Phylogenetic and phylogenomic analyses
Strains TMQ4T and TMQ2 yielded genomes of 5,609,735 bp and 5,589,985 bp in length, separately. The calculated G + C mol% of the two strains were 64.4 mol% and 65.0 mol% as calculated from the draft genome sequences, which were similar with two related strains (Table 1). There were 184 and 148 contigs in the genomes of strains TMQ4T and TMQ2, respectively. All contigs in two genomes were larger than 500 bp, of which the largest were of 917,808 bp and 568,134 bp. Genome of strain TMQ2 encoded 4,316 genes, including 53 tRNAs and 16 rRNAs, and 4,229 genes accompanied with 53 tRNAs and 12 rRNAs were encoded by strain TMQ4T. N50 values were 568,134 and 97,137, and the sequencing depths of coverage were 346 × and 120 × for the type strain and another strain, respectively. The genomic information of two strains were both listed in Table 1. It seemed that most culturable members in Bradymonadales yielded similar size of genomes except Persicimonas caeni YN101T (Wang et al. 2020), which is more closely related to Bradymonas sediminis FA350T (Table 1). Similar number of genes, tRNA and G + C mol% were also found in these predatory genomes belonged to different genera (Table 1).
Nearly complete 16S rRNA gene sequences of strains TMQ4T (1,501 bp) and TMQ2 (1,500 bp) obtained by amplification were included in the 16S rRNA gene sequences assembled from genomic sequences (1,524 bp and 1,536 bp, separately). Only one complete 16S rDNA were identified, even if there were three pieces in the genome of strain TMQ4T, and all of four 16S rDNA in the genome of strain TMQ2 were found to be partial. The 16S rRNA gene sequence annotated from genome sequences was submitted to GenBank databases, and similar sequences were searched using BLAST algorithm. The EzTaxon-e server (http://eztaxone.ezbiocloud.net/) (Kim et al. 2012) was used to achieve the similarity values of sequences. Based on the 16S rRNA gene sequence (from genome sequences), the strain closely related to Lujinxingia sediminis SEH01T with the highest similarity values of 98.9 %, following Lujinxingia litoralis B210T with the similarity values of 98.1 %. Additionally, the similarity value between strains TMQ4T and TMQ2 was 99.8 %.
Strains TMQ4T and TMQ2 had the OrthoANIu value of 87.7 %, and Lujinxingia sediminis SEH01T had the OrthoANIu value of 85.7 % with strain TMQ4T and 87.0 % with strain TMQ2. Moreover, the ANIb and ANIm values between each pair of strains were all below 90.0 %. However, the Tetra value between strains TMQ4T and TMQ2 was 0.998, higher than the threshold of 0.990, which meant the strain TMQ4T and TMQ2 belonged to the same species (Richter and Rosselló-Móra 2009). The dDDH values between strain TMQ4T and the two members in Lujinxingia were both under the threshold of 70 %, illustrating each of the pair formed deep lineages (Li et al. 2010). Due to an ANI value of 95.0 % and a Tetra value of 0.990, as well as a dDDH value of 70 %, have been proposed as species boundaries, our data indicates that strain TMQ4T (TMQ2) represents a novel species distinct from L. sediminis SEH01T (Richter and Rosselló-Móra 2009).
As the topology shown in Fig. 1, two strains were clustered with L. sediminis SEH01T at the bootstrap confidence level of 92 % and 99 % (Fig. 1). Furthermore, the same taxonomic status with phylogenetic trees was also demonstrated by two phylogenomic trees (Fig. 2).
Genomic analyses
According to the genetic analyses using RAST (http://rast.nmpdr.org) and NCBI genome database (https://www.ncbi.nlm.nih.gov/genome), strains TMQ4T and TMQ2 lost biosynthesis pathway of several amino acids, including glycine, serine, threonine, valine, leucine, isoleucine and lysine. Thiamine and vitamin B6 biosynthesis, as well as biotin metabolism were absent in the two genomes. All above indicated that uptakes of these substrates maybe occurred during their predations. As the genetic annotations using antiSMASH (https://antismash.secondarymetabolites.org), lassopeptide (berninamycin) and NRPS-like (VEPE/AEPE/TG-1/crocaginA/crocaginB) gene clusters were encoded by the genome (Medema et al. 2011; Wang et al. 2019). Predatory index of two strains were 2 (higher than 0), which indicated these two strains were hypothetic bacterial predators. Predatory index of “2” was common among culturable bradymonabacteria (Supplementary Table S2). Actually, all of the genomic features described above could also be found in the other culturable members of Bradymonadales (Mu et al. 2020). Furthermore, as one qualitative and predictive parament, predatory index could not be criteria to evaluate predation of bacterial predators. It was also not relevant to the taxonomic status, actually, it reflected the genomic features affiliated with predation more.
Morphological, physiological and biochemical analysis
No visible colonies but transparent lawn formed for both strains on modified MA after incubation at 37 oC for 144 h. Cells of two strains were both Gram-stain-negative rods without flagella, 0.3–0.5 × 1.0–4.5 µm for strain TMQ4T and 0.3–0.5 × 1.0–3.5 µm for strain TMQ2 in size (Fig. S3, available with the online Supplementary Material). Cells were motile through gliding for they could spread into a big circle culture from a spot inoculation with modified MB with 0.3 % agar.
The optimal NaCl concentration was defined as the one under which the lawn in the first arear on agar medium grew taking least time. No growth happened in salt-free modified MA. Optimal growth of strains TMQ4T and TMQ2 was both observed with 3–4 % (w/v) NaCl (range, 1–8 % [w/v]), at 37°C (range, 25–45°C) and pH 7.5–8.0 (range, pH 6.5–8.0). The physiological characteristics of two strains and phylogenetic neighbours were list in Table 2.
For two strains, nitrate but not nitrite, thiosulfate or sulfate could be used as electron acceptors when in anaerobic growth, and nitrate reductions were positive. Strains TMQ4T and TMQ2 were positive for catalase, DNase and starch, Tweens 20, 40 and 60 hydrolases, but negative for oxidase and alginate hydrolase. Additionally, strain TMQ4T was also positive for Tween 80 hydrolase.
Consequently, cells of strains TMQ4T and TMQ2 were both sensitive to lincomycin (2 µg), norfloxacin (10 µg), neomycin (30 µg), clindamycin (30 µg), kanamycin (30 µg), tetracycline (30 µg), ceftriaxone (30 µg) and erythromycin (15 µg), and both resisted to penicillin (10 µg), vancomycin (30 µg) and tobramycin (10 µg).
Both strains showed positive activities for the reactions of alkaline phosphatase, esterase lipase (C8), leucine arylamidase, cystine arylamidase and naphthol-AS-BI-phosphohydrolase in API ZYM kits (Supplementary Table S3). Additionally, strain TMQ4T was positive for esterase (C4), which was different from other bradymonabacteria. Moreover, negative for acid phosphatase distinguished strain TMQ4T from strain TMQ2 and two members in Lujinxingia. According to the API 20E tests, both two strains and L. sediminis SEH01T produced tryptophane deaminase and gelationase. However, acetoin production occurred in L. litoralis B210T rather than other bradymonabacteria. The results of API 50CHB indicated that acids could be produced by the both two strains with d-ribose, esculin, d-tagatose and potassium 5-ketogluconate. However, acids were not produced by strain TMQ4T with l-sorbose but by strain TMQ2 and the two members in Lujinxingia. According to Biolog GEN III kits, strains TMQ2 and TMQ4T oxidized l-fucose, d-fructose-6-PO4, l-glutamic acid, glucuronamide, mucic acid and α-keto-glutaric acid. However, oxidations of d-arabitol, N-acetyl-d-galactosamine and d-glucose-6-PO4 occurred in strain TMQ2, and d-raffinose, d-lactose, d-salicin, N-acetyl-d-galactosamine oxidized by strain TMQ4T. Strains TMQ4T, SEH01T and B210T but not strain TMQ2 oxidized the substrate of acetoacetic acid. Phenotypic characteristics that differentiate two strains from the closest phylogenetic neighbours are given in Table 2.
Chemotaxonomic analyses
Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) were detected in strains TMQ4T and TMQ2, as well as two members in Lujinxingia as the major polar lipids. These three lipids were also main components in other culturable members in Bradymonadales. Moreover, unidentified lipids existed as the moderate and or minor components in strain TMQ4T (L1, L2, L3 and L4) and strain TMQ2 (L1, L2, L4 and L5). What made strains TMQ4T and TMQ2 differed from the two species of the genus Lujinxingia was the different contents of unidentified lipids (the absence of L2, L3 and L4 in strain SEH01T and the presence of L7 and L8 in strain B210T). Therefore, the polar lipid profile supports that the two strains represented one novel species of the genus Lujinxingia. Further details of contents of polar lipids of strain TMQ4T from L. litoralis B210T and L. sediminis SEH01T were shown in Fig. S4 (available with the online Supplementary Material).
The sole quinone of strains TMQ4T and TMQ2 were menaquinone MK-7, which was consistent with all culturable bradymonabacteria.
High content of iso-C15:0 was detected in strains TMQ4T (75.3 %) and TMQ2 (71.5 %) in conjunction with the members in the genus Lujinxingia. Iso-C17:0 contained by strain TMQ4T (11.9 %) tested as one major component in two of the three repeats (one in three repeats for strain TMQ2), which was higher than that in two members of the genus Lujinxingia. Four minor components, C16:0, C18:0, iso-C16:0 and C18:1 ω9c, existed in all four strains. Moreover, sum in feature 3 detected in strain SEH01T was absent in strain TMQ4T and strain B210T. All the variances referred suggested that strain TMQ4T (accompanied with strain TMQ2) presented a novel species in the genus Lujinxingia, and the details of the discrepancy between strains TMQ4T and TMQ2, as well as the related strains were listed in Table S4 (available with the online Supplementary Material).
Predatory assays
Preys of strain TMQ4T distributed in different category of bacterial species isolated from marine and saltern environments, which included Gram-stain-negative bacteria and Gram-stain-positive bacteria (data not shown). Paraliobacillus ryukyuensis DSM 15140T and Brumimicrobium aurantiaca N62T were further analysed as representative strains in this study. After cross-streaking incubation of predators and the prey, plates were incubated until the growth of both bacteria was indicated by visible lawn. Predatory bacteria could be detected by 16S rRNA gene sequencing in the second line, if the test strain was not prey (Fig. S2E). However, when the predator fed on the test bacteria, such as P. ryukyuensis DSM 15140T, strain TMQ4T could be detected in the second line (Fig. S2B, C and D). Fig. S2D and E indicated that the growth of predators increased when provided with appropriate prey strains. For most culturable bradymonabacteria, the similar predation pattern could be observed. Results of cross-streaking tests incubating strain TMQ4T and the test bacteria are also shown in Fig. S2.