Comparative genomic analyses of four novel Ramlibacter species and the cellulose-degrading properties of Ramlibacter cellulosilyticus sp. nov.

Chemotaxonomic characteristics

The predominant respiratory quinone for all novel strains was ubiquinone 8 (Q-8), consistent with other Ramlibacter species. C_16:0 and summed feature 3 (consisting of C_16:1 ω7c and/or C_16:1 ω6c) were identified as the common major fatty acids (> 10%) of the novel strains USB13^T, AW1^T, GTP1^T, and HM2^T. Other than the aforementioned fatty acids, strain USB13^T had C_10:0 3-OH additionally as its major fatty acid, whereas strains AW1^T and HM2^T shared C_17:0 cyclo and summed feature 8 (consisting of C_18:1 ω7c and/or C_{18: 1} ω6c) as its additional fatty acids. Detailed comparisons of the fatty acid profiles of the novel strains and their reference strains are summarized in Table S1.

Strains USB13^T, AW1^T, GTP1^T, and HM2^T shared major polar lipids diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE), which was consistent with the major polar lipids of the reference strains. Additionally, the polar lipid profile of USB13^T consisted of one unidentified phosphoaminolipid, two unidentified phosphoglycoaminolipids, and six unidentified polar lipids while the polar lipid profile of AW1^T had one unidentified lipid, one unidentified phosphoglycolipid, and three unidentified glycolipids in addition. The polar lipid profile of strain GTP1^T additionally consisted of two unidentified phosphoaminolipids, and that of strain HM2^T additionally had one unidentified phosphoaminolipid, one unidentified phosphoglycolipid, one unidentified phosphoglycoaminolipid, and two unidentified phospholipids. Polar lipid profiles of the novel strains USB13^T, AW1^T, GTP1^T, and HM2^T are shown in Figure S1.

Physiological, morphological characteristics, and screening of cellulose-degrading strains

When grown on R2A agar, strain USB13^T produced reddish white and flat colonies while strain AW1^T produced orange, convex colonies, strain GTP1^T produced white, convex colonies, and strain HM2^T produced cream-colored, flat, transparent colonies. Under TEM, monotrichous flagella were observed only in strain HM2^T, and when tested for motility, strain USB13^T and AW1^T showed gliding motility, whereas strain GTP1^T was non-motile. Strains USB13^T and HM2^T showed positive results for both catalase and oxidase activities; strain AW1^T showed positive results for catalase and negative results for oxidase activity, and strain GTP1^T showed negative results for catalase and positive results for oxidase activity. All strains were identified to be strictly aerobic, while showing negative results for urea, gelatin, starch, chitin, and DNA hydrolysis and positive results for hydrolysis of Tween 80. In addition, strain USB13^T was the only strain to produce iron-chelating siderophores. When tested for NaCl tolerance, growth of strain USB13^T was observed in NaCl concentrations of 0–7% (w/v), possibly due to the fact the strain was isolated from a marine environment. A detailed comparison of physiological and morphological characteristics between the novel species and its closely related Ramlibacter strains is presented in Table 1, while TEM images of the novel strains are shown in Figure S2. Results of the reference strains in Table 1 coincided with the data from the original literature^1,3,4,5,7,8.

Strains: 1, USB13^T; 2, AW1^T; 3, GTP1^T; 4, HM2^T; R. monticola KACC 19175^T; 6, R. alkalitolerans KACC 19305^T; 7, R. ginsenosidimutans KACC 17527^T; 8, R. humi KCTC 52922^T; 9, R. henchirensis KACC 11925^T; 10, R. tataouinensis KACC 11924^T; 11, R. rhizophilus KCTC 52083^T. All strains are positive for esterase lipase (C8), while all strains are negative for chitin hydrolysis. All data were obtained from this study unless indicated otherwise. + , Positive; w + , weakly positive; -, negative.

R2A agar plates supplemented with 1% (w/v) CMC were stained with Congo red dye after 7 days of incubation. Clear zones only formed around colonies of strain USB13^T, indicating that strain USB13^T solely possessed CMC-hydrolyzing activity among the four novel strains. When inoculated in basal salt medium, filter paper from the USB13^T sample underwent degradation, whereas samples containing strains AW1^T, GTP1^T, and HM2^T did not show any signs of degradation.

Phylogenetic and genomic analyses

EzBioCloud search results and BLASTn searches revealed that the novel strains belonged to the family Comamonadaceae and genus Ramlibacter. Using BLASTn, 16S rRNA gene sequence similarities were determined where strain USB13^T was closest to strain GTP1^T (98.5%), followed by strain HM2^T (98.1%) and strain AW1^T (97.1%). Strain AW1^T shared the highest similarity with strain GTP1^T (97.3%), followed by strain HM2^T (97.1%), while strain GTP1^T shared a similarity of 98.2% with strain HM2^T. Phylogenetic analysis based on the MP method (Fig. 1) showed the clustering of the novel strains USB13^T, AW1^T, GTP1^T, and HM2^T with strains such as R. monticola G-3-2^T, R. ginsenosidimutans BXN5-27^T, R. alkalitolerans CJ661^T, and R. rhizophilus YS3.2.7^T. Similar topologies were observed in trees reconstructed by ML (Figure S3) and MP methods. The UBCG phylogenomic tree (Fig. 2), which was reconstructed using whole genome sequences, also showed close clustering of the selected reference strains and novel strains.

Draft genome sequences of the novel strains USB13^T, AW1^T, GTP1^T, and HM2^T were deposited in the GenBank database under the accession numbers JACORT000000000, JAEQNA000000000, JACORU000000000, and JADDIV000000000, respectively. In addition, the draft genome sequences of R. monticola KACC 19175^T, R. alkalitolerans KACC 19305^T, and R. ginsenosidimutans KACC 17527^T were also deposited in GenBenk under the accession numbers JAEQNE000000000, JAEQND000000000, and JAEPWM000000000, respectively. The assembled genome size of the novel strains USB13^T, AW1^T, GTP1^T, and HM2^T was 5.53 Mbp, 5.11 Mbp, 6.15 Mbp, 4.31 Mbp, respectively. G + C content ranged from 67.9% to 69.9%, which was similar to those of the reference strains. The genomic features of the novel strains and their closely related Ramlibacter strains are presented in Table S2. CheckM analysis showed the following estimations for each strain: USB13^T, had a 99.84% completeness and 0.68% contamination; AWI^T, had a 99.84% completeness and 0.86% contamination; GTP1^T, had a 99.38% completeness and 1.32% contamination; HM2^T, had a 97.51% completeness and 0.16% contamination. These results indicated that the draft genome results for all strains were reliable. ANI values between the novel strains and reference strains ranged from 76.5–83.4% while dDDH values ranged from 20.7–26.7%, and AAI values ranged from 65.7–80.4%. All values were below the threshold for delineation of a new species⁵⁴. ANI values between the novel strains and their reference strains are presented in Fig. 3, while a detailed comparison of GGDC and AAI values are shown in Table 2.

Based on NCBI PGAP annotation and CAZyme prediction results, strain USB13^T, which was the only strain to show cellulolytic activity, possessed a total of four protein CDs encoding CAZymes, namely, two GH15 proteins, one glycosyl hydrolase protein, and one GH99-like domain-containing protein. Despite not showing any cellulolytic activity, strain AW1^T possessed eight CAZyme CDs; the most amount among the novel strains. The enzymes include, two GH2 proteins, one GH5 protein, three GH15 proteins, one glycoside hydrolase protein, and one cellulase family glycosyl hydrolase. Strain GTP1^T possessed two CDs encoding one GH15 protein and one GH16 protein; strain HM2^T possessed three CDs encoding one GH2, one GH15, and one GH18 protein. All strains possessed GH15, which is known for its glucoamylase activity in fungi⁵⁵. A detailed summary of the novel strains CAZymes are presented in Table S3 and a comparison of CAZyme numbers between strains USB13^T, AW1^T, GTP1^T, and HM2^T is summarized in Table S4. The presence of these genes may suggest the cellulolytic activity of strain USB13^T, while it is uncertain why GH families responsible for endoglucanase (GH 5–8, 12, 16, 44, 45, 48, 51, 64, 71, 74, 81, 87, 124, and 128), exoglucanase (GH 5–7, and 48), and β-glucosidase (GH 1, 3, 4, 17, 30, and 116) were not present in the genome¹¹.

COG predictions (Fig. 4) revealed that the majority of the core genes of the four novel strains accounted for genes belonging to the functional categories C (energy production and conversion), E (amino acid transport and metabolism), I (lipid transport and metabolism), T (signal transduction mechanisms), and K (transcription). Meanwhile, the number of core genes belonging in category G, carbohydrate transport and metabolism, was the highest for strain USB13^T (258), followed by GTP1^T (230), HM2^T (212), and AW1^T (181). The high number of genes in strain USB13^T may be a contributing factor in the strain’s cellulolytic activity. A comparison of COG gene count distribution of the novel strains is presented in Table S5.

AntiSMASH analysis results showed four gene clusters within the genome of strain USB13^T: ribosomally synthesized and post-translationally modified peptides (RIPP)-like cluster (989,516–1,000,916 nt; JACORT010000001), terpene synthesis (8,622–30,347 nt; JACORT010000003), RIPP precursor peptide recognition element (RRE)-containing cluster (311,469–333,619 nt; JACORT010000004), and redox-cofactor (281,860–303,948 nt; JACORT010000007). Among the clusters, the RRE-containing cluster showed 11% similarity to streptobactin, a tricatechol-type siderophore isolated from Streptomyces sp. YM5-799⁵⁶. Strain AW1^T had a total of eight gene clusters which encoded for: arylpolyene (165,946–207,130 nt), terpene (618,322–640,854 nt), RIPP-like proteins (804,411–819,137 nt), non-ribosomal peptide synthetase cluster (NRPS)-like (61,798–104,764 nt), betalactone (323,399–348,739 nt), N-acetylglutaminylglutamine amide (NAGGN; 106,834–121,648 nt), type I polyketide synthase (T1PKS; 56,584–107,578 nt), and heterocyst glycolipid synthase-like polyketide synthase (hglE-KS; 75,419–113,566 nt). Strain GTP1^T possessed four gene clusters that encoded for RRE-containing cluster (175,155–199,102 nt), homoserine lactone (110,293–130,892 nt), a signaling molecule known for its involvement in bacterial quorum sensing, the RIPP-like cluster (38,002–48,856 nt), and terpene synthesis (47,942–69,701 nt). Strain HM2^T had two gene clusters that encoded for resorcinol (403,967–445,901 nt), an organic compound known for its antiseptic properties, and terpene (697,660–721,242 nt), which showed 100% similarity for carotenoid synthesis. BRIG analysis results showed that a majority of the regions within the four analyzed genomes were conserved with at least 70% similarity (Figure S4).

Cellulolytic potential and FE-SEM analysis of strain USB13^T

A USB13^T-inoculated basal salt medium sample containing degraded filter paper was examined under FE-SEM to observe the morphological interactions between cellulose fibers and USB13^T cells. Images in Fig. 5 show individual rod cells of strain USB13^T surrounding filter paper fibers, indicating bacterial adherence.

The enzymatic assay results showed endoglucanase, exoglucanase, β-glucosidase, and filter paper cellulase (FPCase) activities of strain USB13^T, wherein activities for endoglucanase was the highest and β-glucosidase was the lowest in all experiments. As seen in Fig. 6A, enzyme activity for all cellulolytic enzymes increased along with its cultivation time. In addition, enzyme activities showed the highest results when tested on buffer solutions of pH 6.0 (Fig. 6B), indicating the enzymes’ resistance to moderately acidic conditions. The pH of the buffer solution seemed to be an important factor in enzyme activity, as activity of endoglucanase, exoglucanase, and FPCase drastically decreased when the pH was altered from pH 6.0 to pH 7.0. Meanwhile, β-glucosidase activity was relatively resistant to pH change as its activity decreased less than 50%. On day 7, enzyme activities were measured as 1.91 IU/mL for endoglucanase, 1.77 IU/mL for exoglucanase, 0.76 IU/mL for β-glucosidase, and 1.12 IU/mL for FPCase at pH 6.0. When measured at pH 8.0, where enzyme activity was the lowest, enzyme activities were measured as 0.51 IU/mL for endoglucanase, 0.25 IU/mL for exoglucanase, 0.45 IU/mL for β-glucosidase, and 0.23 IU/mL for FPCase; all values were less than half of the measured activity at pH 6.0. The results of strain USB13^T are comparable to FPCase results of other species such as Mucilaginibacter polytrichastri RG4-7^T (0.98 U/mL) isolated from the moss Polytrichastrum formosum¹⁴, Paenibacillus lautus BHU3 (2.9 U/mL) isolated from a landfill site⁵⁷, and Serratia rubidaea DBT4 (0.5 U/mL) isolated from the gastrointestinal tract of a black Bengal goat⁵⁸.

Despite the absence of the main three cellulolytic enzymes, endoglucanase, exoglucanase, and β-glucosidase, the cellulolytic activity of strain USB13^T was confirmed through SEM images, CMC agar screening, and enzymatic assay results. However, because PGAP annotation results showed that other non-cellulolytic strains also possessed CAZymes, in some cases more than strain USB13^T, further research is necessary to understand the mechanics of how CAZymes and other cellulases interact to degrade cellulose, and how these genes are expressed under certain conditions. Furthermore, the cellulolytic activity of strain USB13^T can be further optimized for commercial use by adjusting growth conditions such as pH, temperature, and growth media.

While cellulolytic bacteria are known to inhabit animal intestinal tracts, the rumen, and soil, they can be found almost everywhere, such as ocean floors, municipal landfills, and even extreme environments such as hot springs⁵⁹. In these habitats, cellulolytic bacteria utilize cellulose while cohabiting with non-cellulolytic bacteria. There have been many studies suggesting the synergistic role non-cellulolytic bacteria play in cellulose degradation, where non-cellulolytic bacteria aid cellulose degradation by neutralizing pH or removing harmful metabolites^60,61,62.

Bacterial cellulases have shown immense value in various industries such as animal feed processing, food and brewery production, and agriculture, not to mention biofuel synthesis through biomass utilization¹¹. Due to the versatile uses of bacterial cellulases, the cellulolytic strain USB13^T has the potential to become an invaluable resource. However, further research of the novel strain’s cellulose-degradation mechanisms is necessary to develop and commercially make use of its bacterial cellulases in the future. In addition, research regarding co-culturing non-cellulolytic bacteria and strain USB13^T may also help in developing effective methods to use an otherwise underutilized bioresource.

Taxonomy of novel Ramlibacter species

While phylogenetic analyses indicated that the novel strains USB13^T, AW1^T, GTP1^T, and HM2^T should be assigned to the genus Ramlibacter, differences in fatty acid compositions, polar lipid profiles, and physiological characteristics suggested that the four novel strains are noticeably distinct from other validly published species of the genus. Additionally, genomic characteristics such as ANI, dDDH, and AAI values further supported the novel strains’ position as a distinct species within the genus Ramlibacter. Therefore, we propose that the strains USB13^T, AW1^T, GTP1^T, and HM2^T represent novel species within the genus Ramlibacter.

Description of the novel Ramlibacter species

The descriptions of the novel species are given according to the standards of the Judicial Commission of the International Committee on Systematic Bacteriology⁶³.

Description of Ramlibacter cellulosilyticus sp. nov

Ramlibacter cellulosilyticus (cel.lu.lo.si.ly’ti.cus. N.L. n. cellulosum, cellulose; N.L. adj. lyticus from Gr. lytikos, dissolving; N.L. masc. adj. cellulosilyticus, cellulose-dissolving).

Cells of strain USB13^T are Gram-negative, rod-shaped, non-flagellated and motile by gliding. The strain is positive for both oxidase and catalase activity, while cells have a width of 0.3–0.5 μm and length of 2.0–2.4 μm. When observed on R2A agar, colonies are reddish white, flat with entire margins, and have a diameter of 1–2 mm. Growth of strain USB13^T is observed at 7–50 °C (optimum, 28–30 °C), at pH 5.0–10.0 (optimum, pH 6.0), and at NaCl concentrations of 0–7% (optimum, 0–3%). The strain is unable to grow in anaerobic conditions. Produces siderophores and hydrolyzes Tween 20, Tween 80, CMC, and esculin. According to the API ZYM results, the strain showed positive results for alkaline phosphatase, esterase lipase (C8), leucine arylamidase, acid phosphatase, β-galactosidase, α-glucosidase, and β-glucosidase. In the API 20NE assay, strain USB13^T showed positive results only for β-galactosidase. The predominant respiratory quinone is ubiquinone 8 (Q-8). The major fatty acids are C_16:0, C_10:0 3-OH, and summed feature 3 (consisting of C_16:1 ω7c and/or C_16:1 ω6c). The polar lipid profile consists of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), one unidentified phosphoaminolipid, two unidentified phosphoglycoaminolipids, and six unidentified polar lipids. The G + C content is 69.7%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the assembled genome sequence of strain USB13^T are MN603953 and JACORT000000000, respectively.

The type strain USB13^T (= KACC 21656^T = NBRC 114839^T) was isolated from shallow coastal water at Haeundae Beach, Busan, Republic of Korea.

Description of Ramlibacter aurantiacus sp. nov

Ramlibacter aurantiacus (au.ran.ti’a.cus. L. masc. adj. aurantiacus, orange-colored, referring to the orange colonies of the strain).

Cells of strain AW1^T are Gram-negative, coccoid to short rod-shaped, non-flagellated, and motile by gliding. The strain is negative for oxidase activity, and positive for catalase activity. When observed on R2A agar, colonies are orange, convex, with entire margins, and 0.5–1.0 mm in diameter. Under TEM cells have and approximate width of 0.3–0.5 μm and length of 0.6–0.8 μm. Growth of strain AW1^T can be observed at 7–45 °C (optimum, 30 °C), at pH 7.0–10.0 (optimum, 7.0–8.0), and at NaCl concentrations of 0–3% (optimum, 0–1%). The strain does not grow under anaerobic conditions but is able to hydrolyze Tween 80. In addition, AW1^T is not able to produce siderophores. In the API ZYM assay, positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, and β-glucosidase. In the API 20NE assay, positive for esculin hydrolysis. The predominant respiratory quinone is ubiquinone 8 (Q-8). The major fatty acids are C_16:0, C_17:0 cyclo, summed feature 3 (consisting of C_16:1 ω7c and/or C_16:1 ω6c), and summed feature 8 (consisting of C_18:1 ω7c and/or C_18:1 ω6c). The polar lipid profile consists of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), one unidentified phosphoglycolipid, one unidentified lipid, and three unidentified glycolipids. The G + C content is 68.6%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the assembled genome sequence of strain AW1^T are MN498045 and JAEQNA000000000, respectively.

The type strain AW1^T (= KACC 21544^T = NBRC 114862^T) was isolated from soil at Aewol, Jeju Island, Republic of Korea.

Description of Ramlibacter albus sp. nov

Ramlibacter albus (al’bus. L. masc. adj. albus, white, referring to the white colonies of the strain).

Strain GTP1^T is non-motile, Gram-negative, strictly aerobic, positive for oxidase activity, and negative for catalase activity. When observed on R2A, colonies are white, convex, with entire margins, and 1–2 mm in diameter. Under TEM, cells lack flagella, are rod-shaped, and have a width of 0.7–0.8 μm and length of 1.6–1.9 μm. Growth of strain GTP1^T can be observed at 10–45 °C (optimum, 30 °C), at pH 5.0–8.0 (optimum, pH 7.0), and at NaCl concentrations of 0–2% (optimum, 0%). The strain shows positive results for Tween 20 and Tween 80 hydrolysis. GTP1^T does not produce siderophores when tested on CAS-blue agar. According to API ZYM results, strain GTP1^T is positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), and leucine arylamidase, while the API 20NE assay results show negative results for all substrates. The predominant respiratory quinone is ubiquinone 8 (Q-8). The major fatty acids are C_16:0 and summed feature 3 (consisting of C_16:1 ω7c and/or C_16:1 ω6c). The polar lipid profile consists of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and two unidentified phosphoaminolipids. The predominant respiratory quinone is ubiquinone 8 (Q-8). The major fatty acids are C_16:0, C_17:0 cyclo, summed feature 3 (consisting of C_16:1 ω7c and/or C_16:1 ω6c), and summed feature 8 (consisting of C_18:1 ω7c and/or C_18:1 ω6c). The polar lipid profile consists of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), one unidentified phosphoaminolipid, one unidentified phosphoglycolipid, one unidentified phosphoglycoaminolipid, and two unidentified polar lipids. The G + C content is 67.9%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the assembled genome sequence of strain GTP1^T are MN498046 and JACORU000000000, respectively.

The type strain GTP1^T (= KACC 21702^T = NBRC 114488^T) was isolated from soil at Seogwipo, Jeju Island, Republic of Korea.

Description of Ramlibacter pallidus sp. nov

Ramlibacter pallidus (pal’li.dus. L. masc. adj. pallidus, pale, referring to the color of the colonies).

Cells of strain HM2^T are Gram-negative, and positive for both oxidase and catalase activities. When observed on R2A agar, colonies are cream-colored, transparent, 1.0–2.5 mm in diameter, and flat with entire margins. Under TEM, monotrichous flagella are observed, and cells are rod-shaped with a width of 0.4–0.78 μm and length of 1.7–1.8 μm. The strain shows the fastest growth at a temperature range of 25–35 °C and at pH values between 8.0 and 9.0. When NaCl is present, growth is observed at concentrations of 0–3% (w/v), with optimal growth was observed at concentrations of 0–1% (w/v). The strain is not able to tolerate anaerobic conditions. Strain HM2^T hydrolyzes Tween 80 and weakly hydrolyzes casein. However, siderophore production cannot be observed when tested on CAS-blue agar. According to API ZYM tests, strain HM2^T shows positive results for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase. In addition, API 20NE tests show positive results for nitrate (NO₃) to nitrite (NO₂^–) reduction and esculin hydrolysis. The G + C content is 69.9%. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the assembled genome sequence of strain HM2^T are MN498047 and JADDIV000000000, respectively.

The type strain HM2^T (= KCTC 82557^T = NBRC 114489^T) was isolated from soil at Seopjikoji, Jeju Island, Republic of Korea.

Source: Ecology - nature.com