| "text": "This is an academic paper. This paper has corpus identifier PMC2529317\nAUTHORS: Yingfeng Luo, Xiaoli Xu, Zonghui Ding, Zhen Liu, Bing Zhang, Zhiyu Yan, Jie Sun, Songnian Hu, Xun Hu\n\nABSTRACT:\nBackgroundPhenylobacterium zucineum is a recently identified facultative intracellular species isolated from the human leukemia cell line K562. Unlike the known intracellular pathogens, P. zucineum maintains a stable association with its host cell without affecting the growth and morphology of the latter.ResultsHere, we report the whole genome sequence of the type strain HLK1T. The genome consists of a circular chromosome (3,996,255 bp) and a circular plasmid (382,976 bp). It encodes 3,861 putative proteins, 42 tRNAs, and a 16S-23S-5S rRNA operon. Comparative genomic analysis revealed that it is phylogenetically closest to Caulobacter crescentus, a model species for cell cycle research. Notably, P. zucineum has a gene that is strikingly similar, both structurally and functionally, to the cell cycle master regulator CtrA of C. crescentus, and most of the genes directly regulated by CtrA in the latter have orthologs in the former.ConclusionThis work presents the first complete bacterial genome in the genus Phenylobacterium. Comparative genomic analysis indicated that the CtrA regulon is well conserved between C. crescentus and P. zucineum.\n\nBODY:\nBackgroundPhenylobacterium zucineum strain HLK1T is a facultative intracellular microbe recently identified by us [1]. It is a rod-shaped Gram-negative bacterium 0.3–0.5 × 0.5–2 μm in size. It belongs to the genus Phenylobacterium [2], which presently comprises 5 species, P. lituiforme (FaiI3T) [3], P. falsum (AC49T) [4], P. immobile (ET) [2], P. koreense (Slu-01T) [5], and P. zucineum (HLK1T) [1]. They were isolated from subsurface aquifer, alkaline groundwater, soil, activated sludge from a wastewater treatment plant, and the human leukemia cell line K562, respectively. Except for P. zucineum, they are environmental bacteria, and there is no evidence that these microbes are associated with eukaryotic cells. The HLK1T strain, therefore, represents the only species so far in the genus Phenylobacterium that can infect and survive in human cells. Since most, if not all, of the known microbes that can invade human cells are pathogenic, we proposed that HLK1T may have pathogenic relevance to humans [1]. Unlike the known intracellular pathogens that undergo a cycle involving invasion, overgrowth, and disruption of the host cells, and repeating the cycle by invading new cells, HLK1T is able to establish a stable parasitic association with its host, i.e., the strain does not overgrow intracellularly to kill the host, and the host cells carry them to their progeny. One cell line (SW480) infected with P. zucineum has been stably maintained for nearly three years in our lab (data not shown).In this report, we present the complete genome sequence of P. zucineum.ResultsGenome anatomyThe genome is composed of a circular chromosome (3,996,255 bp) and a circular plasmid (382,976 bp) (Figure 1; Table 1). The G + C contents of chromosome and plasmid are 71.35% and 68.5%, respectively. There are 3,861 putative protein-coding genes (3,534 in the chromosome and 327 in the plasmid), of which 3,180 have significant matches in the non-redundant protein database. Of the matches, 585 are conserved hypothetical proteins and 2,595 are proteins with known or predicted functions. Forty-two tRNA genes and one 16S-23S-5S rRNA operon were identified in the chromosome.Table 1Genome summary of P. zucineum Strain HLK1TGenomic ElementChromosomeplasmidLength (bp)3,996,255382,976GC content (%)71.3568.54Proteins3, 534327Coding region of genome (%)88.85%81.94%Proteins with known or predicted function2,394(67.75%)201(61.47%)Conserved hypothetical proteins560(15.84%)25(7.65%)Hypothetical proteins580(16.41%)101(30.88%)rRNA operon10tRNAs420Proteins in each[J] Translation, ribosomal structure and biogenesis185 (5.24%)3 (1.21%)COG category[K] Transcription210 (5.94%)22 (8.91%)[L] Replication, recombination and repair139 (3.93%)23 (9.31%)[D] Cell cycle control, cell division, chromosome partitioning27 (0.76%)0[V] Defense mechanisms51 (1.44%)3 (1.21%)[T] Signal transduction mechanisms166 (4.7%)24 (9.72%)[M] Cell wall/membrane/envelope biogenesis195 (5.52%)15 (6.07%)[N] Cell motility62 (1.75%)4 (1.62%)[U] Intracellular trafficking, secretion, and vesicular transport96 (2.72%)13 (5.26%)[O] Posttranslational modification, protein turnover, chaperones151 (4.27%)32 (12.96%)[C] Energy production and conversion188 (5.32%)16 (6.48%)[G] Carbohydrate transport and metabolism161 (4.56%)15 (6.07%)[E] Amino acid transport and metabolism293 (8.29%)5 (2.02%)[F] Nucleotide transport and metabolism58 (1.64%)3 (1.21%)[H] Coenzyme transport and metabolism116 (3.28%)3 (1.21%)[I] Lipid transport and metabolism215 (6.09%)12 (4.86%)[P] Inorganic ion transport and metabolism223 (6.31%)24 (9.72%)[Q] Secondary metabolites biosynthesis, transport and catabolism152(4.3%)9 (3.64%)[R] General function prediction only444 (12.57%)28 (11.34%)[S] Function unknown307 (8.69%)20 (8.10%)Figure 1Circular representation of the P. zucineum strain HLK1T chromosome and plasmid (smaller circle). Circles indicate (from the outside): (1) Physical map scaled in megabases from base 1, the start of the putative replication origin. (2) Coding sequences transcribed in the clockwise direction are color-coded according to COG functional category. (3) Coding sequences transcribed in the counterclockwise direction are color-coded according to COG functional category. (4) Proteins involved in establishment of intracellular niche are TonB-dependent receptors (orange) and pilus genes (sienna). (5) Functional elements responsible for environmental transition are extracytoplasmic function sigma factors (royal blue), transcriptional regulators (violet red), two-component signal transduction proteins (deep sky blue), heat shock molecular chaperons (spring green), type IV secretion systems (plum), chemotaxis systems (green yellow) and flagellum proteins (gray). (6) G + C percent content (10-kb window and 1-kb incremental shift for chromosome; 300 bp window and 150 bp for incremental shift for plasmid); values larger than average (71.35% in chromosome and 68.5% in plasmid) are in red and smaller in medium blue. (7) GC skew (10-kb window and 1-kb incremental shift for chromosome; 300 bp window and 150 bp for incremental shift for plasmid); values greater than zero are in gold and smaller in purple. (8) Repeat families, repeats 01-08 are in dark salmon, dark red, wheat, tomato, light green, salmon, dark blue and gold, respectively.There are 7 families of protein-coding repetitive sequences and a family of noncoding repeats in the genome (Table 2). Notably, identical copies of repeats 02–04 were found in both the chromosome and the plasmid, suggesting their potential involvement in homologous recombination.Table 2Repetitive elements in the P. zucineum genomeRepeat IDLength bpDR1Number of copiesPosition of insertionIdentity (%)Coding informationComplete2PartialChromosomePlasmidRepeat0132,58773104>99TransposaseRepeat0241,26233122100TransposaseRepeat0351,392NA4242100TransposaseRepeat0461,257NA10073100TransposaseRepeat051,554NA2020>98Hypothetical proteinRepeat061,136NA2020>90Isovaleryl-CoA dehydrogenaseRepeat071,077NA2020>982-nitropropane dioxygenaseRepeat08≈130NA130130>90Noncoding repeats1Size in base pairs of the consensus and the direct repeat (DR) generated by insertion into the genome target site.2A copy is complete if the length of the repeat is ≥ 90% of the consensus, otherwise, the copy is partial.3One complete copy which harbors a 7 bp direct repeat (TCCTAAC) that disrupts the VirD4.4The partial copy is located in the plasmid.5Two partial copies are located in the chromosome, of which a \"partial\" copy with full length is inserted by a copy of repeat04.6repeats 01–04 are IS elementsOn the basis of COG (Cluster of Orthologous Groups) classification, the chromosome is enriched in genes for basic metabolism, such as categories E (amino acid transport and metabolism) and I (lipid transport and metabolism), accounting for 8.29% and 6.09% of the total genes in the chromosome, respectively. On the other hand, the plasmid is enriched for genes in categories O (posttranslational modification, protein turnover, chaperones) and T (signal transduction mechanisms), constituting 12.96% and 9.72% of the total genes in the plasmid, respectively.As to genes in the plasmid that cope with environmental stimuli, about half of the genes in category O are molecular chaperones (17/32), including 2 dnaJ-like molecular chaperones, 2 clusters of dnaK and its co-chaperonin grpE (PHZ_p0053-0054 and PHZ_p0121-122), a cluster of groEL and its co-chaperonin groES (PHZ_p0095-0096), and 9 heat shock proteins Hsp20. Of 23 genes in category T, there is one cluster (FixLJ, PHZ_p0187-0188), which is essential for the growth of C. crescentus under hypoxic conditions [6].General metabolismThe enzyme sets of glycolysis and the Entner-Doudoroff pathway are complete in the genome. All genes comprising the pentose phosphate pathway except gluconate kinase were identified, consistent with our previous experimental result that the strain cannot utilize gluconate [1]. The genome lacks two enzymes (kdh, alpha ketoglutarate dehydrogenase and kgd, alpha ketoglutarate decarboxylase), making the oxidative and reductive branches of the tricarboxylic acid cycle operate separately. The genome has all the genes for the synthesis of fatty acids, 20 amino acids, and corresponding tRNAs. Although full sets of genes for the biosynthesis of purine and pyrimidine were identified, enzymes for the salvage pathways of purine (apt, adenine phosphoribosyltransferase; ade, adenine deaminase) and pyrimidine (cdd, cytidine deaminase; codA, cytosine deaminase; tdk, thymidine kinase; deoA, thymidine phosphorylase; upp, uracil phosphoribosyltransferase; udk, uridine kinase; and udp, uridine phosphorylase) were absent. The plasmid encodes some metabolic enzymes, such as those participating in glycolysis, the pentose phosphate pathway, and the citric acid cycle. However, it is worth noting that the plasmid has a gene (6-phosphogluconate dehydrogenase) that is the only copy in the genome (PHZ_p0183).Like most other species in the genus Phenylobacterium, the strain is able to use L-phenylalanine as a sole carbon source under aerobic conditions [1]. A recent study revealed that phenylalanine can be completely degraded through the homogentisate pathway in Pseudomonas putida U [7]. P. zucineum may use the same strategy to utilize phenylalanine, because all the enzymes for the conversion of phenylalanine through intermediate homogentisate to the final products fumarate and acetoacetate are present in the chromosome (Table 3).Table 3Phenylalanine-degrading enzymes in the P. zucineum genomeGeneP. zucineum LocusLength (bp)Alignment coverage (%)ScoreAmino acid Identity (%)Gene nameP. putidaP. zucineumP. putidaP. zucineumphhAPHZ_c140926230883.5971.7521948.65phenylalanine-4-hydroxylasephhBPHZ_c00771189779.6693.8138.526.32carbinolamine dehydratasetryBPHZ_c164439840660.0557.3933.921.86tyrosine aminotransferasehpdPHZ_c283335837498.3293.5839857.984-hydroxyphenylpyruvate dioxygenasehmgAPHZ_c283143337760.2867.6453.522.3homogentisate 1,2-dioxygenasehmgBPHZ_c03134302269.7718.1427.739.53fumarylacetoacetate hydrolasehmgCPHZ_c031421021298.198.1121351.67maleylacetoacetate isomeraseFunctional elements responding to environmental transitionHLK1T is able to survive intracellularly and extracellularly. Consistently, the genome contains the fundamental elements to support the life cycle in different environments. The genome contains abundant two-component signal transduction proteins, transcriptional regulators, and heat shock response proteins, enabling the strain to respond to extra- and intra-cellular stimuli at transcriptional and post-translational levels. Among the total of 102 two-component signal transduction proteins (91 in the chromosome and 11 in the plasmid), there are 36 histidine kinases, 48 response regulators, and 18 hybrid proteins fused with histidine kinase and response regulator. Sixteen pairs of histidine kinase and response regulator (1 in the plasmid) are adjacently aligned and may act as functional operons. These tightly linked modules make two-component signal transduction systems respond to environmental changes efficiently. The genome encodes 170 transcriptional regulators (16 in the plasmid) (Table 4). Notably, we annotated the proteins of 93 bacteria (see methods – comparative genomics) with the same annotation criteria used for P. zucineum and found that the fraction of two-component signal transduction proteins and transcriptional regulators was positively correlated with the capacity for environmental adaptation (Figure 2). The genome contains 17 extracytoplasmic function (ECF) sigma factors (3 in the plasmid) (Table 5). ECFs are suggested to play a role in environmental adaptation for Pseudomonas putida KT2440, whose genome contains 19 ECFs [8]. P. zucineum has 3 heat shock sigma factors rpoH (2 in the plasmid) and 33 heat shock molecular chaperons (17 in the plasmid) (Table 6), which can cope with a variety of stresses, including cellular energy depletion, extreme concentrations of heavy metals, and various toxic substances. [9].Table 4Transcriptional regulators in the P. zucineum genomeFamily nameAction typeChromosomePlasmidProposed rolesAsnC familyActivator/repressor80Amino acid biosynthesisAraC familyActivator101Carbon metabolism, stress response and pathogenesisArsR familyRepressor80Metal resistanceBlaI familyRepressor20Penicillin resistanceCold shock familyActivator60Low-temperature resistanceCro/CI familyRepressor92Unknown2Crp/Fnr familyActivator/repressor72Global responses, catabolite repression and anaerobiosisGntR familyRepressor70General metabolismLacI familyRepressor40Carbon source utilizationLuxR familyActivator51Quorum sensing, biosynthesis and metabolism, etc.LysR familyActivator/repressor151Carbon and nitrogen metabolismMarR familyActivator/repressor60Multiple antibiotic resistanceMerR familyRepressor92Resistance and detoxificationTetR familyRepressor220Biosynthesis of antibiotics, efflux pumps, osmotic stress, etc.XRE familyRepressor22Unknown (initial function is lysogeny maintenance)Other types2-345-Total-15416-1Initial function is related to controlling the expression of phage gene2\"Other types\" include the transcriptional regulators with only one member in the P. zucineum genome or transcriptional regulators that could not be classified into any known family.Table 5Extracytoplasmic function (ECF) sigma factors in the P. zucineum genomeLocusLocation of proteinsCOG categoryGenomic element5'-end3'-endPHZ_p0151Plasmid171,032170,316COG15951PHZ_p0174Plasmid208,703208,053COG1595PHZ_p0192Plasmid229,133228,516COG1595PHZ_c0249Chromosome249,840250,553COG1595PHZ_c0301Chromosome296,299295,706COG1595PHZ_c1475Chromosome1,676,9201,677,492COG1595PHZ_c1529Chromosome1,730,7831,731,403COG1595PHZ_c1531Chromosome1,732,2191,732,800COG1595PHZ_c1907Chromosome2,134,9712,135,507COG1595PHZ_c2171Chromosome2,447,5812,448,396COG1595PHZ_c2233Chromosome2,526,8362,527,369COG1595PHZ_c2394Chromosome2,724,7592,725,307COG1595PHZ_c2577Chromosome2,965,2502,964,390COG1595PHZ_c2585Chromosome2,970,3682,969,811COG1595PHZ_c2684Chromosome3,077,2723,076,727COG1595PHZ_c0569Chromosome605,441604,233COG49412PHZ_c3154Chromosome3,582,0103,583,269COG49411COG1595, DNA-directed RNA polymerase specialized sigma subunit, sigma24 homolog;2COG4941, predicted RNA polymerase sigma factor containing a TPR repeat domainTable 6Distribution of heat shock related proteins in P. zucineum and representative alphaproteobacteria with different living habitatsContent\\SpeciesS. melilotiB. suisC. crescentusP. zucineumR. conoriiG. oxydansChromosomePlasmidrpoH, heat shock sigma factor12211211dnaK, molecular chaperone2 (Hsp70)1111211grpE, molecular chaperone (co-chaperonin of Hsp70)1111211dnaK-like molecular chaperone1111011dnaJ, molecular chaperone1111011dnaJ-like molecular chaperone4336213groEL, molecular chaperone (hsp60)5111111groES, molecular chaperone (Hsp10, co-chaperonin of Hsp60)3111111molecular chaperone Hsp205223903molecular chaperone Hsp3311110011rpoH may be responsible for the expression of some or all heat shock proteins2The function of molecular chaperones is to protect unfolded proteins induced by stress factors through renaturation or degradation in cooperation with protease.Figure 2Comparative analysis of transcriptional regulators and two-component signal transduction proteins in 6 groups of bacteria classified according to their habitats. (A): The mean number of transcriptional regulators in each megabase pair of the genomes. (B): The mean number of two-component signal transduction proteins in each megabase pair of the genomes. The fraction of transcriptional regulators and two-component signal transduction proteins (solid black circle) of P. zucineum were 41.56 genes/Mb and 23.30 genes/Mb, respectively. Error bars represent standard errors. O: Obligate (26 species), S: Specialized (5 species), AQ: Aquatic (4 species), F: Facultative (28 species), M: Multiple (27 species), T: Terrestrial (3 species).The genes for cell motility include 3 chemotaxis operons, 7 MCP (methyl-accepting chemotaxis) genes, 15 other genes related to chemotaxis (Table 7), and 43 genes for the biogenesis of the flagellum (Table 8).Table 7Chemotaxis proteins in the P. zucineum genomeLocus P. zucineum5'-end3'-endNameOrthologs C. crescentusOperonBest BLAST matchPHZ_c0690753,270753,812chemotaxis protein CheW-1M. magneticum AMB-1PHZ_c0691753,812755,218chemotaxis protein methyltransferase CheR-1M. magnetotacticum MS-1PHZ_c0692755,240755,836chemotaxis signal transduction protein-1Rhodospirillum centenumPHZ_c0693755,836757,488methyl-accepting chemotaxis protein-1M. magneticum AMB-1PHZ_c0694757,501759,642chemotaxis histidine kinase CheA-1M. magnetotacticum MS-1PHZ_c0695759,642760,709chemotaxis response regulator CheB-1Rhodospirillum centenumPHZ_c32303,661,5143,661,050CheE protein-2C. crescentus CB15PHZ_c32313,662,0993,661,527chemotaxis protein CheYIIICC04402C. crescentus CB15PHZ_c32333,662,8603,662,477chemotaxis protein CheYIICC05912R. palustris CGA009PHZ_c32343,663,1863,666,188chemotaxis histidine kinase CheACC05942Azospirillum brasilensePHZ_c32353,666,1883,666,733chemotaxis protein CheWCC05952Rhodospirillum centenumPHZ_c32363,666,7863,669,191methyl-accepting chemotaxis protein McpHCC33492R. palustris CGA009PHZ_c32373,670,1663,669,336chemotaxis protein methyltransferase CheRCC05982R. palustris HaA2PHZ_c32383,671,2423,670,166chemotaxis response regulator CheBCC05972M. magneticum AMB-1PHZ_c33713,820,1213,819,669CheE proteinCC04413C. crescentus CB15PHZ_c33723,820,7293,820,124chemotaxis protein CheYIII-3C. crescentus CB15PHZ_c33733,821,0343,820,729CheU proteinCC04393C. crescentus CB15PHZ_c33743,821,6513,821,082chemotaxis protein CheDCC04383C. crescentus CB15PHZ_c33753,822,0373,821,651chemotaxis protein CheYIICC04373C. crescentus CB15PHZ_c33763,823,0683,822,040chemotaxis response regulator CheBCC04363C. crescentus CB15PHZ_c33773,823,9553,823,068chemotaxis protein methyltransferase CheRCC04353A. cryptum JF-5PHZ_c33783,824,4103,823,946chemotaxis protein CheWCC04343Rhizobium etli CFN 42PHZ_c33793,826,6143,824,422chemotaxis histidine kinase CheACC04333A. cryptum JF-5PHZ_c33803,826,9973,826,635chemotaxis protein CheYICC04323Caulobacter vibrioidesPHZ_c33813,827,2993,826,997CheX proteinCC04313Sinorhizobium melilotiPHZ_c33823,829,2343,827,306methyl-accepting chemotaxis protein McpACC04303A. cryptum JF-5PHZ_c010194,22093,750CheE protein-scattedC. crescentus CB15PHZ_c010294,79594,220chemotaxis protein CheYIII-scattedC. crescentus CB15PHZ_c0297292,469292,864chemotaxis protein CheYIVCC3471scattedC. crescentus CB15PHZ_c0298292,867293,679chemotaxis protein methyltransferase CheRCC3472scattedC. crescentus CB15PHZ_c0732803,383804,876methyl-accepting chemotaxis protein McpBCC0428scattedC. crescentus CB15PHZ_c09611,057,1341,058,720methyl-accepting chemotaxis protein McpICC2847scattedR. palustris CGA009PHZ_c11981,380,8831,383,294methyl-accepting chemotaxis protein McpU-scattedA. cryptum JF-5PHZ_c11991,383,2971,383,758chemotaxis protein CheW1-scattedSinorhizobium melilotiPHZ_c16871,890,2741,891,176chemotaxis MotB proteinCC1573scattedC. crescentus CB15PHZ_c19362,169,6342,169,939chemotactic signal response protein CheLCC2583scattedC. crescentus CB15PHZ_c22112,499,7442,499,274chemotaxis protein CheYIII-scattedO. alexandrii HTCC2633PHZ_c23922,720,6112,720,144chemotaxis protein CheYIII-scattedC. crescentus CB15PHZ_c27413,142,7503,143,238chemotaxis protein CheYIIICC3155scattedC. crescentus CB15PHZ_c31233,549,1503,550,016chemotaxis MotA proteinCC0750scattedC. crescentus CB15PHZ_c34013,848,8113,850,766methyl-accepting chemotaxis protein McpA-scattedC. vibrioidesTable 8Flagella genes in the P. zucineum genomeLocus5'-end3'-endNameGene symbolProposed rolePHZ_c008075,41376,462flagellin modification protein FlmAflmAregulatorPHZ_c008176,46777,621flagellin modification protein FlmBflmBregulatorPHZ_c0745816,772818,034flagellar hook-length control protein FliKfliKflagellar structurePHZ_c0787868,051866,696flagellar hook protein FlgEflgEflagellar structurePHZ_c0788868,860868,171flagellar hook assembly protein FlgDflgDflagellar structurePHZ_c0789870,604868,865flagellar hook length determination proteinflageregulatorPHZ_c0790870,819872,918flagellar hook-associated proteinflaNflagellar structurePHZ_c0791872,933873,862flagellin and related hook-associated proteins-flagellar structurePHZ_c0853945,008946,354flagellum-specific ATP synthase FliIfliIprotein export ATPasePHZ_c0854946,354946,758fliJ proteinfliJflagellar structurePHZ_c0857950,714948,621flagellar biosynthesis protein FlhAflhAexport apparatusPHZ_c0859952,470952,138flagellar motor switch protein FliNfliNmotorPHZ_c0860953,126952,479flbE proteinflbEregulatorPHZ_c0861954,151953,126flagellar motor switch protein FliGfliGmotorPHZ_c0862955,794954,151flagellar M-ring protein FliFfliFflagellar structurePHZ_c09131,007,7531,006,992flagellar L-ring protein FlgHflgHflagellar structurePHZ_c09141,008,5081,007,753distal basal-body ring component protein FlaDflaDflagellar structurePHZ_c09151,009,3001,008,515flagellar basal-body rod protein FlgGflgGflagellar structurePHZ_c09161,010,0521,009,318flagellar basal-body rod protein FlgFflgFflagellar structurePHZ_c09171,010,2721,010,874flagellar basal body-associated protein FliLfliLflagellar structurePHZ_c09181,010,9101,011,983flagellar motor switch protein FliMfliMmotorPHZ_c09221,017,0851,016,351flagellar biosynthesis protein FliPfliPexport apparatusPHZ_c09231,017,4201,017,151flagellar protein FliOfliOexport apparatusPHZ_c09241,017,5021,017,918flagellar basal-body rod protein FlgBflgBflagellar structurePHZ_c09251,017,9421,018,355flagellar basal-body rod protein FlgCflgCflagellar structurePHZ_c09261,018,3701,018,678flagellar hook-basal body complex protein FliEfliEflagellar structurePHZ_c09301,021,7961,022,056flagellar biosynthesis protein FliQfliQexport apparatusPHZ_c09311,022,0791,022,837flagellar biosynthesis protein FliRfliRexport apparatusPHZ_c09321,022,8371,023,913flagellar biosynthesis protein FlhBflhBexport apparatusPHZ_c13801,563,2811,562,745putative flagella accessory protein FlaCEflaCEflagellar structurePHZ_c13811,565,1451,563,358flagellin modification protein FlmGflmGregulatorPHZ_c13821,565,3431,565,765flagellar repressor protein FlbTflbTregulatorPHZ_c13831,565,7821,566,093flagellar biosynthesis regulator FlaFflaFregulatorPHZ_c13841,566,3751,567,202flagellin FljMfljMflagellar structurePHZ_c13851,567,4691,568,314flagellin FljMfljMflagellar structurePHZ_c13861,568,4341,568,724flagellin FlaGflaGflagellar structurePHZ_c13871,568,8871,569,720flagellin FljLfljLflagellar structurePHZ_c19352,168,5222,169,634flagellar P-ring protein FglIfglIflagellar structurePHZ_c19372,169,9422,170,382flagellar basal-body protein FlbYflbYflagellar structurePHZ_c25952,982,5502,983,593flagellin modification protein FlmDflmDregulatorPHZ_c25972,984,8742,986,508flagellin modification protein FlmGflmGregulatorPHZ_c25992,989,3152,989,974flmC; flagellin modification protein FlmCflmCregulatorPHZ_c26002,990,5492,989,977flagellin modification protein FlmHflmHregulatorThe genome contains sec-dependent, sec-independent, typical type II (Table 9) and IV secretion systems (Table 10), which are known to play important roles in adapting to diverse conditions [10,11].Table 9Distributions of proteins involved in environmental adaptation in P. zucineum and representative alphaproteobacteria with different living habitatsSpeciesS. melilotiB. suisC. crescentusP. zucineumR. conoriiG. oxydansGenome size (Mb)6.693.324.024.381.272.92GC content (%)62.257.367.271.132.460.8HabitatMultiple1Facultative1Aquatic1Facultative2Obligate1Multiple3ECF, extracytoplasmic function sigma factor (/Mb)11 (1.6)2 (0.6)15 (3.7)17 (3.9)0 (0)2 (0.7)Transcriptional regulator (/Mb)433 (64.7)149(44.9)183 (45.5)170 (38.8)11 (8.7)89 (30.1)Two-component signal transduction protein (/Mb)113 (16.9)44 (13.3)111 (27.6)102 (23.3)7 (5.5)41 (14.1)molecular chaperone23121433814Flagellar protein413742431040Chemotaxis protein4244841011Pilus protein13491624Sec-dependent secretion system111111111112Sec-independent secretion system444434Type II secretory protein2081303Type IV secretory protein989311511The habitats of S. meliloti, B. suis, and R. conorii were indicated in a recent publication [42].2According to our recent publication [1], P. zucineum was classified as \"facultative\". 3Given that G. oxydans is often isolated from sugary niches (such as flowers and fruits) and associated soil (such as garden soil and baker's soil) [43], we classified G. oxydans as \"multiple\".Table 10Type IV secretion systems in the P. zucineum genomeLocusLocation of proteinNameGenomic element5'-end3'-endPHZ_p0007Plasmid6,7867,445type IV secretion protein, VirB1PHZ_p0008Plasmid7,4837,800type IV secretion protein, VirB2PHZ_p0009Plasmid7,8168,148type IV secretion protein, VirB3PHZ_p0010Plasmid8,14410,546type IV secretion protein, VirB4PHZ_p0011Plasmid10,54611,298type IV secretion protein, VirB5PHZ_p0012Plasmid11,55312,488type IV secretion protein, VirB6PHZ_p0013Plasmid12,81613,493type IV secretion protein, VirB8PHZ_p0014Plasmid13,49314,320type IV secretion protein, VirB9PHZ_p0015Plasmid14,32015,543type IV secretion protein, VirB10PHZ_p0016Plasmid15,54316,538type IV secretion protein, VirB11PHZ_c1506Chromosome1,709,4811,709,999type IV secretion protein, TraFPHZ_c1508Chromosome1,711,0581,712,773type IV secretion protein, VirD2PHZ_c1509Chromosome1,712,7901,714,763type IV secretion protein, VirD4PHZ_c1512Chromosome1,716,2621,717,242conjugal transfer protein, TrbBPHZ_c1513Chromosome1,717,2421,717,559conjugal transfer protein, TrbCPHZ_c1514Chromosome1,717,5621,717,828conjugal transfer protein, TrbDPHZ_c1515Chromosome1,717,8361,720,283conjugal transfer protein, TrbEPHZ_c1516Chromosome1,720,2831,721,014conjugal transfer protein, TrbJPHZ_c1517Chromosome1,721,2381,722,398conjugal transfer protein, TrbLPHZ_c1518Chromosome1,722,4011,723,084conjugal transfer protein, TrbFPHZ_c1519Chromosome1,723,0871,724,064conjugal transfer protein, TrbGPHZ_c1520Chromosome1,724,0701,725,212conjugal transfer protein, TrbIPHZ_c2348Chromosome2,660,5172,660,813type IV secretion protein, VirB2PHZ_c2349Chromosome2,660,8092,661,144type IV secretion protein, VirB3PHZ_c2350Chromosome2,661,1192,663,497type IV secretion protein, VirB4PHZ_c2352Chromosome2,664,3742,665,309type IV secretion protein, VirB6PHZ_c2353Chromosome2,665,4822,666,159type IV secretion protein, VirB8PHZ_c2354Chromosome2,666,1592,667,004type IV secretion protein, VirB9PHZ_c2355Chromosome2,667,0042,668,041type IV secretion protein, VirB10PHZ_c2356Chromosome2,668,0462,669,035type IV secretion protein, VirB11PHZ_c2357Chromosome2,669,0912,670,872type IV secretion protein, VirD4To better understand the roles of proteins responsible for environmental transition, we computed the distributions of those proteins in 5 representative alphaproteobacteria with typical habitats (see methods – comparative genomics). Like other multiple bacteria and facultative bacteria, which can survive in multiple niches, P. zucineum encodes a higher fraction of ECFs, transcriptional regulators and two-component signal transduction proteins than obligate bacteria (Table 9). Notably, P. zucineum has the largest number of heat shock related proteins (Table 6), in comparison to the 5 representative alphaproteobacteria and 93 bacteria (data not shown). Among the plasmid-encoded heat shock related proteins are 2 RpoH (PHZ_p0049 and PHZ_p0288) and 2 DnaK-GrpE clusters (PHZ_p0053-0054 and PHZ_p0121-0122). Further phylogenetic analysis suggested that the plasmid-encoded DnaK-GrpE clusters may have undergone a genus-specific gene duplication event (Figure 3C &3D).Figure 3Neighbor-joining trees of 5 representative alphaproteobacteria and P. zucineum, inferred from (A) 16S rRNA genes, (B) RpoH proteins, (C) DnaK proteins and (D) GrpE proteins. The node labels are bootstrap values (100 replicates). The plasmid-encoded DnaK and GrpE of P. zucineum may have undergone a genus-specific gene duplication event (C &Adaptation to an intracellular life cycleTo survive intracellularly, P. zucineum must succeed in adhering to and subsequently invading the host cell [12], defending against a hostile intracellular environment [13-16], and capturing iron at very low concentration [17].It is well known that the pilus takes part in adhering to and invading a host cell [12]. We identified one pili biosynthesis gene (pilA) and 2 operons for pili biosynthesis (Table 11).Table 11Pilus proteins in the P. zucineum genomeLocus5'-end3'-endNameGene symbolPHZ_c0356362,116362,289pilus subunit protein PilApilAPHZ_c29923,412,8003,413,318Flp pilus assembly protein TadGtadGPHZ_c29953,415,2203,415,468Flp pilus assembly protein, pilin Flp-PHZ_c29963,415,5323,416,023Flp pilus assembly protein, protease CpaAcpaAPHZ_c29973,416,0393,416,899pilus assembly protein CpaBcpaBPHZ_c29983,416,8993,418,350pilus assembly protein CpaCcpaCPHZ_c29993,418,3553,419,587pilus assembly protein CpaEcpaEPHZ_c30003,419,5943,420,991pilus assembly protein CpaFcpaFPHZ_c30013,421,0303,421,944Flp pilus assembly protein TadBtadBPHZ_c30023,421,9443,422,903Flp pilus assembly protein TadCtadCPHZ_c30273,451,6373,452,566Flp pilus assembly protein CpaBcpaBPHZ_c30283,452,5803,453,893Flp pilus assembly protein, secretin CpaCcpaCPHZ_c30293,453,8933,455,056Flp pilus assembly protein, ATPase CpaEcpaEPHZ_c30303,455,0593,456,489Flp pilus assembly protein ATPase CpaFcpaFPHZ_c30313,456,4893,457,445Flp pilus assembly protein TadBtadBPHZ_c30323,457,4923,458,391Flp pilus assembly protein TadCtadCThe genes involved in defense against oxidative stress include superoxide dismutase (PHZ_c0927, PHZ_c1092), catalase (PHZ_c2899), peroxiredoxin (PHZ_c1548), hydroperoxide reductase (ahpF, alkyl hydroperoxide reductase, subunit f, PHZ_c2725, ahpC, alkyl hydroperoxide reductase, subunit c, PHZ_c2724), and the glutathione redox cycle system (glutathione reductase [PHZ_c1740, PHZ_c1981], glutathione synthetase [PHZ_c3479], and γ-glutamylcysteine synthetase [PHZ_c0446, PHZ_c0523]).Since intracellular free Fe is not sufficient to support the life of bacteria, to survive intracellularly, they must use protein-bound iron, such as heme and transferrin, via transporters and/or the siderophore system. The P. zucineum genome has one ABC type siderophore transporter system (PHZ_c1893-1895), one ABC type heme transporter system (PHZ_c0136, PHZ_c0139, PHZ_c0140), and 60 TonB-dependent receptors which may uptake the iron-siderophore complex (Table 12).Table 12TonB-dependent receptors in the P. zucineum genomeAnnotationChromosomePlasmidCOG categoryTonB-dependent receptor512COG16291TonB-dependent receptor vitamin B1230COG42062TonB-dependent receptor40COG477131COG1629, Outer membrane receptor proteins, mostly Fe transport2COG4206, Outer membrane cobalamin receptor protein3COG4774, Outer membrane receptor for monomeric catecholsComparative genomics between P. zucineum and C. crescentusComparative genomic analysis demonstrated that P. zucineum is phylogenetically the closest to C. crescentus [18] (Figure 4), consistent with the phylogenetic analysis based on 16S RNA gene sequences (Figure 5).Figure 4List of top 10 complete sequenced bacteria closest to P. zucineum. All 10 are alphaproteobacteria. Among all the sequenced bacterial genomes, C. crescentus shares the greatest number of similar ORFs with P. zucineumFigure 5Neighbor-joining tree of the alphaproteobacteria, inferred from 16S rRNA genes. The node labels are bootstrap values (100 replicates). C. crescentus is phylogenetically the closest to P. zucineum.Though the genome size and protein number of P. zucineum (4.37 Mb, 3,861 proteins) are similar to those of C. crescentus (4.01 Mb, 3,767 proteins), no large-scale synteny was found between the genomes. The largest synteny region is only about 30 kb that encodes 24 proteins. The conservation region with the largest number of proteins is the operon encoding 27 ribosomal proteins. In addition, the species share only 57.8% (2,231/3,861) of orthologous proteins. Categories J (translation, ribosomal structure and biogenesis), F (nucleotide transport and metabolism), and L (replication, recombination and repair) are the top 3 conservative COG categories between the species, sharing 88.01%, 81.67%, and 80.65% of the orthologs, respectively.Comparison of cell cycle genes between P. zucineum and C. crescentusSince P. zucineum is phylogenetically closest to C. crescentus, and since the latter is a model organism for studies of the prokaryotic cell cycle [19,20], we compared the genes regulating the cell cycle between these species.The cell cycle of C. crescentus is controlled to a large extent by the master regulator CtrA, which controls the transcription of 95 genes involved in the cycle [19,20]. On the other hand, ctrA is regulated at the levels of transcription, phosphorylation, and proteolytic degradation by its target genes, e.g., DNA methyltransferase (CcrM) regulates the transcription of ctrA, histidine kinases (CckA, PleC, DivJ, DivL) regulate its activity, and ClpXP degrades it. These regulatory 'loops' enable CtrA to precisely control the progression of the cell cycle.P. zucineum has most of the orthologs mentioned above (Table 13). Among the 95 CtrA-regulated genes in C. crescentus, 75 have orthologs in the P. zucineum genome (Additional file 1). The fraction of CtrA-regulated genes with orthologs in P. zucineum (76.9%, 73/95) is significantly greater than the mean level of the whole genome (57.8%, 2,231/3,861), indicating that the CtrA regulatory system is highly conserved. Genes participating in regulating central events of the cell cycle, such as CcrM (CC0378), Clp protease (CC1963) and 14 regulatory proteins, except for one response regulator (CC3286), are present in the P. zucineum genome. The genes without counterparts in P. zucineum are mostly for functionally unknown proteins.Table 13Comparison of the signal transduction pathways regulating CtrA between the P. zucineum and the C. crescentusLocusLengthAmino acid Identity (%)AnnotationC. crescentusP. zucineumC. crescentusP. zucineumCC0378PHZ_c057735535980.00modification methylase CcrMCC1078PHZ_c093369166367.22cell cycle histidine kinase CckACC2482PHZ_c268184260663.78sensor histidine kinase PleCCC1063PHZ_c271259750453.83sensor histidine kinase DivJCC3484PHZ_c021876976967.66tyrosine kinase DivLCC2463PHZ_c130913012189.26polar differentiation response regulator DivKCC1963PHZ_c181720220580.19ATP-dependent protease, ClpP subunitCC1961PHZ_c181442042090.47ATP-dependent protease, ClpX subunitNotably, the sequence of CtrA is strikingly similar between P. zucineum and C. crescentus, with 93.07% identity of amino acid sequence and 89.88% identity of nucleotide sequence. In addition, they share identical promoters (p1 and p2) [21] and the motif (GAnTC) recognized by DNA methyltransferase (CcrM) (Figure 6) [22], suggesting that they probably share a similar regulatory loop of CtrA.Figure 6Nucleotide acid sequence alignment of the ctrA promoter regions (-200 to +21) of C. crescentus and P. zucineum. Blue background: identical nucleotides; \"-\": gaps; red and black box: P1 and P2 promoter; black underline: motif recognized by CcrM; red underline: first 21 nucleotides starting with initial codon \"ATG.\".Consistent with the results from in silico sequence analysis, the CtrA of P. zucineum can restore the growth of temperature-sensitive strain LC2195 (a CtrA mutant) of C. crescentus [23] at 37°C, indicating that the CtrA of P. zucineum can functionally compliment that of C. crescentus in our experimental conditions (data not shown).Taken together, the comparative genomics of P. zucineum and C. crescentus suggests that the cell cycle of the former is likely to be regulated similarly to that of the latter.Presence of ESTs of the strain in humanSince P. zucineum strain HLK1T can invade and persistently live in several human cell lines [1], we were curious about whether this microbe can infect humans. By blasting against the human EST database (dbEST release 041307 with 7,974,440 human ESTs) with the whole genome sequence of P. zucineum, we found 9 matched ESTs (Table 14), of which 3 were from a library constructed from tissue adjacent to a breast cancer, and 6 were from a library constructed from a cell line of lymphatic origin. The preliminary data suggest that P. zucineum may invade humans.Table 14Human ESTs matching the genome sequences of P. zucineumQuery GISample originQuery LengthQuery PositionChromosome PositionScoreE ValueSimilarity (%)BeginEndBeginEnd14251638Breast tissue1226411751,276,9141,277,0482042.00E-5394.078261474Breast tissue11611081,277,0421,276,9371672.00E-4296.3114251634Breast tissue142191341,277,0541,276,9372041.00E-5397.4633194938Lymphatic cell line244184411,029,5751,029,142749096.7733194696Lymphatic cell line65286521,029,5751,028,9311,166097.6733193754Lymphatic cell line65486541,029,5751,028,9291,191098.157117824Lymphatic cell line40574051,558,8311,558,433735098.2533194587Lymphatic cell line63876382,864,4702,863,8381,191098.897114909Lymphatic cell line34763473,498,6243,498,283654099.121All of three sequences come from the library BN0075 containing 182 ESTs; the original dataset was produced by a modification of the EST sequencing strategy ORESTES (open reading frame expressed sequences tags)[44,45]2All six sequences come from the library NIH_MGC_51 containing 2,381 ESTs; the original dataset was produced and released by the \"Mammalian Gene Collection\" project [46].ConclusionThis work presents the first complete bacterial genome in the genus Phenylobacterium. Genome analysis reveals the fundamental basis for this strain to invade and persistently survive in human cells. P. zucineum is phylogenetically closest to C. crescentus based on comparative genome analysis.MethodsBacterial growth and genomic library constructionP. zucineum strain HLK1Twas grown in LB (Luria-Bertani) broth at 37°C and then harvested for the preparation of genomic DNA[1]. Genomic DNA was prepared using a bacterial genomic DNA purification kit (V-Gene Biotech., Hangzhou, China) according to the manufacturer's instructions. Sheared DNA samples were fractionated to construct three different genomic libraries, containing average insert sizes of 2.0–2.5 kb, 2.5–3.0 kb and 3.5–4.0 kb. The resulting pUC18-derived library plasmids were extracted using the alkaline lysis method and subjected to direct DNA sequencing with automated capillary DNA sequencers (ABI3730 or MegaBACE1000).Sequencing and finishingThe genome of P. zucineum was sequenced by means of the whole genome shotgun method with the phred/phrap/consed software packages [24-27]. Sequencing and subsequent gene identification was carried out as described in our earlier publications [28-30]. Briefly, during the shotgun sequence phase, clones were picked randomly from three shotgun libraries and then sequenced from both ends. 44,667 successful sequence reads (>100 bp at Phred value Q13), accounting for 5.47× sequence coverage of the genome, were assembled into 563 sequence contigs representing 60 scaffolds connected by end-pairing information.The finishing phase involved iterative cycles of laboratory work and computational analysis. To reduce the numbers of scaffolds, reads were added into initial contig assembly by using failed universal primers as primers and by using plasmid clones that extended outwards from the scaffolds as sequence reaction templates. To resolve the low-quality regions, resequencing of the involved reads in low quality regions with universal primers and primer walking the plasmid clones were the first choice, otherwise, resequencing with alternate temperature conditions resolved the remaining low-quality regions. New sequence reads obtained from the above laboratory work were assembled into existing contigs, which yielded new contigs and new scaffolds connected by end-pairing information. Then, consed interface helped us to do nest round of laboratory work based on new arisen contig assembly. After about four iterative cycles of the above \"finish\" procedures to close gaps and to resolve the low-quality regions, the PCR product obtained by using total genomic DNA as template was sequenced from both ends to close the last physical gap. In addition, the overall sequence quality of the genome was further improved by using the following criteria: (1) two independent high-quality reads as minimal coverage, and (2) Phred quality value = Q40 for each given base. Collectively, 3,542 successful reads were incorporated into initial assembles during the finishing phase. The final assembly was composed of two circular \"contigs\", of which a smaller one with a protein cluster (including repA, repB, parA and parB) related to plasmid replication was assigned as the plasmid, and the larger one was the chromosome.AnnotationtRNA genes were predicted with tRNAscan-SE [31]. Repetitive sequences were detected by REPuter [32,33], coupled with intensive manual alignment. We identified and annotated the protein profiles of chromosome and plasmid with the same workstream. For the chromosome, the first set of potential CDSs in the chromosome was established with Glimmer 2.0 trained with a set of ORFs longer than 500 bp from its genomic sequence at default settings [34]. The resulting 5,029 predicted CDSs were BLAST searched against the NCBI non-redundant protein database to determine their homology [35]. 1,174 annotated proteins without the word \"hypothetical\" or \"unknown\" in their function description, and without frameshifts or in-frame stop codons, were selected as the second training set. The resulting second set of 4,018 predicted CDSs (assigned as \"predicted CDSs\") were searched against the NCBI non-redundant protein database. Predicted CDSs that accorded with the following BLAST search criteria were considered \"true proteins\": (1) 80% of the query sequence was aligned and (2) E-value ≤ 1e-10. Then, the ORFs extracted from the chromosome region among \"true proteins\" were searched against the NCBI non-redundant protein database. The ORFs satisfying the same criteria as true proteins were considered \"true ORFs\". Overlapping proteins were manually inspected and resolved, according to the principle we described previously [30]. The final version of the protein profile comprised three parts: true proteins, true ORFs, and predicted CDSs located in the rest of the genome. The translational start codon of each protein was identified by the widely used RBS script [36] and then refined by comparison with homologous proteins [30].To further investigate the function of each protein, we used InterProScan to search against the InterPro protein family database [37]. The up-to-date KEGG pathway database was used for pathway analysis [38]. All proteins were searched against the COG database which included 66 completed genomes [39,40]. The final annotation was manually inspected by comprehensively integrating the results from searching against the databases of nr, COG, KEGG, and InterPro.Phylogenetic tree construction16S rRNA genes were retrieved from 63 alphaproteobacteria, P. zucineum and Escherichia coli O157:H7 EDL933. A neighbor-joining tree with bootstrapping was built using MEGA [41]. The gammaproteobacterium E. coli was used as the outgroup to root the tree. To illustrate the evolutionary history of heat shock related proteins (RpoH, DnaK and GrpE), neighbor-joining trees based on the 16S rRNA genes and the above three proteins of 5 representative alphaproteobacteria (Sinorhizobium meliloti 1021, Brucella suis 1330, C. crescentus CB15, Rickettsia conorii str. Malish 7, Gluconobacter oxydans 621H), P. zucineum and E. coli O157:H7 EDL933 were constructed.Comparative genomicsSequence data for comparative analyses were obtained from the NCBI database . The database has 520 completely sequenced bacterial genomes (sequences downloaded on 2007/06/05). All P. zucineum ORFs were searched against the ORFs from all other bacterial genomes with BLASTP. The number of P. zucineum ORFs matched to each genome with significance (E value = 1e-10) was calculated.To illustrate the contribution of transcriptional regulators and two-component signal transduction proteins to environmental adaptation, we compared the mean fraction of these two types of proteins in bacteria living in 6 different habitats, as described by Merav Parter [42]. These are: (1) obligate bacteria that are necessarily associated with a host, (2) specialized bacteria that live in specific environments, such as marine thermal vents, (3) aquatic bacteria that live in fresh or seawater, (4) facultative bacteria, free-living bacteria that are often associated with a host, (5) multiple bacteria that live in many different environments, and (6) terrestrial bacteria that live in the soil. For bacteria with more than one sequenced strain, we chose only one strain for the comparative study. The numbers of bacterial species in each group were: 26 obligate, 5 specialized, 4 aquatic, 28 facultative, 27 multiple, and 3 terrestrial. We annotated the proteins of these 93 species with the same workflow used for P. zucineum and calculated the mean fraction of transcriptional regulators and two-component signal transduction proteins.In addition, we annotated the ORFs of 5 representative alphaproteobacteria with different habitats (multiple bacteria S. meliloti 1021 and G. oxydans 621H, facultative bacterium B. suis 1330, aquatic bacterium C. crescentus CB15, and obligate bacterium R. conorii str. Malish 7) using the same workflow and computed the distributions of proteins involved in environmental adaptation.Ortholog identificationAll proteins encoded by one genome were BLASTP searched against a database of proteins encoded by another genome [35], and vice versa. The threshold used in these comparisons was 1e-10. Orthology was identified if two proteins were each other's best BLASTP hit (best reciprocal match).Data accessibilityThe sequences reported in this paper have been deposited in the GenBank database. The accession numbers for chromosome and plasmid are CP000747 and CP000748, respectively.AbbreviationsEST: Expressed Sequence Tag; KEGG: Kyoto Encyclopedia of Genes and Genomes.Authors' contributionsXH and SH designed the project; YL, XX, ZD, ZL, ZY and JS performed the research; SH and BZ contributed new reagents\\analytical tools; YL, XX, and ZD analyzed the data; and XH, YL, and SH wrote the paper. All authors read and approved the final manuscript.Supplementary MaterialAdditional file 1Supplemental Table 1 Comparison of genes directly regulated by CtrA between P. zucineum and C. crescentus.Click here for file\n\nREFERENCES:\nNo References" |
