Complete genome of <it>Phenylobacterium zucineum </it>– a novel facultative intracellular bacterium isolated from human erythroleukemia cell line K562

1471-2164-9-386 1471-2164 Research article Complete genome of <it>Phenylobacterium zucineum </it>– a novel facultative intracellular bacterium isolated from human erythroleukemia cell line K562 Luo Yingfeng luoyf@big.ac.cn Xu Xiaoli jessie_xuxiaoli@hotmail.com Ding Zonghui dingzonghui@yahoo.com.cn Liu Zhen liuzhen@cancer.ac.cn Zhang Bing zhangbing@big.ac.cn Yan Zhiyu yanzhiyu1982@yahoo.com.cn Sun Jie sunjie1030@yahoo.com.cn Hu Songnian husn@big.ac.cn Hu Xun huxun@zju.edu.cn

Cancer Institute (Key Laboratory for Cancer Intervention and Prevention, National Ministry of Education, PR China; Key Laboratory of Molecular Biology in Medical Sciences, Zhejiang Province, PR China), the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, PR China

James D. Watson Institute of Genome Sciences, Zhejiang University, Hangzhou, PR China

Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, PR China

BMC Genomics 1471-2164 2008 9 1 386 http://www.biomedcentral.com/1471-2164/9/386 18700039 10.1186/1471-2164-9-386 21 8 2007 13 8 2008 13 8 2008 2008 Luo et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Phenylobacterium 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.

Results

Here, we report the whole genome sequence of the type strain HLK1^T. 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.

Conclusion

This 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.

Background

Phenylobacterium zucineum strain HLK1^Tis 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 (HLK1^T) 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 HLK1^Tstrain, 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 HLK1^Tmay 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, HLK1^Tis 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.

Results

Genome anatomy

The 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 1

Genome summary of P. zucineum Strain HLK1^T

Genomic Element

Chromosome

plasmid

Length (bp)

3,996,255

382,976

GC content (%)

71.35

68.54

Proteins

3, 534

327

Coding region of genome (%)

88.85%

81.94%

Proteins with known or predicted function

2,394(67.75%)

201(61.47%)

Conserved hypothetical proteins

560(15.84%)

25(7.65%)

Hypothetical proteins

580(16.41%)

101(30.88%)

rRNA operon

tRNAs

Proteins in each

[J] Translation, ribosomal structure and biogenesis

185 (5.24%)

3 (1.21%)

COG category

[K] Transcription

210 (5.94%)

22 (8.91%)

[L] Replication, recombination and repair

139 (3.93%)

23 (9.31%)

[D] Cell cycle control, cell division, chromosome partitioning

27 (0.76%)

[V] Defense mechanisms

51 (1.44%)

3 (1.21%)

[T] Signal transduction mechanisms

166 (4.7%)

24 (9.72%)

[M] Cell wall/membrane/envelope biogenesis

195 (5.52%)

15 (6.07%)

[N] Cell motility

62 (1.75%)

4 (1.62%)

[U] Intracellular trafficking, secretion, and vesicular transport

96 (2.72%)

13 (5.26%)

[O] Posttranslational modification, protein turnover, chaperones

151 (4.27%)

32 (12.96%)

[C] Energy production and conversion

188 (5.32%)

16 (6.48%)

[G] Carbohydrate transport and metabolism

161 (4.56%)

15 (6.07%)

[E] Amino acid transport and metabolism

293 (8.29%)

5 (2.02%)

[F] Nucleotide transport and metabolism

58 (1.64%)

3 (1.21%)

[H] Coenzyme transport and metabolism

116 (3.28%)

3 (1.21%)

[I] Lipid transport and metabolism

215 (6.09%)

12 (4.86%)

[P] Inorganic ion transport and metabolism

223 (6.31%)

24 (9.72%)

[Q] Secondary metabolites biosynthesis, transport and catabolism

152(4.3%)

9 (3.64%)

[R] General function prediction only

444 (12.57%)

28 (11.34%)

[S] Function unknown

307 (8.69%)

20 (8.10%)

Figure 1

Circular representation of the P. zucineum strain HLK1^Tchromosome and plasmid (smaller circle)

Circular representation of the P. zucineum strain HLK1^Tchromosome 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 2

Repetitive elements in the P. zucineum genome

Repeat ID

Length bp

DR¹

Number of copies

Position of insertion

Identity (%)

Coding information

Complete²

Partial

Chromosome

Plasmid

Repeat01³

2,587

>99

Transposase

Repeat02⁴

1,262

100

Transposase

Repeat03⁵

1,392

100

Transposase

Repeat04⁶

1,257

100

Transposase

Repeat05

1,554

>98

Hypothetical protein

Repeat06

1,136

>90

Isovaleryl-CoA dehydrogenase

Repeat07

1,077

>98

2-nitropropane dioxygenase

Repeat08

≈130

>90

Noncoding repeats

¹Size 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 elements

On 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 metabolism

The 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 3

Phenylalanine-degrading enzymes in the P. zucineum genome

Gene

P. zucineum Locus

Length (bp)

Alignment coverage (%)

Score

Amino acid Identity (%)

Gene name

P. putida

P. zucineum

P. putida

P. zucineum

phhA

PHZ_c1409

262

308

83.59

71.75

219

48.65

phenylalanine-4-hydroxylase

phhB

PHZ_c0077

118

79.66

93.81

38.5

26.32

carbinolamine dehydratase

tryB

PHZ_c1644

398

406

60.05

57.39

33.9

21.86

tyrosine aminotransferase

hpd

PHZ_c2833

358

374

98.32

93.58

398

57.98

4-hydroxyphenylpyruvate dioxygenase

hmgA

PHZ_c2831

433

377

60.28

67.64

53.5

22.3

homogentisate 1,2-dioxygenase

hmgB

PHZ_c0313

430

226

9.77

18.14

27.7

39.53

fumarylacetoacetate hydrolase

hmgC

PHZ_c0314

210

212

98.1

98.11

213

51.67

maleylacetoacetate isomerase

Functional elements responding to environmental transition

HLK1^Tis 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 4

Transcriptional regulators in the P. zucineum genome

Family name

Action type

Chromosome

Plasmid

Proposed roles

AsnC family

Activator/repressor

Amino acid biosynthesis

AraC family

Activator

Carbon metabolism, stress response and pathogenesis

ArsR family

Repressor

Metal resistance

BlaI family

Repressor

Penicillin resistance

Cold shock family

Activator

Low-temperature resistance

Cro/CI family

Repressor

Unknown²

Crp/Fnr family

Activator/repressor

Global responses, catabolite repression and anaerobiosis

GntR family

Repressor

General metabolism

LacI family

Repressor

Carbon source utilization

LuxR family

Activator

Quorum sensing, biosynthesis and metabolism, etc.

LysR family

Activator/repressor

Carbon and nitrogen metabolism

MarR family

Activator/repressor

Multiple antibiotic resistance

MerR family

Repressor

Resistance and detoxification

TetR family

Repressor

Biosynthesis of antibiotics, efflux pumps, osmotic stress, etc.

XRE family

Repressor

Unknown (initial function is lysogeny maintenance)

Other types²

Total

154

¹Initial function is related to controlling the expression of phage gene

²"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 5

Extracytoplasmic function (ECF) sigma factors in the P. zucineum genome

Locus

Location of proteins

COG category

Genomic element

5'-end

3'-end

PHZ_p0151

Plasmid

171,032

170,316

COG1595¹

PHZ_p0174

Plasmid

208,703

208,053

COG1595

PHZ_p0192

Plasmid

229,133

228,516

COG1595

PHZ_c0249

Chromosome

249,840

250,553

COG1595

PHZ_c0301

Chromosome

296,299

295,706

COG1595

PHZ_c1475

Chromosome

1,676,920

1,677,492

COG1595

PHZ_c1529

Chromosome

1,730,783

1,731,403

COG1595

PHZ_c1531

Chromosome

1,732,219

1,732,800

COG1595

PHZ_c1907

Chromosome

2,134,971

2,135,507

COG1595

PHZ_c2171

Chromosome

2,447,581

2,448,396

COG1595

PHZ_c2233

Chromosome

2,526,836

2,527,369

COG1595

PHZ_c2394

Chromosome

2,724,759

2,725,307

COG1595

PHZ_c2577

Chromosome

2,965,250

2,964,390

COG1595

PHZ_c2585

Chromosome

2,970,368

2,969,811

COG1595

PHZ_c2684

Chromosome

3,077,272

3,076,727

COG1595

PHZ_c0569

Chromosome

605,441

604,233

COG4941²

PHZ_c3154

Chromosome

3,582,010

3,583,269

COG4941

¹COG1595, DNA-directed RNA polymerase specialized sigma subunit, sigma24 homolog;

²COG4941, predicted RNA polymerase sigma factor containing a TPR repeat domain

Table 6

Distribution of heat shock related proteins in P. zucineum and representative alphaproteobacteria with different living habitats

Content\Species

S. meliloti

B. suis

C. crescentus

P. zucineum

R. conorii

G. oxydans

Chromosome

Plasmid

rpoH, heat shock sigma factor¹

dnaK, molecular chaperone²(Hsp70)

grpE, molecular chaperone (co-chaperonin of Hsp70)

dnaK-like molecular chaperone

dnaJ, molecular chaperone

dnaJ-like molecular chaperone

groEL, molecular chaperone (hsp60)

groES, molecular chaperone (Hsp10, co-chaperonin of Hsp60)

molecular chaperone Hsp20

molecular chaperone Hsp33

¹rpoH may be responsible for the expression of some or all heat shock proteins

²The function of molecular chaperones is to protect unfolded proteins induced by stress factors through renaturation or degradation in cooperation with protease.

Figure 2

Comparative analysis of transcriptional regulators and two-component signal transduction proteins in 6 groups of bacteria classified according to their habitats

Comparative 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 7

Chemotaxis proteins in the P. zucineum genome

Locus P. zucineum

5'-end

3'-end

Name

Orthologs C. crescentus

Operon

Best BLAST match

PHZ_c0690

753,270

753,812

chemotaxis protein CheW

M. magneticum AMB-1

PHZ_c0691

753,812

755,218

chemotaxis protein methyltransferase CheR

M. magnetotacticum MS-1

PHZ_c0692

755,240

755,836

chemotaxis signal transduction protein

Rhodospirillum centenum

PHZ_c0693

755,836

757,488

methyl-accepting chemotaxis protein

M. magneticum AMB-1

PHZ_c0694

757,501

759,642

chemotaxis histidine kinase CheA

M. magnetotacticum MS-1

PHZ_c0695

759,642

760,709

chemotaxis response regulator CheB

Rhodospirillum centenum

PHZ_c3230

3,661,514

3,661,050

CheE protein

C. crescentus CB15

PHZ_c3231

3,662,099

3,661,527

chemotaxis protein CheYIII

CC0440

C. crescentus CB15

PHZ_c3233

3,662,860

3,662,477

chemotaxis protein CheYII

CC0591

R. palustris CGA009

PHZ_c3234

3,663,186

3,666,188

chemotaxis histidine kinase CheA

CC0594

Azospirillum brasilense

PHZ_c3235

3,666,188

3,666,733

chemotaxis protein CheW

CC0595

Rhodospirillum centenum

PHZ_c3236

3,666,786

3,669,191

methyl-accepting chemotaxis protein McpH

CC3349

R. palustris CGA009

PHZ_c3237

3,670,166

3,669,336

chemotaxis protein methyltransferase CheR

CC0598

R. palustris HaA2

PHZ_c3238

3,671,242

3,670,166

chemotaxis response regulator CheB

CC0597

M. magneticum AMB-1

PHZ_c3371

3,820,121

3,819,669

CheE protein

CC0441

C. crescentus CB15

PHZ_c3372

3,820,729

3,820,124

chemotaxis protein CheYIII

C. crescentus CB15

PHZ_c3373

3,821,034

3,820,729

CheU protein

CC0439

C. crescentus CB15

PHZ_c3374

3,821,651

3,821,082

chemotaxis protein CheD

CC0438

C. crescentus CB15

PHZ_c3375

3,822,037

3,821,651

chemotaxis protein CheYII

CC0437

C. crescentus CB15

PHZ_c3376

3,823,068

3,822,040

chemotaxis response regulator CheB

CC0436

C. crescentus CB15

PHZ_c3377

3,823,955

3,823,068

chemotaxis protein methyltransferase CheR

CC0435

A. cryptum JF-5

PHZ_c3378

3,824,410

3,823,946

chemotaxis protein CheW

CC0434

Rhizobium etli CFN 42

PHZ_c3379

3,826,614

3,824,422

chemotaxis histidine kinase CheA

CC0433

A. cryptum JF-5

PHZ_c3380

3,826,997

3,826,635

chemotaxis protein CheYI

CC0432

Caulobacter vibrioides

PHZ_c3381

3,827,299

3,826,997

CheX protein

CC0431

Sinorhizobium meliloti

PHZ_c3382

3,829,234

3,827,306

methyl-accepting chemotaxis protein McpA

CC0430

A. cryptum JF-5

PHZ_c0101

94,220

93,750

CheE protein

scatted

C. crescentus CB15

PHZ_c0102

94,795

94,220

chemotaxis protein CheYIII

scatted

C. crescentus CB15

PHZ_c0297

292,469

292,864

chemotaxis protein CheYIV

CC3471

scatted

C. crescentus CB15

PHZ_c0298

292,867

293,679

chemotaxis protein methyltransferase CheR

CC3472

scatted

C. crescentus CB15

PHZ_c0732

803,383

804,876

methyl-accepting chemotaxis protein McpB

CC0428

scatted

C. crescentus CB15

PHZ_c0961

1,057,134

1,058,720

methyl-accepting chemotaxis protein McpI

CC2847

scatted

R. palustris CGA009

PHZ_c1198

1,380,883

1,383,294

methyl-accepting chemotaxis protein McpU

scatted

A. cryptum JF-5

PHZ_c1199

1,383,297

1,383,758

chemotaxis protein CheW1

scatted

Sinorhizobium meliloti

PHZ_c1687

1,890,274

1,891,176

chemotaxis MotB protein

CC1573

scatted

C. crescentus CB15

PHZ_c1936

2,169,634

2,169,939

chemotactic signal response protein CheL

CC2583

scatted

C. crescentus CB15

PHZ_c2211

2,499,744

2,499,274

chemotaxis protein CheYIII

scatted

O. alexandrii HTCC2633

PHZ_c2392

2,720,611

2,720,144

chemotaxis protein CheYIII

scatted

C. crescentus CB15

PHZ_c2741

3,142,750

3,143,238

chemotaxis protein CheYIII

CC3155

scatted

C. crescentus CB15

PHZ_c3123

3,549,150

3,550,016

chemotaxis MotA protein

CC0750

scatted

C. crescentus CB15

PHZ_c3401

3,848,811

3,850,766

methyl-accepting chemotaxis protein McpA

scatted

C. vibrioides

Table 8

Flagella genes in the P. zucineum genome

Locus

5'-end

3'-end

Name

Gene symbol

Proposed role

PHZ_c0080

75,413

76,462

flagellin modification protein FlmA

flmA

regulator

PHZ_c0081

76,467

77,621

flagellin modification protein FlmB

flmB

regulator

PHZ_c0745

816,772

818,034

flagellar hook-length control protein FliK

fliK

flagellar structure

PHZ_c0787

868,051

866,696

flagellar hook protein FlgE

flgE

flagellar structure

PHZ_c0788

868,860

868,171

flagellar hook assembly protein FlgD

flgD

flagellar structure

PHZ_c0789

870,604

868,865

flagellar hook length determination protein

flage

regulator

PHZ_c0790

870,819

872,918

flagellar hook-associated protein

flaN

flagellar structure

PHZ_c0791

872,933

873,862

flagellin and related hook-associated proteins

flagellar structure

PHZ_c0853

945,008

946,354

flagellum-specific ATP synthase FliI

fliI

protein export ATPase

PHZ_c0854

946,354

946,758

fliJ protein

fliJ

flagellar structure

PHZ_c0857

950,714

948,621

flagellar biosynthesis protein FlhA

flhA

export apparatus

PHZ_c0859

952,470

952,138

flagellar motor switch protein FliN

fliN

motor

PHZ_c0860

953,126

952,479

flbE protein

flbE

regulator

PHZ_c0861

954,151

953,126

flagellar motor switch protein FliG

fliG

motor

PHZ_c0862

955,794

954,151

flagellar M-ring protein FliF

fliF

flagellar structure

PHZ_c0913

1,007,753

1,006,992

flagellar L-ring protein FlgH

flgH

flagellar structure

PHZ_c0914

1,008,508

1,007,753

distal basal-body ring component protein FlaD

flaD

flagellar structure

PHZ_c0915

1,009,300

1,008,515

flagellar basal-body rod protein FlgG

flgG

flagellar structure

PHZ_c0916

1,010,052

1,009,318

flagellar basal-body rod protein FlgF

flgF

flagellar structure

PHZ_c0917

1,010,272

1,010,874

flagellar basal body-associated protein FliL

fliL

flagellar structure

PHZ_c0918

1,010,910

1,011,983

flagellar motor switch protein FliM

fliM

motor

PHZ_c0922

1,017,085

1,016,351

flagellar biosynthesis protein FliP

fliP

export apparatus

PHZ_c0923

1,017,420

1,017,151

flagellar protein FliO

fliO

export apparatus

PHZ_c0924

1,017,502

1,017,918

flagellar basal-body rod protein FlgB

flgB

flagellar structure

PHZ_c0925

1,017,942

1,018,355

flagellar basal-body rod protein FlgC

flgC

flagellar structure

PHZ_c0926

1,018,370

1,018,678

flagellar hook-basal body complex protein FliE

fliE

flagellar structure

PHZ_c0930

1,021,796

1,022,056

flagellar biosynthesis protein FliQ

fliQ

export apparatus

PHZ_c0931

1,022,079

1,022,837

flagellar biosynthesis protein FliR

fliR

export apparatus

PHZ_c0932

1,022,837

1,023,913

flagellar biosynthesis protein FlhB

flhB

export apparatus

PHZ_c1380

1,563,281

1,562,745

putative flagella accessory protein FlaCE

flaCE

flagellar structure

PHZ_c1381

1,565,145

1,563,358

flagellin modification protein FlmG

flmG

regulator

PHZ_c1382

1,565,343

1,565,765

flagellar repressor protein FlbT

flbT

regulator

PHZ_c1383

1,565,782

1,566,093

flagellar biosynthesis regulator FlaF

flaF

regulator

PHZ_c1384

1,566,375

1,567,202

flagellin FljM

fljM

flagellar structure

PHZ_c1385

1,567,469

1,568,314

flagellin FljM

fljM

flagellar structure

PHZ_c1386

1,568,434

1,568,724

flagellin FlaG

flaG

flagellar structure

PHZ_c1387

1,568,887

1,569,720

flagellin FljL

fljL

flagellar structure

PHZ_c1935

2,168,522

2,169,634

flagellar P-ring protein FglI

fglI

flagellar structure

PHZ_c1937

2,169,942

2,170,382

flagellar basal-body protein FlbY

flbY

flagellar structure

PHZ_c2595

2,982,550

2,983,593

flagellin modification protein FlmD

flmD

regulator

PHZ_c2597

2,984,874

2,986,508

flagellin modification protein FlmG

flmG

regulator

PHZ_c2599

2,989,315

2,989,974

flmC; flagellin modification protein FlmC

flmC

regulator

PHZ_c2600

2,990,549

2,989,977

flagellin modification protein FlmH

flmH

regulator

The 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 1011.

Table 9

Distributions of proteins involved in environmental adaptation in P. zucineum and representative alphaproteobacteria with different living habitats

Species

S. meliloti

B. suis

C. crescentus

P. zucineum

R. conorii

G. oxydans

Genome size (Mb)

6.69

3.32

4.02

4.38

1.27

2.92

GC content (%)

62.2

57.3

67.2

71.1

32.4

60.8

Habitat

Multiple¹

Facultative¹

Aquatic¹

Facultative²

Obligate¹

Multiple³

ECF, 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 chaperone

Flagellar protein

Chemotaxis protein

Pilus protein

Sec-dependent secretion system

Sec-independent secretion system

Type II secretory protein

Type IV secretory protein

¹The 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 10

Type IV secretion systems in the P. zucineum genome

Locus

Location of protein

Name

Genomic element

5'-end

3'-end

PHZ_p0007

Plasmid

6,786

7,445

type IV secretion protein, VirB1

PHZ_p0008

Plasmid

7,483

7,800

type IV secretion protein, VirB2

PHZ_p0009

Plasmid

7,816

8,148

type IV secretion protein, VirB3

PHZ_p0010

Plasmid

8,144

10,546

type IV secretion protein, VirB4

PHZ_p0011

Plasmid

10,546

11,298

type IV secretion protein, VirB5

PHZ_p0012

Plasmid

11,553

12,488

type IV secretion protein, VirB6

PHZ_p0013

Plasmid

12,816

13,493

type IV secretion protein, VirB8

PHZ_p0014

Plasmid

13,493

14,320

type IV secretion protein, VirB9

PHZ_p0015

Plasmid

14,320

15,543

type IV secretion protein, VirB10

PHZ_p0016

Plasmid

15,543

16,538

type IV secretion protein, VirB11

PHZ_c1506

Chromosome

1,709,481

1,709,999

type IV secretion protein, TraF

PHZ_c1508

Chromosome

1,711,058

1,712,773

type IV secretion protein, VirD2

PHZ_c1509

Chromosome

1,712,790

1,714,763

type IV secretion protein, VirD4

PHZ_c1512

Chromosome

1,716,262

1,717,242

conjugal transfer protein, TrbB

PHZ_c1513

Chromosome

1,717,242

1,717,559

conjugal transfer protein, TrbC

PHZ_c1514

Chromosome

1,717,562

1,717,828

conjugal transfer protein, TrbD

PHZ_c1515

Chromosome

1,717,836

1,720,283

conjugal transfer protein, TrbE

PHZ_c1516

Chromosome

1,720,283

1,721,014

conjugal transfer protein, TrbJ

PHZ_c1517

Chromosome

1,721,238

1,722,398

conjugal transfer protein, TrbL

PHZ_c1518

Chromosome

1,722,401

1,723,084

conjugal transfer protein, TrbF

PHZ_c1519

Chromosome

1,723,087

1,724,064

conjugal transfer protein, TrbG

PHZ_c1520

Chromosome

1,724,070

1,725,212

conjugal transfer protein, TrbI

PHZ_c2348

Chromosome

2,660,517

2,660,813

type IV secretion protein, VirB2

PHZ_c2349

Chromosome

2,660,809

2,661,144

type IV secretion protein, VirB3

PHZ_c2350

Chromosome

2,661,119

2,663,497

type IV secretion protein, VirB4

PHZ_c2352

Chromosome

2,664,374

2,665,309

type IV secretion protein, VirB6

PHZ_c2353

Chromosome

2,665,482

2,666,159

type IV secretion protein, VirB8

PHZ_c2354

Chromosome

2,666,159

2,667,004

type IV secretion protein, VirB9

PHZ_c2355

Chromosome

2,667,004

2,668,041

type IV secretion protein, VirB10

PHZ_c2356

Chromosome

2,668,046

2,669,035

type IV secretion protein, VirB11

PHZ_c2357

Chromosome

2,669,091

2,670,872

type IV secretion protein, VirD4

To 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 3

Neighbor-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

Neighbor-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 cycle

To survive intracellularly, P. zucineum must succeed in adhering to and subsequently invading the host cell 12, defending against a hostile intracellular environment 13141516, 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 11

Pilus proteins in the P. zucineum genome

Locus

5'-end

3'-end

Name

Gene symbol

PHZ_c0356

362,116

362,289

pilus subunit protein PilA

pilA

PHZ_c2992

3,412,800

3,413,318

Flp pilus assembly protein TadG

tadG

PHZ_c2995

3,415,220

3,415,468

Flp pilus assembly protein, pilin Flp

PHZ_c2996

3,415,532

3,416,023

Flp pilus assembly protein, protease CpaA

cpaA

PHZ_c2997

3,416,039

3,416,899

pilus assembly protein CpaB

cpaB

PHZ_c2998

3,416,899

3,418,350

pilus assembly protein CpaC

cpaC

PHZ_c2999

3,418,355

3,419,587

pilus assembly protein CpaE

cpaE

PHZ_c3000

3,419,594

3,420,991

pilus assembly protein CpaF

cpaF

PHZ_c3001

3,421,030

3,421,944

Flp pilus assembly protein TadB

tadB

PHZ_c3002

3,421,944

3,422,903

Flp pilus assembly protein TadC

tadC

PHZ_c3027

3,451,637

3,452,566

Flp pilus assembly protein CpaB

cpaB

PHZ_c3028

3,452,580

3,453,893

Flp pilus assembly protein, secretin CpaC

cpaC

PHZ_c3029

3,453,893

3,455,056

Flp pilus assembly protein, ATPase CpaE

cpaE

PHZ_c3030

3,455,059

3,456,489

Flp pilus assembly protein ATPase CpaF

cpaF

PHZ_c3031

3,456,489

3,457,445

Flp pilus assembly protein TadB

tadB

PHZ_c3032

3,457,492

3,458,391

Flp pilus assembly protein TadC

tadC

The 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 12

TonB-dependent receptors in the P. zucineum genome

Annotation

Chromosome

Plasmid

COG category

TonB-dependent receptor

COG1629¹

TonB-dependent receptor vitamin B12

COG4206²

TonB-dependent receptor

COG4771³

¹COG1629, Outer membrane receptor proteins, mostly Fe transport

²COG4206, Outer membrane cobalamin receptor protein

³COG4774, Outer membrane receptor for monomeric catechols

Comparative genomics between P. zucineum and C. crescentus

Comparative 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 4

List of top 10 complete sequenced bacteria closest to P. zucineum

List 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. zucineum

Figure 5

Neighbor-joining tree of the alphaproteobacteria, inferred from 16S rRNA genes

Neighbor-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. crescentus

Since P. zucineum is phylogenetically closest to C. crescentus, and since the latter is a model organism for studies of the prokaryotic cell cycle 1920, 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 1920. 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.

Additional file 1

Supplemental Table 1 Comparison of genes directly regulated by CtrA between P. zucineum and C. crescentus.

Click here for file

Table 13

Comparison of the signal transduction pathways regulating CtrA between the P. zucineum and the C. crescentus

Locus

Length

Amino acid Identity (%)

Annotation

C. crescentus

P. zucineum

C. crescentus

P. zucineum

CC0378

PHZ_c0577

355

359

80.00

modification methylase CcrM

CC1078

PHZ_c0933

691

663

67.22

cell cycle histidine kinase CckA

CC2482

PHZ_c2681

842

606

63.78

sensor histidine kinase PleC

CC1063

PHZ_c2712

597

504

53.83

sensor histidine kinase DivJ

CC3484

PHZ_c0218

769

67.66

tyrosine kinase DivL

CC2463

PHZ_c1309

130

121

89.26

polar differentiation response regulator DivK

CC1963

PHZ_c1817

202

205

80.19

ATP-dependent protease, ClpP subunit

CC1961

PHZ_c1814

420

90.47

ATP-dependent protease, ClpX subunit

Notably, 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 6

Nucleotide acid sequence alignment of the ctrA promoter regions (-200 to +21) of C. crescentus and P. zucineum

Nucleotide 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 human

Since P. zucineum strain HLK1^Tcan 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 14

Human ESTs matching the genome sequences of P. zucineum

Query GI

Sample origin

Query Length

Query Position

Chromosome Position

Score

E Value

Similarity (%)

Begin

End

Begin

End

14251638

Breast tissue¹

226

175

1,276,914

1,277,048

204

2.00E-53

94.07

8261474

Breast tissue

116

108

1,277,042

1,276,937

167

2.00E-42

96.31

14251634

Breast tissue

142

134

1,277,054

1,276,937

204

1.00E-53

97.46

33194938

Lymphatic cell line²

441

1,029,575

1,029,142

749

96.77

33194696

Lymphatic cell line

652

1,029,575

1,028,931

1,166

97.67

33193754

Lymphatic cell line

654

1,029,575

1,028,929

1,191

98.15

7117824

Lymphatic cell line

405

1,558,831

1,558,433

735

98.25

33194587

Lymphatic cell line

638

2,864,470

2,863,838

1,191

98.89

7114909

Lymphatic cell line

347

3,498,624

3,498,283

654

99.12

¹All 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)4445

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.

Conclusion

This 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.

Methods

Bacterial growth and genomic library construction

P. zucineum strain HLK1^Twas grown in LB (Luria-Bertani) broth at 37°C and then harvested for the preparation of genomic DNA1. 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 finishing

The genome of P. zucineum was sequenced by means of the whole genome shotgun method with the phred/phrap/consed software packages 24252627. Sequencing and subsequent gene identification was carried out as described in our earlier publications 282930. 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.

Annotation

tRNA genes were predicted with tRNAscan-SE 31. Repetitive sequences were detected by REPuter 3233, 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 3940. The final annotation was manually inspected by comprehensively integrating the results from searching against the databases of nr, COG, KEGG, and InterPro.

Phylogenetic tree construction

16S 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 genomics

Sequence data for comparative analyses were obtained from the NCBI database ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/Bacteria/. 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 identification

All 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 accessibility

The sequences reported in this paper have been deposited in the GenBank database. The accession numbers for chromosome and plasmid are CP000747 and CP000748, respectively.

Abbreviations

EST: Expressed Sequence Tag; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Authors' contributions

XH 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.

Acknowledgements

This work was supported in part by the Cheung Kong Scholars Programme (National Ministry of Education, China, and the Li Ka Shing Foundation, Hong Kong) to XH, a Natural Science Foundation of China grant (30672382) to XH, and a Zhejiang Natural Science Foundation, China, grant (R204204) to XH. We thank Dr. Lucy Shapiro (Department of Developmental Biology, Stanford University) for the gifts of the C. crescentus temperature sensitive strain LC2195 and the plasmid pSAL14. We are grateful to Dr. Iain Bruce (Department of Physiology, Zhejiang University School of Medicine) for English editing.

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