Bradymonabacteria are efficient predators of diverse prey bacteria
Totally, 9 strains of bacteria with in the novel order Bradymonadales have been isolated by using enrichment culture method [22]. Among these strains, strain FA350T [17, 18] and B210T [23] were the two type strains within different genera in Bradymonadales. And both type strains were used to investigate the predator-prey range of Bradymonabacteria. A total of 281 isolated bacteria were co-cultured with Bradymonabacteria FA350T [17, 18] or B210T [23] as lawns on Petri dish, respectively (Fig. 1a, Table S1). Zones of predation were measured (Fig. 1b), and results showed Bradymonabacteria preyed on diverse bacteria but a high preference on Bacteroidetes (90% of tested bacteria could be preyed) and Proteobacteria (71% of tested bacteria could be preyed) (Fig. 1c). Predation on bacteria in the orders Flavobacteriales, Caulobacterales, Propionibacteriales, and Pseudomonadales were broadly distributed, with the mean predation percentage greater than 90%, while, predation of Micrococcales and Enterobacteriales were less efficient.
Transmission electronic microscopy (TEM) and scanning electronic microscopy (SEM) analyses were done to understand the mechanism of predation of strain FA350T on subcellular level. Lysis of the prey cells can be detected nearby strain FA350T in both TEM and SEM analyses (Fig. 2). Strain FA350T was detected to have pili (Figs. 2b and 2 g) and the outer membrane vesicles (OMVs) like structures (Figs. 2d, 2e, 2f, and 2 h). In addition, FA350T cells contained intracellular particles with low electron-dense (Figs. 2b, 2c, 2d, and 2f), which was polyhydroxyalkanoates (PHAs) tested by Nile blue A staining. Meanwhile, FA350T cells also contained several electron-dense intracellular granules (black granules) (Fig. 2b, 2c, 2d, and 2f), which indicated the presence of intracellular polyphosphate granules [24]. Both these particles were significantly accumulated during predation (Fig. 2).
Bradymonabacteria are multiple auxotrophs
To explore the metabolic capabilities and predation mechanism of this novel group, we analyzed 13 genomes of Bradymonadales (9 high-quality genomes sequenced from cultured strains and 4 reconstructed from published studies [25]). Genome size of Bradymonabacteria ranged from 5.0 Mb to 8.0 Mb. Average Nucleotide Identity (ANI) analysis of the 9 cultured strains of Bradymonadales revealed 7 different species [26] (Fig. S1b). Other General features of genomes were described in Supplementary Materials (Supplementary Materials Results and Fig. S1a).
Almost all strains (except FA350T) possessed a minimal Pentose Phosphate Pathway, which lacked key steps for the synthesis of ribose 5-phosphate (Fig. 3, Table S2) [27]. Most of the Bradymonabacteria genomes lacked key enzymes for pyrimidine synthesis, such as aspartate carbamoyltransferase, which catalyzes the first step in the pyrimidine biosynthetic pathway. All genomes lacked the complete purine de novo pathway, which was missing the phosphoribosylaminoimidazole carboxylase catalytic subunit or even completely missing the whole pathway.
Beside the auxotrophs in synthesis of pentose and nucleotide, all the genomes lacked complete pathways for the synthesis of many amino acids, such as serine, methionine, valine, leucine, isoleucine, histidine, tryptophan, tyrosine, and phenylalanine (Fig. 3). For example, all the genomes encoded potential D-3-phosphoglycerate dehydrogenase for the conversion of glycerate-3P into 3-phosphonooxypyruvate for amino-acid synthesis (Fig. 3). However, this pathway appeared to be blocked at the subsequent step because of the absence of phosphoserine aminotransferase in all members of Bradymonabacteria, despite that Bradymonabacteria can continue the subsequent pathways to complete the biosynthesis of cysteine and glycine. Additionally, many cofactors and vitamins that promote the growth of bacteria [22], such as biotin, thiamin, ubiquinone, VB12, and VB6, can not be synthesized by the de novo pathway in almost all the genomes. Notably, all the genomes had an incomplete pathway for type II fatty acid biosynthesis, lacking the key enzymes 3-oxoacyl-[acyl-carrier-protein] synthase I/II (FabB/F) and Enoyl-[acyl-carrier-protein] reductase (FabI/L).
Dual-transcriptome analysis of potential predation mechanism of Bradymonabacteria
To further determine the genes involved in predation, we did dual-transcriptome analysis which Bradymonas sediminis FA350T with/without preying on Algoriphagus marinus am2 (Fig. S2). Like obligate predators, one way that Bradymonabacteria killed their prey bacteria was likely by using contact-dependent mechanisms. Bradymonabacterial genomes possessed complete Type IV pili (T4P) (Fig. 3), and the attached areas showed more type IV pili than the non-attached areas (SEM, Fig. 2g and 2 h). Dual-transcriptome analysis showed that genes encoding type IV pili twitching motility protein PilT (DN745_17255) were significantly up-regulated during predation (Fig. S3), suggesting these genes may be involved in predation. Bradymonabacteria also had T4b pilins relative homology with those in Bdellovibrio bacteriovorus HD100, in which T4b pilins were necessary for predation [28, 29] (Fig. S4), so T4b pilins may participate in regulating predation. In addition, the group of bacteria had type II and type III secretion systems (The YscRSTUV proteins form a membrane-embedded complex known as ‘‘export apparatus’’ [30]). Dual-transcriptome analysis also supported the prediction that genes encoding type III secretion system inner-membrane protein complex (DN745_01900, DN745_10315, DN745_17280, DN745_03325, and DN745_00480) were significantly up-regulated during predation (Fig. S3), implying these genes may be involved in predation.
Another way that Bradymonabacteria killed their prey bacteria was likely by using secreting antimicrobial substances into the surrounding environment. Similar with most facultative bacterial predators, there were a few potential antimicrobial clusters of secondary metabolites synthesis, such as Lassopeptide [31], in almost all genomes of Bradymonabacteria (Fig. 3). Genes involved in OMVs-like biosynthesis were also detected in most genomes, such as ompA (cell envelope biogenesis protein), envC (Murein hydrolase activator) and tolR (envelope stability) [32]. It was detected that vesicle membrane related genes (DN745_03865, DN745_02930, and DN745_07125) were significantly up-regulated during predation (Table S4, Fig. S3).
Bradymonabacteria are novel predators different from obligate or facultative predators
Comparative genomic analysis with other bacterial predators was done to explore whether Bradymonabacteria had unique living strategy. Two-way cluster analysis showed that Bradymonabacterial genomes features were different from either obligate or facultative predators, which phylogenetically located in a different branch (Fig. 4). The specific multiple metabolic deficiencies of Bradymonabacteria had some similarities with most obligate predators. For example, both of Bradymonabacteria and obligate predators possessed minimal Pentose Phosphate Pathway, lacked key enzymes for pyrimidine synthesis, and lacked complete pathways for the synthesis of many amino acids, cofactors, and vitamins (Fig. 4). However, Bradymonabacteria with multiple auxotrophs could grow on common media (such as marine agar medium) though at a low growth rate [33], which was different from obligate predators.
Unlike most obligate predators, the polyphosphate accumulation pathway, containing a pair of genes (Polyphosphate kinase and Exopolyphosphatase) associated with both polyphosphate formation and degradation [34], was present in most Bradymonabacteria (Fig. 4). Polyphosphate accumulation was also detected in FA350T cells during predation (Fig. 2). Different from most of the other predators, potential polyhydroxyalkanoates (PHAs) synthesis from 𝛃-Oxidation of fatty acids [35] were observed in most Bradymonabacterial genomes (Fig. 3). In this study, TEM analysis shows strain FA350T could significantly accumulate PHAs during predation compared with pure culture (Fig. 2). In spite of an incomplete pathway for fatty acid biosynthesis, all the Bradymonabacteria had a high copy number of long-chain fatty acid transporters (fadL) compared to other predators to gain the fatty acids from environments (Fig. 4). In addition, genes associated with alkane synthesis, which was important for maintaining cell membrane integrity and adapt to cold environment [36], were present in most genomes of Bradymonabacteria (Figs. 3 and 4). As a result, we proposed that Bradymonabacteria could be categorized into novel predators different from the so-called obligate or facultative predators (Table 1).
Table 1
The features of 3 different types of bacterial predators
Current predators type | Redefine predators type | Metabolic pathways deficiencies | Pure-cultivable | Storing nutrients as polymers | Predation strategy | Predation specificity |
Obligate | Highly prey-dependent | High | Extremely Difficult | None | Contact-dependent | Gram-negative |
Bradymonabacteria | Facultative prey-dependent | High | Difficult | Polyhydroxyalkanoates, polyphosphate, and alkane | Contact-dependent | Gram-negative and Gram-positive |
Facultative | Prey-independent | Low | Normal | Polyphosphate* | Mostly contact-independent | Gram-negative and Gram-positive |
* Polyphosphate accumulation pathway was fund in genomes, but not determined by experiments. |
Bradymonadales are mainly distributed in saline environments with a high diversity
To evaluate the global prevalence of the Bradymonadales order, we surveyed recently published 16S rRNA gene amplicon studies that provided a fine taxonomic resolution along with relative sequence abundances. 16S rRNA gene amplicons from 1552 samples were grouped into eight types of environments (Fig. 5a and Table S5). A total of 811 samples were from an inland environment, while others were from the marine environment, with each biotope showing a relatively different microbial community (Figs. 5b and S5). Bradymonabacteria was detected in 348 of 741 marine samples (relative abundance > 0.01%), but only 20 of 544 soil samples (Fig. 5a). All samples were sorted into an ordination diagram based on the similarity of communities (Fig. 5b). Saline biotopes were clearly separated from non-saline ones (Fig. S6), suggesting that saline was a significant factor in shaping microbial communities. For each biotope, the relative abundance of Bradymonadales in the saline environments (i.e. seawater and saline lake sediment) was significantly higher than in the non-saline environment (i.e. no-saline soil and non-saline water) (P < = 0.0001, Fig. 5c). The distribution analysis was consistent with the genomic feature analysis (Fig. 2), which several genes encoding sodium symporters and Na+/H+ antiporters were found in genomes, and suggesting a beneficial effect of salinity on Bradymonobacteria.
In addition, we compared the relative abundance of Bradymonadales with another two orders of well-known predatory bacteria, Bdellovibrionales and Myxococcales [12, 37, 38]. We found that Myxococcales and Bdellovibrionales were also globally distributed (Fig. S7); however, Myxococcales were more likely distributed in soil and sediment environments, while Bdellovibrionales were more likely distributed in freshwater and seawater (Fig. S7). The total relative abundances of Bradymonadales, Bdellovibrionales, and Myxococcales ranged from 0.7–6.4% of total prokaryotic microbes in all 1552 samples (Fig. S8a). The mean relative abundance of Bradymonadales (0.51%) was similar to Bdellovibrionales (0.62%) when both were detected in environmental samples (Fig. S8b). In contrast, Bradymonadales was one of the most abundant known predatory bacteria in saline lake sediment and saline lake water (Fig. S8c).
To further determine how salinity affected the relative abundance of Bradymonadales, we used Gaodao multi-pond salterns as a model combined with 16S rRNA gene amplicons, fluorescence in situ hybridization (FISH), and real-time PCR analyses (Figs. S8d and S9). The results showed that Bradymonadales appeared in all tested multi-pond saltern datasets, accounting for an average of 0.74% of all bacterial sequences and more than 1.0% relative abundance within the range of 80 g/L and 265 g/L salinity (Fig. S8d), which was significantly higher than Bdellovibrionales and Myxococcales. The detailed descriptions of effects on the abundance of Bradymonadales were in supplementary materials (Supplementary Materials: Results, Figs. S8d and S9).
To explore the diversity and distinct evolutionary of Bradymonabacteria subgroups in different biotopes, we performed a phylogenetic analysis of nearly full-length 16S rRNA gene sequences of diverse origin by maximum likelihood inferences (Table S6). A total of 187 OTUs were detected and formed six sequence clusters (Fig. 6a). Almost 87.2% representative sequences originated from saline biotopes (such as seawater, marine sediments, salterns, corals, and saline lake). Since Bradymonabacterial subgroups may be selectively distributed in local biotopes, we investigated the relative abundance of each subgroup throughout the 127 representative samples, in which the relative abundance of Bradymonadales was above 1% (Fig. 6b). Five of 6 Bradymonabacterial subgroups showed significantly high abundance patterns in saline environments. Cluster-2 and cluster-6 were mainly observed in seawater biotopes, whereas cluster-3 was mainly observed in marine sediment and saline lake sediment (Fig. 6b), consistent with the environment of the cultured strains. The cluster-5 lineages tended to occur in both freshwater and seawater (Fig. 6b).