Studies On The Interactions Of Two Marine Roseobacters And Their Phages

Phages are the most abundant microbial group in the ocean.They play importantroles in dominating the mortality of prokaryotic cells,structuring the microbialcommunity,influencing the microbial food web process and promoting thebiogeochemical (such as C and N etc.) cycles in the ocean.Since phage infection hasan important impact on the bacterial physiological and biochemical process,for acomprehensively investigation and evaluation of the kinetic process and ecologicalsignificance of one microbial group,the substantial existence of phages in thesurrounding environment and their interactions with these bacteria would be anindispensable factor to be considered.Studies on the interactions between bacteria andphages are of great ecological meanings.In this study,the two bacteria,Roseobacterdenitrificans OCh114 and Silicibacterpomeroyi DSS-3,who both have an importantecological status in the ocean,were served as the host bacteria to isolate theirsusceptive phages and to set up their specific host-phage systems,then using thesehost-phage systems,the interactions of host bacteria and their phages wereinvestigated.Firstly,by using R.denitrificans OCh114 as the host to challenge viralconcentrate collected from the South China Sea,we isolated the roseophage RDJLΦ1and then characterized this phage from several aspects,such as phage morphology,growth characteristics,host range,nucleic acids and proteomic compostion,etc.Transmission electric microscopy revealed that RDJLΦ1 has an isometric head (ca.69 nm in diameter) and a long,flexible,non-contractile tail (ca.170 nm long and ca.9nm wide),morphologically belonging to the Siphoviridae family.The strongresistance to chloroform indicated that RDJLΦ1 is a lipid-free phage.It is capable ofrapid lysis of host cells with a burst size of ca.200 phages per cell and a latent periodof ca.80 min.Host-range test revealed a highly specific infectivity of RDJLΦ1 whichcan only infect R.denitrificans OCh114 among 24 tested bacterial strains.Its double-stranded genomic DNA is refractory to several commonly used restrictionenzymes,suggesting that it contains some modified nucleic acid bases.Sequencealignment analysis of a 1,651-bp phage DNA fragment,which was revealed byrandomly amplified polymorphic DNA PCR coupled with cloning and sequencing ofthe amplicons,indicated a close relatedness of RDJLΦ1 with another marinesiphophageΦJL001.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis ofthe virion proteins revealed that RDJLΦ1 is composed of at least 12 proteins.Surprisingly,4 of them were identifed as the host cellular proteins via matrix assistedlaser desorption/ionization time-of-flight mass spectrometry/mass spectrometry(MALDI-TOF MS/MS) method.While since no homologues of the other 8 proteinswere found in the NCBI database,they were considered to be novel proteins.R.denitrificans OCh114 is the first discovered aerobic anoxygenic phototrophicbacterium (AAPB),and RDJLΦ1 isthe firstphage isolated from the AAPBfunctional group.They represent the first host-phage model for studying theinteractions between AAPB and its susceptive phages.Secondly,we employed atomic force microscopy and proteomic techniques toinvestigate the host responses of R.denitrificans OCh114 during the whole course ofinfection by RDJLΦ1.In situ real-time atomic force microscopic imaging of theinfected R.denitrificans OCh114 cells revealed the morphological dynamic changesin the host surface following phage infection.Notably,several valuable atomic forcemicroscopic images revealing the highly dynamic phage lysis process were obtainedfor the first time.It showed that several large hollows with increasing diameter anddepth were abruptly and successively formed on the host surface,finally leading tothe collapse of the host cell.Then,using the two-dimensional polyacrylamide gelelectrophoresis method,at a global scale,we investigated the dynamic proteinexpression profiles of the host ceils at different timepoints (0,0.5,1.5,3 and 4.5 hpostinfection) following phage infection.Interestingly,the host protein responseswere found to be almost completed in only 30 min,which is much shorter than thelatent period ofRDJLΦ1(ca.80 min).For a preliminary investigation of the responsemechanism,several response proteins with significant changes in expression were identified using MALDI-TOF MS/MS method and are discussed separately in thisarticle.From a new perspective,this study contributes to our knowledge about theinteractions of bacteria and their phages,especially those which exist in the marinesystem.Under phage infection,sometimes some prokaryotic cells can survive byobtaining a mutation conferring upon themselfthe anti-phage ability.M1 is just such aphageresistant mutant of the marine roseobacter Silicibacter pomeroyi DSS-3.In thelast part of this paper,the phage resistance mechanisms of the mutant strain M1 wereinvestigated.Upon establishment of phage resistant strain M1 of S.pomeroyi DSS-3,our results excluded the possibilities of several phage resistance mechanisms,including abortive infection,adsorption inhibition,and the clustered regularlyinterspaced short palindromic repeats (CRISPRs).The comparison of proteinexpression profiles between the wild type strain (DSS-3) and mutant strain (M1)identified a potential phage resistance response due to the modification of four highlyexpressed proteins.One of these four proteins was successfully identified via massspectrometry,and matched a hypothetical protein (SPOA0343) annotated from the S.pomeroyi DSS-3 genome.Our results suggest that the modification of these proteinslike the protein SPOA0343 involves in the development of resistance to phageinfection.To the best of our knowledge,this work constitutes the first application ofcomparative proteomics to the study of phage resistance.As a result,it expands ourunderstanding of the anti-phage mechanisms of bacteria.