| Since Bradymonadales was first proposed in 2015,three species(two genera)were validly published,including Bradymonas sediminis FA350T Lujinxingia litoralis B210T,and Lujinxingia sediminis SEH01T.Based on qualitative results of phenotypic tests,members of Bradymonadales could predate a variety of bacteria.In our view,the term predator should be used for those bacteria that hunt actively to kill their prey and consume their macromolecules as nutrients.Accordingly,in this review we will apply the term predator to any bacteria that obtain energy and biosynthetic materials after killing other living cells.Bacterial predation has now been proposed as an evolutionary driving force.The structure and diversity of the predatory bacterial community is beginning to be recognized as an important factor in biodiversity due to its potential role in controlling and modelling bacterial populations in the environment.Physiological and biochemical characteristics,predation cycle and predation characteristics,and metabolic characteristics of the classical predatory bacteria have been comprehensively reported.According to the dependence on prey,predatory bacteria can be divided into obligate predatory groups and facultative predator groups.The former depends highly on prey and the latter not.In the basics of studies in representative bacterial predators,man can clearly know the mechanism of predation bacteria at the level of cell population,single cell and intracellular structure.According to the interaction between predators and prey during the process of predation,hunting strategies can be classified into three types:epibiotic predation,endobiotic predation or direct invasion,and group attack.Bdellovibiro,Bdellovibiro and like organisms(BALOs)and myxobacteria were representative bacterial predators.Bdellovibiro and BALOs belong to obligate predator groups,and myxobacteria belong to facultative predator groups,respectively.Through in-depth studies by means of omics analyses,the predation mechanisms of these predatory bacteria were deeper understood from the genomic or molecular level.However,the present developments of omics are still unmatured,and its wide applications are unavailable and limited.As a new-found bacterial predator,Bradymonabacteria has great significance in studies and potential application values.In addition,large blank in studies about bradymonabacteria still existed.Therefore,this study tries to explore the predatory characteristics of bradymonabacteria through the combination of phenotypic experiments and omics analyses,like comparative genomic analyses with other predatory bacteriaMethod of prey-trap was introduced into the isolation of bradymonabacteria.Combined with the direct isolation method,six isolates were purely cultured.However,based on polyphasic taxonomic technologies,only four isolates were identified as novel taxa,including four novel species-Venatimonas marina(V1718T),Persicimonas caeni(YN101T),Lujinxingia celatus(TMQ2T)and Lujinxingia vulgaris(TMQ4T),two novel genera—Venatimonas and Persicimonas,and one novel family—Venatimonadaceae.Cells of P.caeni YN101T motile by flagellar but not sliding,which is different from the fimbria movement of other members in Bradymonadales.This also shows that the cell of P.caeni YN101T is more capable in prey-taxis.Bradymonabacteria prefers to live in saline environments,according to the analyses of 16S rRNA high-throughput sequencing results from 8 environmental samples.Genomes of all known cultivable bradymonabacteria,accompanied with 4 genomes spliced from environmental metagenomic data,were selected for comparative genomic analyses.Combining the results of phenotypic tests with comparative genomics analyses,genomes of these predators have similar characteristics with known predators,such as absence of metabolic pathways of several important compounds,synthesis of a variety of transporters and transporter systems,and presence of type tad IVb pili or flagella and type III secretory system.By comparing with the predatomes summarized with classical predators,genomes of Bradymonadales conformed to predatory genomic features.The results revealed that all of bradymonabacteria were trophic bacteria.Their pentose phosphate pathways were incomplete,and purine and pyrimidine could not be de novo synthesized.Meanwhile,many amino acids and growth factor synthesis pathways were deficient,and fatty acid could not be synthesized.Compared with other 24 predatory genomes reported,deficiencies in metabolic pathway of bradymonabacteria were similar to obligate predatory bacteria.However,they were relatively easy to be purely cultivated,which was different from obligate predatory.In addition,bradymonabacteria synthesized polyhydroxy fatty acid esters and hydrocarbons as a carbon source storage,which was different from facultative predators.Judging from the physiological characteristics and genomic analyses,bradymonabacteria was a new type of predator group different from obligate and facultative predators.As a result,predatory bacteria can be re-classified into three types,including obligate-prey-dependent type,facultative-prey-dependent type,and prey-independent type,according to their dependence on prey.In order to further explore the predating cycle and predatory characteristics of bradymonabacteria,the type species B.sediminis FA350T was selected for predating assays.Inoculating B.sedinzinis FA350T and 107 type species with cross-streaking method,the predating ability showed by the predator in different assays was summarized.Based on wide category of prey bacteria,Algoriphagus iarius am2T was picked up as prey for its cell morphology different from B.sediminis FA350T.Moreover,B.sedinmnis FA350T predated A.marius am2T well,and the two species had distant evolutionary relationship.Predating cycle was measured through predatory curve measured by various quantitative means,including plate-counting,absolute quantification PCR,and fluorescence microscope counting.The cycle was divided into three periods,including early-stage,mid-stage,and late-stage,to easily perform further experiments.Based on the results of cross-streaking experiments,it can be clearly seen that salinity had great impact on the predating and growth states of B.sediminis FA350T under different salinity conditions.The presence of prey can weaken the negative influence from salinity to B.sediminis FA350T.Additionally,according to toxin detection of fennentation of B.sediminis FA350T,no contact between predators and prey cells would not induce the unnatural death of prey.Contact-depended predation was proved by Transwell and filter-membrane separation tests.Results of section transmission electron microscopy also demonstrated the cell morphology at different predation periods and contact-depended predation.In order to further investigate the predation mechanism of B.sediminis FA350T,transcriptomic analyses were introduced.It showed that prey A.marius am2T induced B.sediminis FA350T to enter a special metabolism stage different from the normal growth stage.Clusters built on the basic of differentially expressed genes sequences showed the up-regulated genes in mid-stage and late-stage were affiliated with ABC transport system,energy transfer,signal peptidase,DNA uptake,protein transport,membrane protein synthesis,type III secretion system and type IV pili synthesis.Genes affiliated with fatty acid metabolism,transcriptional regulation,protein folding and heterogeneity,and DNA repair were up-regulated expressed in late-stage but not mid-stage,and down-regulated expressed genes included amino acid and growth factor synthesis.In addition,fluorescence quantitative PCR was performed to demonstrate the transcriptome analysis results,and 6 genes were selected for experiments.Their functions were related with membrane proteins,transport systems,secretory systems and type IV pili,and these genes showed a trend of significantly up-regulated expression during mid-stage and late-stage.Moreover,metabolic pathways of B.sediminis FA350T during predating cycle were predicted:B.sediminis FA350T sensed the presence of prey cells signaling molecules from A.marius am2T and moved towards prey by type IV pili.Contact with prey cell induced B.sediminis FA350T entering predating cycle,meanwhile,type IV pili inserted into the prey cell wall and fixed predator cell to prey cell.Type III secretory system inserted into prey cells and injected toxin molecules into periplasm,or some antibiotics secreted by other elffux pumps,which made prey cells broken and died immediately.After that,B.sediminis FA350T produced a large amount of protease and nuclease transported extracellular by the secretion systems,which help degrade these macromolecules into small molecules.ABC transport systems and other transporters opened to transport extracellular small molecules into periplasm for the cell growth and life activities of B.sediminis FA350T,including cell membrane synthesis and DNA replication during cell division.In summary,as a new group of predatory bacteria,bradymonabacteria have unique genomic characteristics and metabolic pathways,which are different from obligate and facultative predator groups.Moreover,according to studies and analyses above,which can be speculated are their roles in the evolution of predation bacteria groups,as well as influences on other bacterial groups in the ecological environments.Considering that bradymonabacteria mainly distributes in saline environments,such as relatively high abundance in offshore and marine sediments,its predation may affect the structure of microbial communities,and then play certain ecological roles.As bacterial predators,bradymonabacteria has a well applicable prospects in aquaculture,animal husbandry and pharmaceutical industry.Bradymonabacteria can also be used as a biological agent for the improvement and regulation of intestinal microbes;some of them have the potential to produce antibiotics.This study enriches our understanding about the diversity of predatory bacterial groups and lays the foundation for a comprehensive evaluation of the roles of bacterial predators in ecosystems in the future.Certainly,there are still some deficiencies in the current work,and some issues need to be further explored if no time limitation exists.For example:(i)Results of high-throughput sequencing analyses show that bradymonabacteria distribute widely and are abundant in the saline environments.More bradymonabacteria can be isolated through prey-trap method designed by our laboratory.(ii)C isotope(13C)tracing detection can also be used to further demonstrate the flow direction of material composition from the cells of prey to verify the predating ability of bradymonabacteria.(iii)Gene knockout should be introduced to verify genes affiliated with key enzymes in important metabolic pathways during predation.(iv)Analyses of different resistant mechanisms between prey and non-prey bacteria should be performed.(5)Explore absolute eutrophication culture conditions to make predation of bradymonabacteria completely eliminated. |
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