Introduction

After annotating a genome, we can generally group these genes into three types: first type are known genes which have been functionally characterized; second type are conserved hypothetical genes which are conserved in many organisms; third type are hypothetical genes which are not found in other organisms. Generally, a newly sequenced genome contains about thirty percents of genes that are annotated poorly or even wrongly [1]. Hence, these poorly characterized hypothetical genes might play an important role in our understanding of life and biology [2]. Given that so little are known about these unknown genes, it is a great challenge for scientists to select a research target worth spending years of time. Here we designed a scoring method to give each candidate protein a score based on different criteria ranking result and applied it to hypothetical proteins of two major oral pathogens, Porphyromonas gingivalis W83 and Streptococcus mutans UA159.

Materials and methods

• Definition
We define our candidates as proteins with "hypothetical protein" in their annotations, from NCBI.


• Hypothetical protein candidates
There are 709 hypothetical proteins in P. gingivalis W83 and 730 in S. mutans UA159. Plasmids are excluded.


• Criteria
We use 7 criteria in this analysis:
• Protein Size [3]
• Gene clustering number (inter-ORF length < 300 bp [4])
• Taxonomy distribution at strain, species, family level (blastp criteria: e-value < 0.0001, coverage > 30%)
• Protein domain scan, hmmpfam [5] (Default parameter)
• Predicted protein solubility [6]
• Length of disordered region (Hotloop catagory [7]; divided by total protein length)
• Upstream sequence TFBS detection (RegulonDB 5.0 [8]; PWM normalized by different GC% of P. gingivalis W83 and S. mutans UA159; upstream sequences, -400 to -1, are retrieved from RSAT [9])


• Scoring formula
For more detailed information, please contact Ching-Hung Tseng.

Results

Porphyromonas gingivalis W83 top-20, [Full ranking table]. (Click on the table header for sorting)

Rank	GI	LANL ID	TIGR ID	Size (aa)	Gene Cluster Size	Domain (#)	Solubility (%)	Disordered Region Length (aa)	Disordered Region Length Percentage (%)	Bacteria Strain (#)	Bacteria Species (#)	Bacteria Family (#)	TFBS (#)	Definition
1	34541330	PG1479	PG_1694	364	5	4	48.7	78	21.4	40	34	19	45	conserved hypothetical protein
2	34541597	PG1750	PG_2004	330	7	1	13.8	14	4.2	40	33	16	43	conserved membrane protein
3	34540102	PG0232	PG_0257	481	7	2	10.7	71	14.8	138	115	51	36	ABC transporter subunit
4	34539940	PG0058	PG_0069	504	11	2	6.8	50	9.9	101	87	36	33	conserved hypothetical protein (possible sugar kinase)
5	34540161	PG0301	PG_0325	209	8	2	41.9	31	14.8	50	43	28	32	conserved hypothetical protein (possible serine cycle enzyme,formiminotransferase cyclodeaminase)
6	34541437	PG1588	PG_1817	271	13	3	41	15	5.5	25	23	15	24	cytochrome c-type synthesis protein (cytochrome c biogenesis protein)
7	34539921	PG0042	PG_0048	367	4	2	28	68	18.5	31	30	44	37	conserved GTP-binding protein
8	34541242	PG1390	PG_1591	365	1	2	41	56	15.3	27	27	21	47	conserved hypothetical protein
9	34540104	PG0235	PG_0259	447	7	1	22.8	65	14.5	47	44	24	29	conserved hypothetical protein (possible ABC transporter related membrane protein)
10	34540684	PG0826	PG_0927	138	13	2	58.9	34	24.6	104	93	41	31	conserved hypothetical protein
11	34540774	PG0920	PG_1033	248	7	2	5.3	9	3.6	94	90	44	36	conserved hypothetical protein/possible ABC element with MSD domain
12	34541294	PG1443	PG_1653	407	7	7	9.4	48	11.8	33	24	10	34	conserved hypothetical protein
13	34540453	PG0590	PG_0652	100	11	1	46.5	0	0	19	17	14	30	conserved hypothetical protein
14	34540561	PG0699	PG_0778	239	8	1	3.8	33	13.8	87	75	35	43	possible glycoprotein endopeptidase
15	34540098	PG0228	PG_0253	152	7	1	46.4	45	29.6	31	31	21	43	conserved hypothetical protein
16	34541477	PG1627	PG_1868	173	5	1	50.7	0	0	21	16	10	32	conserved hypothetical protein
17	34540740	PG0890	PG_0996	302	8	7	12.1	110	36.4	26	23	41	47	conserved hypothetical protein
18	34540228	PG0368	PG_0401	513	7	3	15	156	30.4	106	93	37	32	conserved hypothetical protein(possible CTP synthase)
19	34541630	PG1784	PG_2043	364	3	2	17.9	32	8.8	64	50	25	31	conserved hypothetical protein
20	34540629	PG0769	PG_0859	312	7	3	20.1	61	19.6	16	14	11	35	conserved hypothetical protein

Streptococcus mutans UA159 top-20, [Full ranking table]. (Click on the table header for sorting)

Rank	GI	LANL ID	TIGR ID	Size (aa)	Gene Cluster Size	Domain (#)	Solubility (%)	Disordered Region Length (aa)	Disordered Region Length Percentage (%)	Bacteria Starin (#)	Bacteria Species (#)	Bacteria Family (#)	TFBS (#)	Definition
1	24379036	SMu0505	NTL02SM0504	86	17	1	51.4	0	0	79	71	26	58	conserved hypothetical protein
2	24380160	SMu1634	NTL02SM1628	247	20	2	64.6	34	13.8	56	44	12	57	conserved hypothetical protein; possible methyltransferase
3	24379999	SMu1473	NTL02SM1467	164	11	2	95.3	41	25	92	84	33	58	conserved hypothetical protein
4	24378760	SMu0228	NTL02SM0228	471	10	1	20.8	82	17.4	134	126	66	57	ABC transporter permease
5	24378552	SMu0020	NTL02SM0020	391	18	1	21.4	12	3.1	84	72	24	56	aspartate or aromatic amino acid aminotransferase
6	24379271	SMu0740	NTL02SM0739	378	8	3	25.9	24	6.3	36	30	17	59	aminotransferase
7	24380037	SMu1511	NTL02SM1505	288	14	1	16.3	23	8	116	93	37	57	conserved hypothetical protein, tetrapyrrole methylase family
8	24378835	SMu0303	NTL02SM0303	271	10	2	55.1	26	9.6	99	78	28	55	inner membrane protein
9	24378920	SMu0388	NTL02SM0388	275	4	4	38.5	11	4	61	46	14	58	conserved hypothetical protein, Cof family
10	24379860	SMu1332	NTL02SM1328	262	9	2	20.8	38	14.5	82	64	28	58	conserved hypothetical protein, NIF3-related
11	24380470	SMu1942	NTL02SM1938	657	9	2	27.5	127	19.3	44	36	8	60	conserved hypothetical protein (DHH family protein)
12	24380093	SMu1567	NTL02SM1561	82	10	1	80.8	0	0	40	39	12	58	conserved hypothetical protein
13	24379851	SMu1323	NTL02SM1319	327	7	10	91.9	25	7.6	23	15	4	59	conserved hypothetical protein, tetratricopeptide repeat family
14	24380155	SMu1629	NTL02SM1623	238	20	1	49.5	90	37.8	100	87	30	57	conserved hypothetical protein
15	24378740	SMu0208	NTL02SM0208	555	7	1	32	100	18	75	60	21	57	conserved hypothetical protein (possible kinase)
16	24378693	SMu0161	NTL02SM0161	200	12	1	29.9	15	7.5	21	19	30	59	conserved hypothetical protein (possible oxidoreductase)
17	24379602	SMu1073	NTL02SM1070	246	6	1	58.2	0	0	35	31	20	56	conserved hypothetical protein
18	24379206	SMu0675	NTL02SM0674	273	4	5	40.8	54	19.8	65	49	14	58	hydrolase, haloacid dehalogenase-like family
19	24379498	SMu0969	NTL02SM0966	110	10	7	78.2	32	29.1	63	54	19	58	DNA-binding protein
20	24379348	SMu0817	NTL02SM0816	92	16	1	60.2	13	14.1	31	27	20	59	conserved hypothetical protein

Remarks

In this analysis, we provide a ranked list of those hypothetical proteins in Porphyromonas gingivalis W83 and Streptococcus mutans UA195, both are oral pathogens of great interest. Those hyperlinks provide some evidence about that these hypothetical proteins might possess possible functions. This analysis is not an end but a start for further experiments and one day we might fulfill annotations of these hypothetical proteins.

References

1. Bork P. Powers and pitfalls in sequence analysis: the 70% hurdle. (2000) Genome Res. Vol. 10, 398-400.
2. Michael Y Galperin. Conserved 'hypothetical' proteins: new hints and new puzzles. (2001) Comparative and Functional Genomics Vol. 2, 14-18.
3. Taison Tan, et al. Length, protein-protein interaction, and complexity. (2005) Physica A Vol. 350, 52-62.
4. Ross Overbeek, et al. The use of gene clusters to infer functional coupling. (1999) Proc. Natl. Acad. Sci. USA Vol. 9, 2896-2901.
5. Sean Eddy. HMMER. http://hmmer.wustl.edu/
6. David L. Wilkinson, et al. Predicting the solubility of recombinant proteins in Escherichia coli. (1991) Bio/Technology Vol. 9, 443-448.
7. Rune Linding, et al. Protein Disorder Prediction: Implications for Structural Proteomics. (2003) Structure Vol. 11, 1453-1459.
8. Salgado H., et al. RegulonDB (version 5.0): Escherichia coli K-12 transcriptional regulatory network, operon organization, and growth conditions. (2006) Nucleic Acids Res. Database issue 34, D394-397.
9. van Helden, J. Regulatory sequence analysis tools. (2003) Nucleic Acids Res. Vol. 31, 3593-3956.

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