was examined in MB at 4, 15, 20, 25, 28, 32, 35, 37 and 40°C, respectively. Anaerobic growth was evaluated on modified TYS broth (0.5% tryptone, 0.1% yeast extract, 3.0% sea salt) supplemented with 0.15% (w/v) cysteine hydrochloride and 1‰ (w/v) resazurin for 1 month in an anaerobic jar. Catalase activity was determined by bubble production in 3% (v/v) H₂O₂, and oxidase activity was assessed using commercial test strips (Merck). Hydrolysis of milk (0.5%, w/v), casein (0.5%, w/v), gelatin (1%, w/v), starch (1%, w/v), cellulose (1%, w/v), algin (1%, w/v) and Tweens 20, 40, 60, and 80 (1%, v/v) were carried out as described by Smibert and Krieg (1981). Antibiotic susceptibility was tested by the disc diffusion method (Hudzicki 2009) on MA plates using the following antibiotics: ampicillin (10 µg), cephalexin (30 µg), erythromycin (15 µg), roxithromycin (15 µg), oxytetracycline (30 µg), streptomycin (10 µg), vancomycin (30 µg), polymyxin B (300 IU), sulfadiazine (25 µg), sulfamethoxazole (25 µg), sulfafurazole (25 µg), norfloxacin (10 µg), levofloxacin (5 µg), lincomycin (2 µg) and chloramphenicol (30 µg). Additional enzyme activities and biochemical properties were examined by API ZYM and API 20NE test strips (bioMérieux), following standard protocols with a two-week extended incubation. Bacterial suspensions were prepared in 3% NaCl for API ZYM and in AUX medium for API 20NE, respectively.

Chemotaxonomic analysis

For cellular fatty acid analysis, the cells of strain ASW11-125^T and the reference strains were cultured in marine broth (MB) at 28°C and harvested during the early exponential growth phase (12–24 h). Fatty acid methyl esters were prepared from lyophilized cells by saponification, methylation, and extraction following the standard protocol of the Sherlock Microbial Identification System (MIDI). They were then analysed using the Microbial Identification System (MIS) software for identification and quantification. Polar lipids and isoprenoid quinones were extracted from cells collected at the late exponential phase under the same culture conditions. Polar lipids were extracted according to the method of Komagata and Suzuki (1988) and separated by two-dimensional thin-layer chromatography. Specific lipids were detected by spraying with the following reagents: phosphomolybdic acid (for total lipids), ninhydrin (for aminolipids), molybdenum blue (for phospholipids) (Collins and Jones 1980). Respiratory quinones were analysed from lyophilized cells using a chloroform/methanol (2:1, v/v) extraction system. The extract was examined using a Dionex Ultimate 3000 HPLC system coupled to a Bruker impact HD mass spectrometer for identification.

Genomic sequencing and analysis

The genome of strain ASW11-125^T was sequenced on an Illumina HiSeq X-ten platform (OE Biotech, Shanghai, PR China). Raw reads were quality-controlled using Trimmomatic and assembled using ABySS version 2.0. The assembled draft genome was submitted to the GenBank database. The DNA G + C content was calculated from the genome sequence. Genome relatedness was assessed by the average nucleotide identity (ANI) using the EzBioCloud tool (https://www.ezbiocloud.net/tools/ani) (Yoon et al. 2017), and digital DNA-DNA hybridization (dDDH), estimated through the GGDC web server (http://ggdc.dsmz.de/ggdc.php) (Meier-Kolthoff et al. 2013).Functional annotation was performed using the RAST server (Aziz et al. 2008). To assess the polysaccharide-degrading potential, carbohydrate-active enzymes (CAZymes) were identified using the HMMER tool in the dbCAN3 server (Zheng et al. 2023). Polysaccharide-utilization loci (PULs) were further predicted by searching the dbCAN-PUL database. Variation in glycoside hydrolase (GH) composition across different genera was visualised using non-metric multidimensional scaling (NMDS) based on Bray-Curtis dissimilarities.

Phylogenetic analysis

The nearly complete 16S rRNA gene sequence of strain ASW11-125^T (1491 bp; GenBank accession no. PV134465) was obtained in this study. Comparative analysis revealed that strain ASW11-125ᵀ belongs to the genus Christiangramia, showing the highest sequence similarity to C. portivictoriae MCCC 1A00585^T (98.8%), followed by C. aquimixticola KCTC 42706^T (98.8%), C. marina KCTC 12366^T (98.6%), and other validly published type strains of this genus (93.3%–98.3%). In the ML, NJ and ME phylogenetic trees (Figs. 1, S1, and S2), strain ASW11-125^T consistently formed a robust, distinct clade together with C. portivictoriae MCCC 1A00585^T, C. aquimixticola KCTC 42706^T and C. marina KCTC 12366^T. This subclade was stably positioned within a larger clade comprising all recognised species of the genus Christiangramia, with strong bootstrap support (> 89%). Phylogenomic trees based on 1542 single-copy OCs confirmed that strain ASW11-125ᵀ, C. marina KCTC 12366^T and C. portivictoriae MCCC 1A00585^T formed a subclade, which further clustered with C. aquimixticola KCTC 42706^T into a larger clade (Figs. 2, S3, and S4). Genomic relatedness analysis indicated that the average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between strain ASW11-125ᵀ and the closely related strains were 91.5%, 85.2%, and 74.6% (ANI), and 44.3%, 28.7%, and 18.6% (dDDH), respectively (Table S1). All values fell well below the accepted species demarcation thresholds (ANI < 95–96%; dDDH < 70%), supporting the recognition of strain ASW11-125ᵀ as a novel species (Meier-Kolthoff et al. 2013; Palmer et al. 2020).

Phenotypic and biochemical characterization

Strain ASW11-125ᵀ was determined to be Gram-stain-negative, strictly aerobic, and capable of gliding motility. Flexirubin-type pigments were not produced. Transmission electron microscopy showed that the cells were short rods, approximately 0.5–0.8 µm wide and 1.0–3.0 µm long, and lacked flagella (Fig. S5). After 48 h of incubation on MA at 28°C, colonies were round, smooth, yellow, and 0.5–1.0 mm in diameter. The strain grew at 4–35°C (optimum 28°C), pH 6.0–8.0 (optimum pH 7.0) and in the presence of 0.5–16.0% NaCl (optimum 2.5–3.0%). In antibiotic susceptibility tests, the isolate was resistant to ampicillin, cephalexin, erythromycin, streptomycin, sulfadiazine, and sulfafurazole, but susceptible to roxithromycin, oxytetracycline, sulfamethoxazole, norfloxacin, levofloxacin, vancomycin, polymyxin B, lincomycin, and chloramphenicol. A detailed comparison of the physiological and biochemical characteristics of strain ASW11-125ᵀ and its phylogenetic relatives is provided in the species description and in Table 1.

Table 1
Differential characteristics of strains ASW11-125^T and type strains of closely related species
Characteristic	1	2	3	4	5	6
Growth range of:
Temperature (optimum, ℃)	4–35 (28)	4–37 (28–30)^†	4–36 (28–30)^†	10–40 (30)^†	4–37 (23–25)	4–40 (30)
NaCl (optimum, %)	0.5–16 (2.5–3)	1–15 (2–5)^†	1–6^†	0.5–8 (1–2)^†	1–15 (4–5)^*	0–9 (2)
pH (optimum)	6.0–8.0 (7.0)	ND^†	6.0–10.0 (7.0–8.0)^†	6.0–ND (7.0–8.0)^†	5–9 (7–7.5)^*	6–ND (7.0–8.0)
Acid production from:
Glucose	–	+	–	–	+	ND
Utilization of:
Arginine	–	–	–	–	ND	ND
Arabinose	–	+	+	–	+	–
Mannose	–	–	–	–	–	–
Maltose	–	+	–	–	+	–
Malate	+	+	–	–	ND	–
Urea	–	–	–	–	–	–
Hydrolysis of:
Tween20	+	+	+	–	–	ND
Tween40	+	+	+	–	+	ND
Tween80	W	+	+	–	+	+
Starch	+	+	+	–	+	+
Enzyme activity (API ZYM):
Lipase (C14)	+	+	+	+	+^*	–
Trypsin	+	+	+	+	w^*	–
β-Galactosidase	–	+	–	–	+	–
α-Glucosidase	–	+	+	+	+^*	–
N-acetyl glucosaminidase	–	+	–	–	+^*	+
DNA G + C content (%)	38.3	40.0^†	39.9^†	48.0^†	39.6	39.2
Strains: 1, C. qingdaonensis ASW11-125^T; 2, C. marina KCTC 12366^T; 3, C. portivictoriae MCCC 1A00585^T; 4, C. aquimixticola KCTC 42706^T; 5, C. echinicola KMM 6050^T; 6, C. sabulilitoris HSMS-1^T.
All strains were Gram-negative and tested positive for catalase activities; hydrolysis of gelatin; and enzyme activities of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase. All strains were negative for citrate utilization. Data for columns 1–4 were obtained in this study, while data for columns 5–6 were taken from Nedashkovskaya et al. (2005), Park et al. (2020), respectively. +, positive; –, negative; ND, no data available; W, weakly positive; *, Data from Li et al. 2018; †, Data from Lau et al. 2005, Nedashkovskaya et al. 2010, Park et al. 2015b.

Chemotaxonomic characteristics

The major fatty acids (≥ 10%) of strain ASW11-125ᵀ were iso-C_15:0 (22.7%) and iso-C_17:0 3-OH (10.0%), which are consistent with the general profile of the genus Christiangramia. However, notable differences were observed in the proportions of several fatty acids compared to closely related strains. For example, strain ASW11-125ᵀ exhibited significantly lower proportion of C_16:0 (5.5%) than C. portivictoriae MCCC 1A00585ᵀ (11.5%). Similarly, its content of anteiso-C_15:0 (7.5%) was substantially lower than that in C. aquimixticola KCTC 42706ᵀ (14.8%) (Table 2). Polar lipid analysis indicated that strain ASW11-125^T, along with C. portivictoriae MCCC 1A00585^T, C. aquimixticola KCTC 42706^T and C. marina KCTC 12366^T, all included phosphatidylethanolamine (PE) and two unidentified lipids (L1, L2). An additional unidentified lipid (L3) was detected in both ASW11-125ᵀ and C. portivictoriae MCCC 1A00585ᵀ, while an aminolipid (AL) was present only in C. marina KCTC 12366ᵀ (Fig. S6). The respiratory quinone of strain ASW11-125^T was MK-6, which aligns with the typical quinone system of the genus Christiangramia.

Table 2
Cellular fatty acid compositions (%) of strain ASW11-125^T and closely related strains of the genus *Christiangramia*
Fatty acid	1	2	3	4
Straight-chain
C_16:0	5.5	9.3	11.5	6.0
C_18:0	2.7	3.6	6.0	2.8
Branched
iso-C_14:0	1.3	1.0	ND	ND
iso-C_15:0	22.7	21.8	19.2	14.8
iso-C_15:1	1.2	1.5	2.4	0.8
iso-C_16:0	9.2	6.8	4.1	4.9
iso-C_16:1 H	3.2	1.5	1.6	2.0
anteiso-C_15:0	7.5	7.8	8.2	14.8
Hydroxy
C_15:0 2-OH	2.1	2.3	1.9	2.1
C_17:0 2-OH	3.2	3.7	3.5	7.3
iso-C_15:0 3-OH	1.9	1.7	1.8	1.0
iso-C_16:0 3-OH	4.0	2.4	2.2	1.7
iso-C_17:0 3-OH	10.0	11.7	10.6	7.8
Unsaturated
C_15:1ω6c	1.5	0.5	1.5	0.8
C_17:1ω6c	2.8	1.2	1.2	1.8
C_17:1ω8c	0.8	1.0	0.5	ND
anteiso-C_17:1ω9c	2.0	1.1	1.9	8.3
Summed features*
3	8.9	9.7	8.6	9.3
9	5.4	6.0	5.2	8.7
Strains: 1, C. qingdaonensis ASW11-125^T; 2, C. marina KCTC 12366^T; 3, C. portivictoriae MCCC 1A00585^T; 4, C. aquimixticola KCTC 42706^T.
All data were obtained from this study. Major fatty acids in each strain are shown in bold.
*Summed features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The MIDI system reports these as a combined feature with a single percentage of the total; Summed feature 3 contains C_16:1ω7c and/or C_16:1ω6c and/or iso-C_15:0 2-OH; Summed feature 9 contains iso-C_17:1ω9c and/or C_16:0 10-methyl. ND, no data available.

Genome features

The draft genome of strain ASW11-125^T consists of 3,203,245 bp with 19 contigs, an N50 value of 880,254 bp, and a DNA G + C content of 38.3%. A total of 3,010 genes were predicted, including 2981 protein-coding genes, 6 pseudogenes, and 23 RNA genes (3 rRNA genes, 16 tRNA genes, and 4 other RNA genes) (Table S2). Genomic comparisons indicated that strain ASW11-125ᵀ had significantly higher gene abundances for amino acid/derivative metabolism (21.4%) and cofactor/vitamin/pigment metabolism (15.2%), but maintained comparable levels for protein (11.5%), carbohydrate (9.1%), and nucleoside/nucleotide (7.2%) metabolism relative to its close relatives (Table S3). Based on dbCAN3 analysis, 131 carbohydrate-active enzymes (CAZymes) were identified, accounting for 4.35% of the protein-coding sequences (Table S4). These included 68 glycosyltransferases (GTs, 16 families), 40 glycoside hydrolases (GHs, 25 families), 14 carbohydrate esterases (CEs, 4 families), 2 auxiliary activities (AAs, 2 families), and 7 carbohydrate-binding modules (CBMs, 6 families). NMDS analysis based on GH profiles revealed that the genus Christiangramia clustered within a group of known polysaccharide-degrading Bacteroidota, including Polaribacter, Flavobacterium, Bacteroides, and Algibacter, and was clearly separated from the non-saccharolytic genus Staphylococcus (ANOSIM, r = 0.66, P = 0.001; Fig. 3a). Furthermore, Christiangramia exhibited significantly greater similarity to Algibacter and Polaribacter than to other genera (p < 0.05) (Fig. 3b), indicating a closely aligned potential for polysaccharide degradation.

Polysaccharide-utilization loci for starch, laminarin and fructan

Strain ASW11-125ᵀ harbors three polysaccharide utilization loci (PULs) that target starch, laminarin, and fructan. Each locus encodes a canonical PUL architecture, consisting of CAZymes, SusC/D-like transporters, and transcriptional regulators—an arrangement that is conserved across Christiangramia species (Fig. 4). Synteny across 18 Christiangramia genomes showed the laminarin locus in every strain and starch loci in 94%, whereas the fructan locus is confined to one-third (33%) (Fig. 4). This distribution suggests that laminarin and starch are genus-wide metabolic pillars; fructan utilization constitutes a facultative trait likely maintained in specific ecological contexts. Collectively, these PULs deliver a rapid high-molecular-weight carbon uptake module that confers ecological plasticity and competitive supremacy throughout marine ecosystems.

Taxonomic conclusion

The phenotypic, phylogenetic, biochemical, genomic and chemotaxonomic features all support that strain ASW11-125^T represents a novel species of the genus Christiangramia, for which the name Christiangramia qingdaonensis sp. nov. is proposed here.

Description of Christiangramia qingdaonensis sp. nov.

Christiangramia qingdaonensis [qing.dao.nen’sis. N.L. fem. adj. qingdaonensis, pertaining to Qingdao, China, where the type strain was isolated]

Cells are strictly aerobic, Gram-stain-negative, non-spore-forming, rod-shaped, non-flagellated and motile by gliding, measuring 0.5–0.8 µm in width and 1.0–3.0 µm in length. After 48 h on marine agar at 28°C, colonies are yellow, smooth, and 0.5–1.0 mm in diameter. Growth occurs at 4–35°C (optimally at 28°C), pH 6.0–8.0 (optimally at pH 7.0) and in the presence of 0.5–16.0% (w/v) NaCl (optimum 2.5–3.0%). Flexirubin-type pigments are not produced. Cells are catalase-positive but oxidase-negative. Hydrolytic activities are observed for Tweens 20, 40, 60, and 80, starch, aesculin, casein, and gelatin, but not for cellulose or milk. Nitro-β-D-methylgalactose and malate are assimilated, while arabinose, mannose, mannitol, maltose, and citrate are not. Enzymatic activities are positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cysteine arylamidase, trypsin, chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, and β-glucosidase. However, activities are negative for urease, arginine dihydrolase, β-galactosidase, α-glucosidase, and β-fucosidase. Acid production from glucose, nitrate reduction, and indole production are negative. The major polar lipid is phosphatidylethanolamine, and the predominant fatty acids are iso-C_15:0 (22.7%) and iso-C_17:0 3-OH (10.0%). The respiratory quinone is menaquinone-6 (MK-6). The genomic DNA G + C content of the type strain is 38.3%. The type strain, ASW11-125^T (= KCTC 102340^T = MCCC 1K09555^T), was isolated from intertidal sediment of Qingdao, PR China. The GenBank accession numbers for the 16S rRNA gene and whole-genome sequence are PV134465 and NZ_JBLWIM000000000, respectively.

MCCC

Marine culture collection of China

KCTC

Korean collection for type cultures

MEGA

Molecular evolutionary genetics analysis

RAST

Rapid annotations using subsystems technology

ANI

Average nucleotide identity

dDDH

Digital DNA-DNA hybridization

Competing interests

The authors declare no competing interests.

Ethics approval

This article does not contain any studies conducted by the authors involving human participants or animals.

Funding

This work was funded by the National Natural Science Foundation of China (32300115).

Author Contribution

ZTC designed the study, performed the data analysis, and wrote the manuscript. BY, YSL and ZWZ conducted genome analysis, contributed to the interpretation of data, and revised the manuscript. YL and XYZ supervised the experimental work, provided feedback on the manuscript, and contributed to the final manuscript revision.

Data Availability

All data obtained in this study are shown within the manuscript and supplemental materials.

Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O (2008) The RAST Server: rapid annotations using subsystems technology. BMC genomics 9:75. https://doi.org/10.1186/1471-2164-9-75
Bernardet J-F, Bowman JP (2006) The Genus Flavobacterium. In: The Prokaryotes: Volume 7: Proteobacteria: Delta, Epsilon Subclass. Dworkin M, Falkow S, Rosenberg E, Schleifer K-HStackebrandt E (eds). Springer New York, New York, NY. pp. 481–531.
Bowman JP (2000) Description of Cellulophaga algicola sp. nov., isolated from the surfaces of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov. International journal of systematic and evolutionary microbiology 50 Pt 5:1861–1868. https://doi.org/10.1099/00207713-50-5-1861
Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic acids research 37:D233–238. https://doi.org/10.1093/nar/gkn663
Collins MD, Jones D (1980) Lipids in the Classification and Identification of Coryneform Bacteria Containing Peptidoglycans Based on 2, 4-diaminobutyric Acid. Journal of Applied Bacteriology 48:459–470. https://doi.org/https://doi.org/10.1111/j.1365-2672.1980.tb01036.x
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic acids research 32:1792–1797. https://doi.org/10.1093/nar/gkh340
Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. Journal of molecular evolution 17:368–376. https://doi.org/10.1007/bf01734359
Felsenstein J (1985) CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP. Evolution; international journal of organic evolution 39:783–791. https://doi.org/10.1111/j.1558-5646.1985.tb00420.x
Gavriilidou A, Gutleben J, Versluis D, Forgiarini F, van Passel MWJ, Ingham CJ, Smidt H, Sipkema D (2020) Comparative genomic analysis of Flavobacteriaceae: insights into carbohydrate metabolism, gliding motility and secondary metabolite biosynthesis. BMC genomics 21:569. https://doi.org/10.1186/s12864-020-06971-7
Hameed A, Shahina M, Lin SY, Liu YC, Lai WA, Young CC (2014) Gramella oceani sp. nov., a zeaxanthin-producing bacterium of the family Flavobacteriaceae isolated from marine sediment. International journal of systematic and evolutionary microbiology 64:2675–2681. https://doi.org/10.1099/ijs.0.059881-0
Hudzicki J (2009) Kirby-Bauer disk diffusion susceptibility test protocol. American society for microbiology 15:1–23.
Jeong SH, Jin HM, Jeon CO (2013) Gramella aestuarii sp. nov., isolated from a tidal flat, and emended description of Gramella echinicola. International journal of systematic and evolutionary microbiology 63:2872–2878. https://doi.org/10.1099/ijs.0.048694-0
Joung Y, Kim H, Jang T, Ahn TS, Joh K (2011) Gramella jeungdoensis sp. nov., isolated from a solar saltern in Korea. J Microbiol 49:1022–1026. https://doi.org/10.1007/s12275-011-1192-0
Kabisch A, Otto A, Konig S, Becher D, Albrecht D, Schuler M, Teeling H, Amann RI, Schweder T (2014) Functional characterization of polysaccharide utilization loci in the marine Bacteroidetes 'Gramella forsetii' KT0803. ISME J 8:1492–1502. https://doi.org/10.1038/ismej.2014.4
Kappelmann L, Kruger K, Hehemann JH, Harder J, Markert S, Unfried F, Becher D, Shapiro N, Schweder T, Amann RI, Teeling H (2019) Polysaccharide utilization loci of North Sea Flavobacteriia as basis for using SusC/D-protein expression for predicting major phytoplankton glycans. ISME J 13:76–91. https://doi.org/10.1038/s41396-018-0242-6
Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of molecular evolution 16:111–120. https://doi.org/10.1007/bf01731581
Komagata K, Suzuki K-I (1988) 4 Lipid and Cell-Wall Analysis in Bacterial Systematics. In: Methods in Microbiology. Colwell RRGrigorova R (eds). Academic Press. pp. 161–207.
Lapébie P, Lombard V, Drula E, Terrapon N, Henrissat B (2019) Bacteroidetes use thousands of enzyme combinations to break down glycans. Nature Communications 10:2043. https://doi.org/10.1038/s41467-019-10068-5
Lau SCK, Tsoi MMY, Li X, Plakhotnikova I, Dobretsov S, Wong PK, Qian PY (2005) Gramella portivictoriae sp. nov., a novel member of the family Flavobacteriaceae isolated from marine sediment. International journal of systematic and evolutionary microbiology 55:2497–2500. https://doi.org/10.1099/ijs.0.63824-0
Lechner M, Findeiß S, Steiner L, Marz M, Stadler PF, Prohaska SJ (2011) Proteinortho: Detection of (Co-)orthologs in large-scale analysis. BMC bioinformatics 12:124. https://doi.org/10.1186/1471-2105-12-124
Li AZ, Han XB, Lin LZ, Zhang MX, Zhu HH (2018) Gramella antarctica sp. nov., isolated from marine surface sediment. International journal of systematic and evolutionary microbiology 68:358–363. https://doi.org/10.1099/ijsem.0.002513
Liu K, Li S, Jiao N, Tang K (2014) Gramella flava sp. nov., a member of the family Flavobacteriaceae isolated from seawater. International journal of systematic and evolutionary microbiology 64:165–168. https://doi.org/10.1099/ijs.0.051987-0
Liu L, Wang S, Zhou S, Sun W, Fu T, Zhang Y, Zhang XH, Yu M (2020) Gramella bathymodioli sp. nov., isolated from a mussel inhabiting a hydrothermal field in the Okinawa Trough. International journal of systematic and evolutionary microbiology 70:5854–5860. https://doi.org/10.1099/ijsem.0.004488
Mann AJ, Hahnke RL, Huang S, Werner J, Xing P, Barbeyron T, Huettel B, Stüber K, Reinhardt R, Harder J, Glöckner FO, Amann RI, Teeling H (2013) The genome of the alga-associated marine flavobacterium Formosa agariphila KMM 3901T reveals a broad potential for degradation of algal polysaccharides. Appl Environ Microbiol 79:6813–6822. https://doi.org/10.1128/aem.01937-13
Meier-Kolthoff JP, Auch AF, Klenk H-P, Göker M (2013) Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC bioinformatics 14:60. https://doi.org/10.1186/1471-2105-14-60
Nedashkovskaya OI, Kim SB, Bae KS (2010) Gramella marina sp. nov., isolated from the sea urchin Strongylocentrotus intermedius. International journal of systematic and evolutionary microbiology 60:2799–2802. https://doi.org/10.1099/ijs.0.013870-0
Nedashkovskaya OI, Kim SB, Lysenko AM, Frolova GM, Mikhailov VV, Bae KS, Lee DH, Kim IS (2005) Gramella echinicola gen. nov., sp. nov., a novel halophilic bacterium of the family Flavobacteriaceae isolated from the sea urchin Strongylocentrotus intermedius. International journal of systematic and evolutionary microbiology 55:391–394. https://doi.org/10.1099/ijs.0.63314-0
Palmer M, Steenkamp ET, Blom J, Hedlund BP, Venter SN (2020) All ANIs are not created equal: implications for prokaryotic species boundaries and integration of ANIs into polyphasic taxonomy. International journal of systematic and evolutionary microbiology 70:2937–2948. https://doi.org/https://doi.org/10.1099/ijsem.0.004124
Panschin I, Becher M, Verbarg S, Sproer C, Rohde M, Schuler M, Amann RI, Harder J, Tindall BJ, Hahnke RL (2017) Description of Gramella forsetii sp. nov., a marine Flavobacteriaceae isolated from North Sea water, and emended description of Gramella gaetbulicola Cho et al. 2011. International journal of systematic and evolutionary microbiology 67:697–703. https://doi.org/10.1099/ijsem.0.001700
Pardi F, Guillemot S, Gascuel O (2010) Robustness of Phylogenetic Inference Based on Minimum Evolution. Bulletin of mathematical biology 72:1820–1839. https://doi.org/10.1007/s11538-010-9510-y
Park J-M, Park S, Won S-M, Jung Y-T, Shin K-S, Yoon J-H (2015a) Gramella aestuariivivens sp. nov., isolated from a tidal flat. International journal of systematic and evolutionary microbiology 65:1262–1267. https://doi.org/10.1099/ijs.0.000093
Park S, Kim IK, Kim W, Yoon JH (2020) Gramella sabulilitoris sp. nov., isolated from a marine sand. International journal of systematic and evolutionary microbiology 70:909–914. https://doi.org/10.1099/ijsem.0.003845
Park S, Kim S, Jung YT, Yoon JH (2015b) Gramella aquimixticola sp. nov., isolated from water of an estuary environment. International journal of systematic and evolutionary microbiology 65:4244–4249. https://doi.org/10.1099/ijsem.0.000567
Romanenko LA, Uchino M, Frolova GM, Mikhailov VV (2007) Marixanthomonas ophiurae gen. nov., sp. nov., a marine bacterium of the family Flavobacteriaceae isolated from a deep-sea brittle star. International journal of systematic and evolutionary microbiology 57:457–462. https://doi.org/10.1099/ijs.0.64662-0
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular biology and evolution 4:406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454
Shahina M, Hameed A, Lin SY, Lee RJ, Lee MR, Young CC (2014) Gramella planctonica sp. nov., a zeaxanthin-producing bacterium isolated from surface seawater, and emended descriptions of Gramella aestuarii and Gramella echinicola. Antonie van Leeuwenhoek 105:771–779. https://doi.org/10.1007/s10482-014-0133-4
Shin SK, Kim E, Yi H (2018) Gramella salexigens sp. nov., isolated from seawater. International journal of systematic and evolutionary microbiology 68:2381–2385. https://doi.org/10.1099/ijsem.0.002850
Smibert RM, Krieg NR (1981) Manual of methods for general bacteriology. American Society for Microbiology, Washington, DC, pp 409–443
Sweet MJ, Croquer A, Bythell JC (2011) Bacterial assemblages differ between compartments within the coral holobiont. Coral Reefs 30:39–52. https://doi.org/10.1007/s00338-010-0695-1
Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Molecular biology and evolution 38:3022–3027. https://doi.org/10.1093/molbev/msab120
Tang K, Lin Y, Han Y, Jiao N (2017) Characterization of Potential Polysaccharide Utilization Systems in the Marine Bacteroidetes Gramella Flava JLT2011 Using a Multi-Omics Approach. Front Microbiol 8:220. https://doi.org/10.3389/fmicb.2017.00220
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic acids research 22:4673–4680. https://doi.org/10.1093/nar/22.22.4673
Wang JX, Wang J, Liu NH, Qin QL, Li PY, Song XY, Chen XL, Zhang YZ, Zhang XY (2025) Christiangramia sediminicola sp. nov., a DNA-hydrolysing bacterium isolated from intertidal sediment. International journal of systematic and evolutionary microbiology 75. https://doi.org/10.1099/ijsem.0.006703
Xing P, Hahnke RL, Unfried F, Markert S, Huang S, Barbeyron T, Harder J, Becher D, Schweder T, Glockner FO, Amann RI, Teeling H (2015) Niches of two polysaccharide-degrading Polaribacter isolates from the North Sea during a spring diatom bloom. ISME J 9:1410–1422. https://doi.org/10.1038/ismej.2014.225
Yang L, Shi H, Li Q, Zheng M, Lai Q, Zheng L (2023) Gramella oceanisediminis sp. nov., isolated from deep-sea sediment of the Indian Ocean. International journal of systematic and evolutionary microbiology 73. https://doi.org/10.1099/ijsem.0.005861
Yoon BJ, Oh DC (2012) Spongiibacterium flavum gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from the marine sponge Halichondria oshoro, and emended descriptions of the genera Croceitalea and Flagellimonas. International journal of systematic and evolutionary microbiology 62:1158–1164. https://doi.org/10.1099/ijs.0.027243-0
Yoon J, Jo Y, Kim GJ, Choi H (2015) Gramella lutea sp. nov., a Novel Species of the Family Flavobacteriaceae Isolated from Marine Sediment. Curr Microbiol 71:252–258. https://doi.org/10.1007/s00284-015-0849-z
Yoon SH, Ha SM, Lim J, Kwon S, Chun J (2017) A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie van Leeuwenhoek 110:1281–1286. https://doi.org/10.1007/s10482-017-0844-4
Zheng J, Ge Q, Yan Y, Zhang X, Huang L, Yin Y (2023) dbCAN3: automated carbohydrate-active enzyme and substrate annotation. Nucleic acids research 51:W115–w121. https://doi.org/10.1093/nar/gkad328
Zhu XY, Li Y, Xue CX, Lidbury I, Todd JD, Lea-Smith DJ, Tian J, Zhang XH, Liu J (2023) Deep-sea Bacteroidetes from the Mariana Trench specialize in hemicellulose and pectin degradation typically associated with terrestrial systems. Microbiome 11:175. https://doi.org/10.1186/s40168-023-01618-7

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Christiangramia qingdaonensis sp. nov., a novel polysaccharide-degrading Bacteroidota bacterium, isolated from intertidal sediment

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Phylogenetic analysis

Phenotypic and biochemical characterization

Chemotaxonomic characteristics

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Polysaccharide-utilization loci for starch, laminarin and fructan

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