Bradymonabacteria are efficient predators of diverse prey bacteria
In total, 9 strains of bacteria in the novel order Bradymonadales were isolated using the enrichment culture method . Among these strains, eight strains were isolated from costal sediment sampled in Weihai, China, while strain YN101 was isolated from a Gaodao saltern (36°54’N, 122°14’E) in Weihai, China. Strains FA350T [17, 18] and B210T  are the two type strains for different genera of Bradymonadales. Both these type strains were used to investigate the predator-prey range of Bradymonabacteria. A total of 281 isolated bacteria were cocultured with Bradymonabacteria FA350T [17, 18] or B210T  as lawns in individual Petri dishes (Fig. 1a, Table S1). Zones of predation were measured (Fig. 1b), and the results showed that the Bradymonabacteria preyed on diverse bacteria but showed a strong preference for Bacteroidetes (90% of tested bacteria could be preyed on) and Proteobacteria (71% of tested bacteria could be preyed on) (Fig. 1c). Predation on bacteria in the orders Flavobacteriales, Caulobacterales, Propionibacteriales, and Pseudomonadales was broadly distributed, with a mean predation percentage greater than 90%, while predation of Micrococcales and Enterobacteriales was less efficient.
Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses were performed to understand the mechanism of predation of strain FA350T on the subcellular level. Lysis of the prey cells was detected near strain FA350T in both the TEM and SEM analyses (Fig. 2). Strain FA350T was found to have pili (Figs. 2b and 2g) and outer membrane vesicle (OMV)-like structures (Figs. 2d, 2e, 2f, and 2h). In addition, FA350T cells contained intracellular particles with low electron density (Figs. 2b, 2c, 2d, and 2f), which were shown to contain polyhydroxyalkanoates (PHAs) by Nile blue A staining. FA350T cells also contained several electron-dense (black) spots (Figs. 2b, 2c, 2d, and 2f), which indicated the presence of intracellular polyphosphate granules . Both of these particle types significantly accumulated during predation (Fig. 2).
Bradymonabacteria are polyauxotrophs
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 ). The 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  (Fig. S1b). Other general features of the genomes are described in the 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) . Most of the bradymonabacterial 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; they were missing the phosphoribosylaminoimidazole carboxylase catalytic subunit or even the whole pathway.
In addition to this auxotrophy in the synthesis of pentose and nucleotides, 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 a potential D-3-phosphoglycerate dehydrogenase for the conversion of glycerate-3P into 3-phosphonooxypyruvate for amino-acid synthesis (Fig. 3). However, in all members of Bradymonabacteria, this pathway appeared to be blocked at the subsequent step because of the absence of phosphoserine aminotransferase, although Bradymonabacteria could continue with subsequent pathways to complete the biosynthesis of cysteine and glycine. Additionally, many cofactors and vitamins that promote bacterial growth , such as biotin, thiamin, ubiquinone, VB12, and VB6, could 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 the potential predation mechanism of Bradymonabacteria
To further determine the genes involved in predation, we performed dual-transcriptome analysis of Bradymonas sediminis FA350T with and without preying on Algoriphagus marinus am2 (Fig. S2). As with obligate predators, one way that Bradymonabacteria kill their prey bacteria is likely by using contact-dependent mechanisms. Here, the bradymonabacterial genomes possessed complete Type IV pili (T4P) (Fig. 3), and the attached areas showed more type IV pili than the unattached areas (SEM, Figs. 2g and 2h). The dual-transcriptome analysis showed that genes encoding the type IV pili twitching motility protein PilT (DN745_17255) were significantly upregulated during predation (Fig. S3), suggesting that these genes may be involved in predation. Bradymonabacteria also had T4b pilins showing homology to those in Bdellovibrio bacteriovorus HD100, in which T4b pilins are necessary for predation [28, 29] (Fig. S4), so T4b pilins may also participate in regulating predation in Bradymonabacteria. In addition, this group of bacteria had type II and type III secretion systems (the YscRSTUV proteins that form a membrane-embedded complex known as the ‘‘export apparatus’’ ). The dual-transcriptome analysis also supported the prediction that genes encoding the type III secretion system inner-membrane protein complex (DN745_01900, DN745_10315, DN745_17280, DN745_03325, and DN745_00480) were significantly upregulated during predation (Fig. S3), implying that these genes may also be involved in predation.
Another way that Bradymonabacteria kill their prey bacteria is likely by secreting antimicrobial substances into the surrounding environment. As in most facultative bacterial predators, a few potential antimicrobial clusters for secondary metabolite synthesis, such as Lasso-peptide , were identified in almost all genomes of Bradymonabacteria (Fig. 3). Genes involved in outer membrane vesicles (OMV) biosynthesis were also detected in most genomes, such as ompA (cell envelope biogenesis protein), envC (Murein hydrolase activator) and tolR (envelope stability) . Vesicle membrane-related genes (DN745_03865, DN745_02930, and DN745_07125) were significantly upregulated during predation (Table S4, Fig. S3). However, the fermentation supernatant of Bradymonabacteria showed no antibacterial activity.
Bradymonabacteria are novel predators different from the so-called obligate or facultative predators
Comparative genomic analysis with other bacterial predators was performed to explore whether Bradymonabacteria have a unique living strategy. Two-way cluster analysis showed that bradymonabacterial genomes contained features different from those of either obligate or facultative predators, which were phylogenetically located in a different branch (Fig. 4). The specific multiple metabolic deficiencies of Bradymonabacteria had some similarities to those of most obligate predators. For example, both Bradymonabacteria and obligate predators possessed a 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 auxotrophies could grow on common media (such as marine agar medium), though at a low growth rate , unlike 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 , was present in most Bradymonabacteria (Fig. 4). Polyphosphate accumulation was also detected in FA350T cells during predation (Fig. 2). In contrast to most of the other predator genomes, potential PHA synthesis from β-oxidation of fatty acids  was observed in most bradymonabacterial genomes (Fig. 3). In the present study, TEM analysis showed that strain FA350T could significantly accumulate PHAs during predation observed in co-culture with pure culture (Fig. 2). Despite their incomplete fatty acid biosynthetic pathway, all Bradymonabacteria had a high copy number of long-chain fatty acid transporters (fadL) compared to those of other predators, allowing them to gather fatty acids from the environment (Fig. 4). In addition, genes associated with alkane synthesis, which is important for maintaining cell membrane integrity and adapting to cold environments , were present in most genomes of Bradymonabacteria (Figs. 3 and 4). Thus, we proposed that Bradymonabacteria could be categorized as novel predators different from so-called obligate or facultative predators (Table 1).
Bradymonadales are mainly distributed in saline environments with high diversity
To evaluate the global prevalence of the Bradymonadales order, we surveyed recently published 16S rRNA gene amplicon studies that provided high taxonomic resolution along with relative sequence abundances. The 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 inland environments, while others were from marine environments, with each biotope showing a somewhat different microbial community (Fig. 5b). Based on the alpha diversity analysis, marine sediment and soil biotopes harbored more OTUs compared with other biotopes (Fig. 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 nonsaline biotopes (Fig. S6), suggesting that salinity 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 that in the nonsaline environments (i.e., nonsaline soil and nonsaline water) (P<=0.0001, Fig. 5c). The distribution analysis was consistent with the genomic feature analysis (Fig. 2), in which several genes encoding sodium symporters and Na+/H+ antiporters were found in the genomes, suggesting a beneficial effect of salinity on Bradymonabacteria.
In addition, we compared the relative abundance of Bradymonadales with those of 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 commonly distributed in soil and sediment environments, while Bdellovibrionales were more likely to be found in freshwater and seawater (Fig. S7). The total relative abundances of Bradymonadales, Bdellovibrionales, and Myxococcales ranged from 0.7% to 6.4% of the total prokaryotic microbes in all 1552 samples (Fig. S8a). The mean relative abundance of Bradymonadales (0.5%) was similar to that of Bdellovibrionales (0.6%) 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 the Gaodao multipond salterns as a model and applied 16S rRNA gene amplicon, fluorescence in situ hybridization (FISH), and real-time PCR analyses (Figs. S8d and S9). The results showed that Bradymonadales appeared in all the tested multipond 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), significantly higher than those of Bdellovibrionales and Myxococcales. Bradymonadales may exhibit different correlations with prey at different abundances, a possibility that will require further study. In addition, fluorescence in situ hybridization (FISH) and real-time PCR experiments were performed, and the results showed a relative cell abundance of Bradymonadales of up to 0.6% and a gene copy numbers ratio as high as 1.96% in sediments with salinity 80 g/L (Fig. S9). These findings support those of the global analysis (Fig. S8c) and suggest that Bradymonadales may be a dominant bacterial predator in some specific saline environments.
To explore the diversity and distinct evolution of bradymonabacterial subgroups in different biotopes, we performed a phylogenetic analysis of nearly full-length 16S rRNA gene sequences of diverse origin by maximum likelihood inference (Table S6). A total of 187 OTUs were detected and found to form six sequence clusters (Fig. 6a). Almost 87.2% of the representative sequences originated from saline biotopes (such as seawater, marine sediments, salterns, corals, and saline lakes). 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% of total 16S rRNA gene reads (Fig. 6b). Five of the 6 bradymonabacterial subgroups showed significantly higher abundance 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 environments of the cultured strains. Cluster-1 and cluster-4 were both detected in marine sediment and seawater biotopes. Cluster-5 lineages tended to occur in both freshwater and seawater biotopes (Fig. 6b).