Genome-wide Prediction Phenylobacterium Zucineum Hlk1 ~ T Efflux Protein

P.zucineum is a novel facultative intracellular bacterium which was recently discovered in the tumor cell line K562 by our group and the type strain was named HLK1~T.Since this microbe is a facultative intracellular microbe which can survive in human cells,it may have a pathogenic relevance with human and mammals, Previously,we found that this microbe invaded cells via a "Zipper" entry mechanism, whereby the microbe expresses some surface proteins,including transmembrane proteins and secreted proteins(exported proteins),which react with surface receptors of eukaryotic cells and trigger alteration and redistribution of the cell skeleton structure,extension of cell membrane,which permits the entrance of the.Thus,it is a pivot to identify the exported proteins.In this study,on the basis of the whole genom of P.zucineum,the gene identification and annotation,protein translation and exported proteins prediction by exported signal sequences identification has been carried out.Using a "whole-genome-shotgun" method,we sequenced P.zucineum's genome.At the step of annotation,"Glimmer" was used to predict ORFs(open reading frame), "RBSfinder" was used to readjust the results of Glimmer and "BLAST" was used to search homologous sequences in non-redundant database of NCBI,in order to predict functions of every CDS(coding sequence).All the proteins were classified by COG and the gene sequences were then translated into amino acid sequences.We predicted the exported proteins using consensus between multiple predictive tools,such as SignalP v3.0,LipoP v1.0,Phobius and TMHMM 2.0 by searching the export signals such as N-terminal signal peptides,α-transmembrane helices,etc.TypeⅣsignal peptides and proteins exported via Tat machinery were searched manually based on their conservative motifs.We sequenced its 4,379,231bp genome,including a 3,996,255 bp ring chromosome and a 382,976 bp plasmid.The GC content of chromosome and plasmid is 71.4%and 68.5%,respectively.We identified 42 tRNA genes and 1 16S-23S-5S rRNA operon in the chromosome.One of the interesting features of the P zucineum is its high GC content,which is the fourth highest in 343 genome-sequenced bacteria.The feature prefigures the complex secondary structure in the genome and possible relationship with actinobacteria which also have high GC content. Sequences of P.zucineum have the highest homology to genome of C.crescentus,a single-cell model system to study cellular differentiation,asymmetric division,and their coordination with cell cycle progression.One cell of C.crescentus can divide into two types of cells.Its master regulator of cell cycle is ctrA gene,which controls one quarter of cell cycle regulators and participates in many progresses in cell cycle. ctrA encodes a key response regulator of two-component signal-transuction system, an important system in recognition,invasion,pathogenesis and symbiosis of pathogens to host.A sequence named ctrZ in P zucineurn has 93%homology to ctrA. It may also be an essential component of two-component-transduction system and may play an important role in the pathogenesis to the human host cells.According to the annotation,the genome of P.zucineum encodes 3861 proteins (3534 proteins encoded by chromosome and 327 proteins encoded by plasmid),of which,3,715 have significant matches to the non-redundant protein database.Of the matches,866 are conserved hypothetical proteins,and 2,849 with known or predicted funtions.The existence of a lot of function unknown sequences indicates that the novel genes and functions of P.zucineum.According to our analysis,the genome of P.zucineurn contains abundant two component systems,transcriptional regulator,and heat shock response proteins, enabling the strain to respond to a plethora of stimuli coordinately at transcriptional and post-translational levels.In the total 102 two component systems transduction proteins(11 in plasmid),there are 36 histidine kinase,48 response regulator,and 18 hybrid proteins fused with histidine kinase and response regulator.16 pairs of histidine kinase and response regulator are adjacently aligned and may act as functional operons.These tightly linked modules make two component systems respond to environmental changes efficiently.We identified 170 transcriptional regulators(154 in the chromosome and 16 in the plasmid),which have the potential to respond to almost all known environmental stimulus,including penicillin resistance, low-temperature resistance,and metal resistance.There are 3 heat shock sigma factors rpoH(2 in the plasmid)and 35 heat shock molecular chaperons in the genome(15 in the chromosome and 20 in the plasmid).It was reported that rpoH helped E.coli to respond to the drastic change of temperature,and that heat shock molecular chaperons could be induced by a variety of stresses,including cellular energy depletion,extreme concentration of heavy metal,and various toxic substances.According to the annotation,the P.zucineum genomes contains sec-dependent and sec-independent,typical typeⅡand typeⅣsecretion systems,which are known to play an important role in adapting to diverse conditions.The see-dependent pathway for protein translocation such as SecA(PHZ_c0758),secB(PHZ_c0006),secD (PHZ_c1954),secE(PHZ_c1211),SecF(PHZ_c1953),secG(PHZ_c 1752),and secY (PHZ_c1250)were identified.Signal peptidases(PHZ_c1781),lipoprotein signal peptidase(PHZ_c3162),and the signal recognition particle srp54(PHZ_c0117),and the receptor(ftsY,PHZ_c0131)are present.The twin arginine motif translocation(Tat) pathway accomplishes the secretion of folded cofactor-containing proteins across the membrane.Of the 5 known Tat proteins,TatABC are present.13 proteins coding a set of typeⅡsecretion system are aligned tightly with each other to form an operon. There are 3 sets of typeⅣsecretion system,one of which is in the plasmid.Therefore, it is possible for any exported proteins to translocate across the membrane no matter which pathway they utilize.The genome of P.zucineum contains machineries to adhering and subsequently invading host cells.We identified 1 pili biosynthesis gene(pilA)and two operons related to pili biosynthesis located in chromosome.The genome also has fundamental compositions against oxidative stress, including superoxide dismutases,and hydroperoxide reductase,etc.In addition,the compositions of GSH-redox cycling,a major defense system against intracellular oxidative stress is also present.To survive in the intracellular environment,P.zucineum encodes various proteins to utilize the host iron source.The genome has one ABC type siderophore transporter system and 60 TonB-dependent(2 in the plasmid)which could uptake iron-siderophore complexes.Among all these proteins,35.4%was predicted to encode exported proteins, most of which(totally 735,19.0%of the proteome and 53.3%of all the exported proteins)utilize uncleavable transmembrane helices.In addition,typeⅠsignal peptides(499,12.9%of the proteome and 36.2%of the exported proteins), lipoproteins(101,2.6%of the proteome and 7.3%of the exported proteins)were also identified.Four proteins contain typeⅣsignal peptides and 12 proteins which exported by Tat pathway were detected as well.There are 25 COGs in NCBI and P.zucineum contains 20 of them.Among them, the most abundant group is "function unknown".Based on our exported protein prediction and the characteristics of facultative intracellular bacterium and its "Zipper" invasion mode,some categories of COGs are more important than the others. These categories are cell wall/membrane/envelope biogenesis;inorganic ion transport and metabolism;amino acid transport and metabolism;lipid transport and metabolism; signal transduction mechanisms;intracellular trafficking,secretion,and vesicular transport.The genome of HLK1~T encodes various types of exported proteins which may play an important role in the interaction with the host cells and facilitate the strain to invade the latter.