Predictive characterization of hypothetical proteins in Staphylococcus aureus NCTC 8325.

Staphylococcus aureus is one of the most common hospital acquired infections. It colonizes immunocompromised patients and with the number of antibiotic resistant strains increasing, medicine needs new treatment options. Understanding more about the proteins this organism uses would further this goal. Hypothetical proteins are sequences thought to encode a functional protein but for which little to no evidence of that function exists. About half of the genomic proteins in reference strain S. aureus NCTC 8325 are hypothetical. Since annotation of these proteins can lead to new therapeutic targets, a high demand to characterize hypothetical proteins is present. This work examines 35 hypothetical proteins from the chromosome of S. aureus NCTC 8325. Examination includes physiochemical characterization; sequence homology; structural homology; domain recognition; structure modeling; active site depiction; predicted protein-protein interactions; protein-chemical interactions; protein localization; protein stability; and protein solubility. The examination revealed some hypothetical proteins related to virulent domains and protein-protein interactions including superoxide dismutase, O-antigen, bacterial ferric iron reductase and siderophore synthesis. Yet other hypothetical proteins appear to be metabolic or transport proteins including ABC transporters, major facilitator superfamily, S-adenosylmethionine decarboxylase, and GTPases. Progress evaluating some hypothetical proteins, particularly the smaller ones, was incomplete due to limited homology and structural information in public repositories. These data characterizing hypothetical proteins will contribute to the scientific understanding of S. aureus by identifying potential drug targets and aiding in future drug discovery.

Background

While Staphylococcus aureus is a natural bacterial inhabitant of nasal passages, it is a major cause of nosocomial infections of surgical wounds particularly involving indwelling medical devices [1]. It can also present as superficial skin lesions or localized abscesses turning into deep-seated infections such as furunculosis if left untreated. S. aureus causes toxic shock syndrome when it goes septic, a huge concern considering the rise of antibiotic resistance the organism has experienced. Other health issues related to internalized infections are heart and lung diseases such as endocarditis and necrotizing pneumonia, which are now being diagnosed in the younger community populations, rather than remaining solely a hospital acquired (HA) infection. Deaths have been reported in relation to these heart and lung infections [2].

Methicillin-resistant Staphylococcus aureus (MRSA) bacteria are resistant to all beta-lactam antibiotics such as penicillin, methicillin, amoxicillin, and oxacillin. In 2011, the Center for Disease Control estimate 80,000 invasive MRSA infections and 11,285 related deaths in the United States annually [3]. Most of these are nosocomial infections, though there are increases in community acquired (CA) MRSA infections, particularly among immunocompromised patients. Others in the community setting that have shown a tendency to acquire MRSA are those of younger age rather than older. According to Casey, the median age for CA-MRSA in 2010 was 24 years versus a median age of 61 years for nosocomial MRSA infections [4]. Another predictor of CA-MRSA infections was an increased number of antibiotics prescribed in the year before infection. There is no significant data showing a link between race and CA-MRSA infection rates, but obesity was determined as a risk factor.

CA-MRSA in the United States is of particular concern due to the USA300 strain gaining momentum globally, relative to its excessive production of exotoxins and its genetics of polyamine-resistance [5]. One of the most prominent mechanisms responsible for the virulence of USA300 is its Arginine Catabolic Mobile Element (ACME), which ultimately inhibits polyamines that perpetuate wound healing in patients [6]. The ACME of this CA-MRSA allows it to survive acidic environments that normally limit its colonization.

Due to documented increases of a global spread of CA-MRSA in just the past 20 years, a worldwide need for innovative therapies that target these divergent strains in new ways is of ultimate concern [2]. This directs attention to prediction work with hypothetical proteins in silico, which allows for further investigation into the S. aureus genome. Upon analyzing the phylogeny of its protein sequencing, prediction of the bacteria’s next mutation is possible, thus enhancing knowledge of its mechanisms of action. By this, science gains insight into receptor targets that inhibits reproduction of such a resistant and virulent species.

Approximately 50% of the S. aureus NCTC 8325 genome is comprised of hypothetical proteins. Hypothetical proteins are protein sequences by nucleic acid sequence only with unknown function [7]. These sequences have little to no experimental evidence for their function’s existence, characterized by a low identity to proteins with known function. Frequently, these nonconserved proteins do not follow established phylogenetic lineage. There are two groups of hypothetical proteins: uncharacterized protein families and domains of unknown function. The latter are experimentally identified proteins with no known structural domains related to function.

Several studies have characterized hypothetical proteins. Mohan and Venugopal examined ten hypothetical plasmid proteins in S. aureus in 2012 [8]. They characterized an ABC transporter ATPbinding protein, export proteins, and a protein related to the multiple antibiotic resistance family among others. In 2015, Varma and colleagues examined one hypothetical protein from S. aureus, selected for its size and Basic Local Alignment Search Tool (BLAST) result, which appears to bind to ribosomal subunits [7]. Shahbaaz and researchers predicted the function of 83 hypothetical proteins in Mycoplasma pneumoniae type 2a strain 309, several of which appear virulent [9]. Islam, et al., characterized six hypothetical proteins in Vibrio cholerae O139 predicting the function of an antibiotic resistance protein, an integrase enzyme, and a restriction endonuclease [10]. All used similar methods to those presented in this study.

With approximately half of all genomic protein sequences currently annotated as hypothetical, great potential exists for the discovery of new drug targets [10]. The pharmaceutical industry is struggling to discover and develop new drugs quickly and cheaply. Increasing the number of available targets that pharmaceutical agents could act on by characterizing hypothetical proteins may alleviate some of the pharmaceutical industry’s pressure. This could lead to novel and improved therapeutic agents for better patient care, increased corporate and hospital profits, and decreased drug prices for consumers.

Methodology

This study randomly selected 35 proteins from the Staphylococcus aureus NCTC 8325 chromosomal protein table that the National Center for Biotechnology Information (NCBI) classified as hypothetical. The protein loci were SAOUHSC_00010, SAOUHSC_00077, SAOUHSC_00082, SAOUHSC_00085, SAOUHSC_00091, SAOUHSC_00136, SAOUHSC_00145, SAOUHSC_00156, SAOUHSC_00219, SAOUHSC_00238, SAOUHSC_00303, SAOUHSC_00307, SAOUHSC_00308, SAOUHSC_00328, SAOUHSC_00423, SAOUHSC_00455, SAOUHSC_00548, SAOUHSC_00751, SAOUHSC_00766, SAOUHSC_00837, SAOUHSC_00972, SAOUHSC_01024, SAOUHSC_01291, SAOUHSC_01306, SAOUHSC_01402, SAOUHSC_01851, SAOUHSC_01931, SAOUHSC_01937, SAOUHSC_02471, SAOUHSC_02570, SAOUHSC_02770, SAOUHSC_02889, SAOUHSC_02901, SAOUHSC_02911, and SAOUHSC_02934.

Several algorithms characterized these hypothetical proteins. Position-Specific Iterative BLAST (PSI-BLAST) at NCBI identified potential homologs through secondary protein structure alignments. ExPASy’s Protparam server computed the number of amino acids, amino acid composition and frequencies, molecular weight, the total number of charged residues (aspartic acid plus glutamic acid for positively charged and the sum of arginine and lysine for negatively charged), theoretical isoelectric point (pI), extinction coefficient, instability index (II), aliphatic index (AI), and grand average hydropathy (GRAVY) [11].

Both Pfam and the conserved domain database BLAST (CDDBLAST) from NCBI, performed protein domain identification. Pfam is a comprehensive collection of multiple sequence alignments and Hidden Markov Models that represent protein domains and families [12]. The CDD-BLAST algorithm uses a PSI-BLAST variant to establish position-specific scoring matrices with the protein sequence [13]. Researchers frequently use Pfam and CDD-BLAST together to characterize parts of the protein involved in binding capability [8,9,10].

Tertiary structure predictions were completed by (PS)2, which is an automatic homology modeling server uses a protein sequence in pair-wise and multiple alignments though unions of PSI-BLAST, integrated molecular pathway level analysis, and multiple sequence alignment methods [14]. This approach combines information on sequence and secondary structure to detect homologous proteins with remote similarity and the target-template alignment. MODELLER software builds the protein’s three-dimensional structure containing all non-hydrogen atoms from homology or comparative modeling. The product is a Protein Data Bank (PDB) file used by 3DLigandSite for identifying potential active sites when possible otherwise 3DLigandSite used Pyre2 to attempt to model proteins from sequence [15].

Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) is a database of known and predicted protein interactions. It draws from genomic context, high-throughput experiments, conserved co-expression, and previous PubMed literature [16]. STRING integrates interaction information on functional and physical relationships. Search Tool for Interactions of Chemicals (STITCH) is a database equal to STRING in its capacity to predict a hypothetical protein’s functional associates in a biological network [17]. STITCH integrates information from scientific literature with several databases to focus on drug-target interactions, binding affinities, and biological pathways to predict chemical and protein associates to the protein query.

Two programs analyzed protein location within the cell. PSortB predicts the location of each protein [18]. The SOSUI server characterized a protein’s solubility and identified potential transmembrane regions [19]. Examining how cysteine forms disulfide bonds to stabilize the protein may be helpful. The DISULFIND predicted disulfide bridges and examined structural and functional properties of hypothetical proteins [20]. Default program settings were used for all analyses except for STITCH where the required confidence (score) was set to highest confidence (0.900).

Discussion:

Thirty-five chromosomal hypothetical proteins from S. aureus NCTC 8325 were randomly selected from 1509 possible hypothetical proteins. Characterization included homolog identification, physiochemical measurements, domain identification, active site description, binding partners, cellular location, and solubility calculations.

Sequence Similarity

PSI-BLAST compares protein secondary structures among proteins. Top PSI-BLAST result for each hypothetical protein is listed in Table 1. All hypothetical proteins matched proteins in S. aureus with 100% query coverage, except for SAOUHSC_01937, as PSIBLAST could not match SAOUHSC_01937. SAOUHSC_00010 fit a protein in S. aureus MRSA131. SAOUHSC_00328 matched a protein in S. aureus A5948. SAOUHSC_001024 hit a protein in S. aureus VRS1. Percent identity ranged from 97% to 100% with e-values of 0.0 to 4e-11, indicating strong matches between hypothetical proteins and their homologs.

Table 1

Top PSI-BLAST result for hypothetical proteins

Locus Tag	PSI-BLAST Match	Identity	E-value
SAOUHSC_00010	azaleucine resistance protein	100%	8.00E-163
SAOUHSC_00077	siderophore biosynthesis protein	99%	0
SAOUHSC_00082	diaminopimelate decarboxylase	99%	0
SAOUHSC_00085	membrane protein	99%	6.00E-146
SAOUHSC_00091	ligase	99%	0
SAOUHSC_00136	sulfonate ABC transporter ATP-binding protein	99%	6.00E-179
SAOUHSC_00145	4'-phosphopantetheinyl transferase	99%	8.00E-157
SAOUHSC_00156	outer surface protein 99%	0
SAOUHSC_00219	galactitol-1-phosphate 5-dehydrogenase	99%	0
SAOUHSC_00238	hypothetical protein	98%	3.00E-20
SAOUHSC_00303	hypothetical protein	97%	6.00E-25
SAOUHSC_00307	deacetylase SIR2	99%	0
SAOUHSC_00308	lipoate-protein ligase A	99%	0
SAOUHSC_00328	twin arginine-targeting protein translocase TatC	99%	2.00E-151
SAOUHSC_00423	methionine ABC transporter ATP-binding protein	99%	0
SAOUHSC_00455	signal peptidase II	99%	0
SAOUHSC_00548	glycosyl transferase family 1	99%	0
SAOUHSC_00751	hypothetical protein	99%	2.00E-69
SAOUHSC_00766	competence protein ComF	100%	1.00E-163
SAOUHSC_00837	hypothetical protein	100%	7.00E+00
SAOUHSC_00972	hypothetical protein	99%	4.00E-57
SAOUHSC_01024	hypothetical protein MQA_00274	100%	1.00E+00
SAOUHSC_01291	hypothetical protein	97%	2.00E+00
SAOUHSC_01306	LSM domain protein	98%	8.00E-34
SAOUHSC_01402	MSA protein	99%	4.00E-83
SAOUHSC_01851	hypothetical protein	97%	4.00E+00
SAOUHSC_01931	NTPase	99%	0
SAOUHSC_02471	hypothetical protein	99%	0
SAOUHSC_02570	AraC family transcriptional regulator	99%	0
SAOUHSC_02770	diaminopimelate epimerase	99%	0
SAOUHSC_02889	hypothetical protein	100%	4.00E-20
SAOUHSC_02901	GTPase	99%	0
SAOUHSC_02911	ATPase or DNA integration/ recombination1	99%	0
SAOUHSC_02934	hypothetical protein	97%	4.00E+00
1Ranked equal in top hit

Physiochemical Characterization

ExPASy calculated the physiochemical parameters listed in Table 2. Number of amino acids ranged from 30 to 1370 with molecular weights from 3544.3 to 163266.7. The theoretical isoelectric point, the pI where the protein would be most stable, was calculated from the number of negative and positive residues (Asp and Glu, Arg and Lys, respectively). The extinction coefficient values are for 280nm because that is the wavelength where proteins absorb light strongly while other substances common to protein solutions do not. The extinction coefficient for two smaller hypothetical proteins, SAOUHSC_01024 and SAOUHSC_01291, could not be determined because there were no Trp, Tyr, or Cys in the protein, so the protein should not be visible by UV spectrophotometry. The instability index (II) predicts if a protein would be stable in a test tube under normal conditions. Proteins with II values over 40 considered unstable. The aliphatic index (AI) represents the protein’s volume taken up by aliphatic side chains (Ala, Val, Leu, and Ile). The higher the AI, the wider the temperature range at which the protein will be stable. GRAVY measures the protein’s hydrophobicity. Values spanned -1.984 to 1.096 with higher scores meaning increased hydrophobicity for the protein.

Table 2

Physiochemical properties of hypothetical proteins

Protein	# AA	MW	pI	# neg	# pos	EC	II	AI	GRAVY
SAOUHSC_00010	231	25147	6.06	10	9	28670	28.84	125.45	1.048
SAOUHSC_00077	584	66433.1	5.1	73	50	75540	37.33	98.18	-0.152
SAOUHSC_00082	400	45759.8	5.86	52	37	53080	41.31	88.45	-0.3
SAOUHSC_00085	208	22980.1	6.9	22	22	13200	23.01	123.7	0.455
SAOUHSC_00091	412	47053.3	8.66	23	27	51020	28.94	138.37	0.966
SAOUHSC_00136	246	28095.3	6.76	28	26	13200	44.53	105	-0.296
SAOUHSC_00145	214	25261.9	8.26	21	23	47120	37.21	86.87	-0.335
SAOUHSC_00156	346	39868.6	6.05	42	35	21360	46.22	91.82	-0.323
SAOUHSC_00219	347	38401.3	5.84	44	37	39350	31.27	93.54	-0.069
SAOUHSC_00238	33	5199.3	9.6	2	5	9970	34.69	150.68	0.766
SAOUHSC_00303	30	3544.3	9.9	3	8	0	15.53	65	-0.8
SAOUHSC_00307	314	36397.1	5.29	44	31	44600	40.63	74.9	-0.505
SAOUHSC_00308	340	38847	5.1	53	42	48040	22.53	91.18	-0.394
SAOUHSC_00328	218	25382.7	9.32	6	13	38640	41.54	121.65	1.096
SAOUHSC_00423	341	38675.7	8.2	41	43	13200	22.69	91.96	-0.199
SAOUHSC_00455	267	30232.8	4.84	47	34	16430	33.71	102.92	-0.244
SAOUHSC_00548	496	58418.3	6.41	61	57	77380	38.68	87.22	-0.422
SAOUHSC_00751	104	12723.7	8.31	13	16	19535	70.95	56.25	-0.645
SAOUHSC_00766	224	26347.7	8.86	27	35	24005	37.86	90.62	-0.373
SAOUHSC_00837	37	4265.2	9.7	2	5	8480	29.62	121.35	0.584
SAOUHSC_00972	95	11193.6	4.59	16	9	10430	26.75	102.53	-0.5
SAOUHSC_01024	44	5162.6	5.05	12	10	None1	50.22	48.86	-1.984
SAOUHSC_01291	36	4267.4	10.47	2	9	None1	30.75	135.28	0.578
SAOUHSC_01306	63	7193.2	5.08	9	8	4470	29.79	117.46	-0.168
SAOUHSC_01402	133	15657.1	6.71	10	10	16390	36.75	152.41	1.021
SAOUHSC_01851	37	4504.4	10.17	0	7	5960	2.76	105.14	0.273
SAOUHSC_01931	1370	163266.7	5.95	201	185	241090	39.01	92.5	-0.481
SAOUHSC_01937	35	4041.9	9.4	1	3	8480	20.97	147.71	0.843
SAOUHSC_02471	468	56151.2	5.91	73	68	64765	33.09	90.51	-0.494
SAOUHSC_02570	651	76005.7	8.3	71	75	66170	40.78	104.62	-0.225
SAOUHSC_02770	273	31004	6.08	26	19	36370	43.01	78.13	-0.301
SAOUHSC_02889	42	5160	6.06	6	6	4595	9.37	88.1	-0.06
SAOUHSC_02901	296	32835.3	5.69	34	26	13910	43.56	101.08	0.118
SAOUHSC_02911	240	27789	8.25	32	35	28350	36.67	76.46	-0.464
SAOUHSC_02934	31	3652.3	8.16	2	3	2980	27.75	106.77	0.245
# AA, number of amino acids; MW, molecular weight; pI, theoretical isoelectric point; # neg, total number of negatively charged residues (Asp + Glu); # pos, total number of positively charged residues (Arg + Lys); EC, extinction coefficient assuming all pairs of Cys residues form cystines; II, instability index; AI, aliphatic index; GRAVY, grand average hydropathy. 1As there are no Trp, Tyr, or Cys in the region considered, protein should not be visible by UV spectrophotometry.

Domain Identification

CDD-BLAST and Pfam identified domains for hypothetical proteins. CDD-BLAST and Pfam results and domain descriptions in Table 3,Table 4, Table 5,Table 6, respectively. The programs could not find domains within proteins not listed. If both programs identified a domain, the CDD-BLAST tables identified and defined it (Table 3 and Table 4) and not repeated in the Pfam tables.

Table 3

CDD-BLAST domain data for hypothetical proteins

Locus Tag	Domains	E-value
SAOUHSC_00010	AzlC	3.45E-67
SAOUHSC_00077	lucA_lucC, FhuF, RhbC	6.96e-63, 4.42e-15, 7.18e-180
SAOUHSC_00082	PLPDE_III_PvsE_like, LysA	0e00, 1.42e-118
SAOUHSC_00085	MFS	1.49E-04
SAOUHSC_00091	O-antigen_lig	1.24E-05
SAOUHSC_00136	ABC_NrtD_SsuB_transporters, TauB	2.95e-106, 7.68e-115
SAOUHSC_00145	ACPS, Sfp	1.85e-10, 2.37e-63
SAOUHSC_00156	COG3589	9.06E-161
SAOUHSC_00219	sugar_DH, Tdh	0e00, 7.15e-97
SAOUHSC_00307	SIR2	1.32E-67
SAOUHSC_00308	LplA, Lip_prot_lig_C, lipoyltrans	4.74e-82, 2.33e-30, and 1.09e-69
SAOUHSC_00328	TatC	1.55E-50
SAOUHSC_00423	ABC_MetN_methionine_transporter, NIL, AbcC	3.27e-138, 6.03e-11, 7.16e-176
SAOUHSC_00455	YaaT	5.26E-102
SAOUHSC_00548	GT1_gtfA_like, DUF1975, TIGR02918	6.56e-146, 6.42e-56, 1.53e-31
SAOUHSC_00751	COG4357	3.47E-50
SAOUHSC_00766	PRTases_typeI, ComFC	2.38e-10, 4.47e-52
SAOUHSC_01931	AAA_16, AAA	5.36e-05, 5.87e-03
SAOUHSC_02570	HTH_AraC, HTH_ARAC	4.36e-04, 6.20e-14
SAOUHSC_02770	DapF	7.31E-74
SAOUHSC_02901	cobW, CobW_C, YejR	1.81e-57, 9.02e-10, 1.06e-77
SAOUHSC_02911	COG1636	3.25E-101

Table 4

Description of CDD-BLAST domains

Superfamily	Description
AzlC	Predicted branched-chain amino acid permease (azaleucine resistance)
IucA_IucC	IucA / IucC family
FhuF	Bacterial ferric iron reductase protein
RhbC	Siderophore synthetase component
PLPDE_III_PvsE_like	Type III Pyridoxal 5-phosphate (PLP)-Dependent Enzyme PvsE
LysA	Diaminopimelate decarboxylase
O-antigen_lig	O-antigen ligase like membrane protein
MFS	Major Facilitator Superfamily
ABC_NrtD_SsuB_transporters	ATP-binding cassette domain of the nitrate and sulfonate transporters
TauB	ABC-type nitrate/sulfonate/bicarbonate transport system, ATPase component
ACPS	4'-phosphopantetheinyl transferase superfamily
Sfp	Phosphopantetheinyl transferase
COG3589	Uncharacterized protein [Function unknown]
sugar_DH	NAD(P)-dependent sugar dehydrogenases
Tdh	Threonine dehydrogenase or related Zn-dependent dehydrogenase
SIR2	NAD-dependent protein deacetylase
Lip_prot_lig_C	Bacterial lipoate protein ligase C-terminus
lipoyltrans	Lipoyltransferase and lipoate-protein ligase
LplA	Lipoate-protein ligase A
TatC	Sec-independent protein secretion pathway component
ABC_MetN_methionine_transporter	ATP-binding cassette domain of methionine transporter
NIL	NIL domain
AbcC	ABC-type methionine transport system, ATPase component
YaaT	Cell fate regulator YaaT, PSP1 superfamily
GT1_gtfA_like	GT1 family of glycosyltransferases
DUF1975	Domain of unknown function
TIGR02918	Accessory Sec system glycosylation protein GtfA
COG4357	Uncharacterized protein, contains Zn-finger domain of CHY type
PRTases_typeI	Phosphoribosyl transferase (PRT)-type I domain
ComFC	Predicted amidophosphoribosyltransferases
AAA_16	AAA ATPase domain
AAA	ATPases associated with a variety of cellular activities
HTH_AraC	Bacterial regulatory helix-turn-helix proteins, AraC family
HTH_ARAC	Helix_turn_helix, arabinose operon control protein
DapF	Diaminopimelate epimerase
cobW	CobW/HypB/UreG, nucleotide-binding domain
CobW_C	Cobalamin synthesis protein cobW C-terminal domain
YejR	GTPase, G3E family
COG1636	Predicted ATPase, Adenine nucleotide alpha hydrolases (AANH) superfamily

Table 5

Pfam domain data for hypothetical proteins

Locus Tag	Pfam Domain	E-value
SAOUHSC_00010	AzlC	1.20E-37
SAOUHSC_00077	lucA_lucC, FhuF	2.1e-54, 5e-15
SAOUHSC_00082	Orn_Arg_deC_N,Orn_DAP_Arg_deC	9.7e-37, 2e-10
SAOUHSC_00091	Wzy_C	2.20E-13
SAOUHSC_00136	ABC_tran	3.90E-30
SAOUHSC_00145	ACPS	9.10E-08
SAOUHSC_00156	DUF871	3.50E-86
SAOUHSC_00219	ADH_N, ADH_zinc_N	8.8e-30, 2.8e-22
SAOUHSC_00308	BPL_LplA_LipB, Lip_prot_lig_C	4.6e-09,4.6e-26
SAOUHSC_00328	TatC	4.20E-41
SAOUHSC_00423	ABC_tran, NIL	2.8e-35, 4.3e-10
SAOUHSC_00455	PSP1	4.10E-26
SAOUHSC_00548	Glycos_transf_1	3.30E-32
SAOUHSC_00751	Zf-CHY	8.40E-12
SAOUHSC_02570	HTH_18	1.90E-14
SAOUHSC_02901	cobW, CobW_C	1.4e-45, 4.4e-09
SAOUHSC_02911	DUF208	8.90E-61

Table 6

Description of Pfam domains

Superfamily	Description
Orn_Arg_deC_N	Pyridoxal-dependent decarboxylase, pyridoxal binding domain
Orn_DAP_Arg_deC	Pyridoxal-dependent decarboxylase, Cterminal sheet domain
Wzy_C	O-Antigen ligase
ABC_tran	ABC transporter
DUF871	Bacterial protein of unknown function
ADH_N	Alcohol dehydrogenase GroES-like domain
ADH_zinc_N	Zinc-binding dehydrogenase
BPL_LplA_LipB	Biotin/lipoate A/B protein ligase family
PSP1	PSP1 C-terminal conserved region
Glycos_transf_1	Glycosyl transferases group 1
Zf-CHY	CHY zinc finger
HTH_18	Helix-turn-helix domain
DUF208	Uncharacterized BCR, COG1636

Active Site and Substrate Characterization

The (PS)2 server attempted to model each hypothetical protein. Template information including percent identity and e-value is in Table 7. Several proteins could not be modeled by (PS)2. Hypothetical proteins, SAOUHSC_01931 and SAOUHSC_02570, yielded an error message of computer language when (PS)2 attempted to model them. Attempted to report the problem to (PS)2 at chieh.bi91g@nctu.edu.tw, but no correction was made. The program could not find significant templates for other hypothetical proteins not listed.

Table 7

(PS)2 model data for hypothetical proteins

Protein	Template Structure	Template %ID	E-value
SAOUHSC_00010	Rh50 in NH3 transport	3B9W	14.46 0.053
SAOUHSC_00077	NRPS Condensation Enzyme	1L5A	11.36 3
SAOUHSC_00082	BTRK decarboxylase	2J66	28.79 2.20E-17
SAOUHSC_00085	Aquaporin-4	2D57	14.89 3.5
SAOUHSC_00091	GltPh transport protein	2NWL	13.46 0.024
SAOUHSC_00136	Multiple sugar binding transport ATP-binding protein	2D62	31.12 4.70E-29
SAOUHSC_00145	4'-phosphopantetheinyl transferase SFP-coenzyme A	1QR0	21.12 4.60E-18
SAOUHSC_00156	Conserved Protein of Unknown Function	2P0O	27.53 5.50E-24
SAOUHSC_00219	NAD+-dependent alcohol dehydrogenase	1RJW	28.73 2.50E-11
SAOUHSC_00307	Sir2 homologue F159A mutant-ADP ribose complex	1M2K	16.55 9.80E-10
SAOUHSC_00308	Lipoate-protein ligase a	1VQZ	35.99 1.20E-28
SAOUHSC_00328	Complex III with bound cytochrome C	3CX5	15.9 0.0051
SAOUHSC_00548	Family GT4 glycosyltransferase	2JJM	16.67 1.60E-06
SAOUHSC_00751	CHY zinc finger domain-containing protein 1 RING finger	2DKT	25.77 6.90E-06
SAOUHSC_00766	Glutamine PRPP amidotransferase	1ECF	15.64 0.024
SAOUHSC_00972	Methane monooxygenase regulatory protein	1CKV	23.33 8.4
SAOUHSC_01291	Conserved Protein of Unknown Function	1RLK	34.29 4.8
SAOUHSC_01402	Acetylcholine receptor pore	1OED	11.85 0.99
SAOUHSC_01937	S-adenosylmethionine decarboxylase	1VR7	31.25 2.5
SAOUHSC_02471	Site-specific DNA nickase	2EWF	12.18 2.7
SAOUHSC_02770	Diaminopimelate epimerase	2OTN	15.18 5.90E-09
SAOUHSC_02889	GTPase	1YRB	28.57 3.5
SAOUHSC_02901	Yija protein	1NIJ	24.61 2.50E-10
SAOUHSC_02911	Isoluecyl-tRNA lysidine synthetase	1WY5	12.24 0.81
SAOUHSC_02934	Human TPP1	2I46	17.61 1.1

3DLigandSite characterized the active site for hypothetical proteins. Figure 1 depicts the predicted active site with binding heterogens for the 12 of 22 proteins with the largest active sites. Table 8 lists predicted residues responsible for forming active sites and heterogens. There were insufficient homologous structures with ligands bound for other hypothetical proteins not listed in the table.

Figure 1

3DLigandSite Models for Proteins with the largest active site. Hypothetical proteins shown in grey with potential metallic heterogens shown as space fill and non-metallic heterogens as wireframe. Residues involved in bindings are blue.

Table 8

3DLigandsite active site predictions

Protein	Predicted Binding Site	Heterogens
SAOUHSC_00077	GLN140, SER263, SER264, SER265, ILE270, HIS278, LYS280, CYS326, ARG354,LYS355, PRO357, SER370, HIS424, GLN426, ASN427, LEU429, ARG443, ASP444	STU, ADP, MG, AMP, FMM, ATP
SAOUHSC_00082	MET49, GLU69, ARG138, HIS185, HIS187, SER190, GLY226, GLY227, GLY228,ILE229, GLU266, CYS267, GLY268, ARG269, PHE270, TYR373	PLP
SAOUHSC_00091	PHE340, PHE344, MET371, MET374	HEM, MG, FAD, FE, CA, FE2, ZN
SAOUHSC_00136	PHE11, VAL16, LYS35, SER36, GLY37, CYS38, GLY39, LYS40, SER41, THR42	ADP, MG, CA, ATP
SAOUHSC_00145	HIS43, ARG72, LYS74, LEU91, SER92, TYR93, ASP110, GLU149, LYS153	COA, MG
SAOUHSC_00156	PHE24, TYR133, PHE165	FMN, MG, CU, ZN
SAOUHSC_00219	CYS38, GLY39, SER40, HIS59, GLU60, GLU144, GLY168, CYS169, GLY170,SER171, ILE172, ASP192, ILE193, LYS197, SER212, SER235, SER236, THR241,ASN287	CHD, NDP, NAP, NAD, ZN
SAOUHSC_00307	CYS154, ARG184, CYS185, PRO186, LYS187, CYS188, ASP189, ALA190	ZN
SAOUHSC_00308	VAL80, ARG127, ASP129, LYS136, SER154, LEU156, VAL187	ADP, MG, AMP, ATP
SAOUHSC_00328	SER2, VAL4, ILE5, THR6, VAL7, ILE8, VAL9, VAL10, VAL12, GLN42, MET46,PHE49, VAL59	HEA
SAOUHSC_00423	PHE11, TYR39, GLY41, ALA42, GLY43, LYS44, SER45, THR46, LEU47, HIS199	ADP, MG, CA, ATP
SAOUHSC_00455	THR108, LYS111, LYS113	GTP, ADP, MG, FAS, CA, ATP, ZN
SAOUHSC_00548	LYS18, HIS246, ARG329, GLY406, PHE408, LEU410, ALA411	F6P, ADP, G6P, G1P, PLP, GLC
SAOUHSC_00751	ASN91, CYS94	ZN
SAOUHSC_00766	SER126, ASP193, ASP194	ADP, MG, AMP, CA
SAOUHSC_01931	GLY359, ILE360, GLY361, LYS362, SER363, HIS364, HIS489, PHE492, GLU496,PRO525, LEU526, LYS529	ADP, MG, ATP
SAOUHSC_02471	GLU1	MG
SAOUHSC_02570	MET15, VAL18, GLU20, ILE26, ILE52	NI, ARA, ZN
SAOUHSC_02770	GLU52	CA
SAOUHSC_02901	LEU10, GLY11, GLY12, GLY13, LYS14, THR15, THR16, GLU90, SER92, ASN154	ADP, MG, ADX, ATP
SAOUHSC_02911	HIS41, CYS43, CYS44, ALA45, PRO46, CYS47, SER48, TYR64, ALA66, SER68,ASN69, ARG79, MET135, ARG136, SER154	GTP, MG, AMP, NAP, ATP, ZN,SAM, G6P, CA, NAD

The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and Search Tool for Interactions of Chemicals (STITCH) predicted interactions with hypothetical proteins. Table 9 shows the top non-hypothetical protein interactions with the highest confidence from STRING. If a protein is not listed in the table, it did not have predicted functional partners or all predicted partners were other hypothetical proteins. Figure 2 illustrates the highest confidence interactions STITCH predicted with multiple proteins. Since the findings between STITCH and STRING were similar, if one non-hypothetical protein was predicted with highest confidence, it is listed in Table 9 but not shown in Figure 2.

Table 9

Top STRING predicted substrates

Protein	Substrate	Score
SAOUHSC_00077	Ornithine cyclodeaminase	0.842
SAOUHSC_00082	Ornithine cyclodeaminase	0.876
SAOUHSC_00085	Acetoin reductase	0.488
SAOUHSC_00091	Superoxide dismutase	0.461
SAOUHSC_00136	Putative DNA-binding/iron metalloprotein/AP endonuclease	0.488
SAOUHSC_00145	D-alanine-poly(phosphoribitol) ligase subunit 1	0.933
SAOUHSC_00156	N-acetylmuramic acid-6-phosphate etherase	0.974
SAOUHSC_00219	PTS system protein	0.968
SAOUHSC_00307	DNA-directed RNA polymerase subunit beta	0.541
SAOUHSC_00308	Lipoyl synthase	0.934
SAOUHSC_00328	mttA/Hcf106 family protein	0.919
SAOUHSC_00423	ABC transporter permease	0.999
SAOUHSC_00455	DNA polymerase III subunit delta	0.939
SAOUHSC_00548	Capsular polysaccharide biosynthesis protein	0.785
SAOUHSC_00751	UDP-N-acetylenolpyruvoylglucosamine reductase	0.805
SAOUHSC_00766	Biotin synthase	0.65
SAOUHSC_00837	Glycine cleavage system protein H	0.657
SAOUHSC_00972	Glycosyl transferase	0.859
SAOUHSC_01402	Cold shock protein	0.498
SAOUHSC_01851	Catabolite control protein	0.638
SAOUHSC_01937	Serine protease	0.636
SAOUHSC_02570	Protein A (spA)	0.762
SAOUHSC_02770	Peptide ABC transporter peptide-binding protein	0.782
SAOUHSC_02901	Imidazole glycerol phosphate synthase subunit hisF	0.441
SAOUHSC_02911	Ribonuclease HII (rnhB)	0.762
SAOUHSC_02934	Betaine aldehyde dehydrogenase	0.648

Figure 2

STITCH Chemical-Protein Interactions. Hypothetical proteins with more than one highest confidence binding partner (0.900) shown.

Cellular Location, Solubility, and Stability

PSortB predicted the cellular location of hypothetical proteins with results summarized in Table 10. PSortB was unable to determine cellular location for unlisted proteins.

Table 10

SOSUI results for transmembrane hypothetical proteins

Protein	Location	Localization Scores
SAOUHSC_00010	cytoplasmic membrane	10
SAOUHSC_00077	cytoplasmic membrane	8.16
SAOUHSC_00082	cytoplasmic	7.5
SAOUHSC_00085	cytoplasmic membrane	10
SAOUHSC_00091	cytoplasmic membrane	10
SAOUHSC_00136	cytoplasmic membrane	8.78
SAOUHSC_00145	cytoplasmic	7.5
SAOUHSC_00156	cytoplasmic	7.5
SAOUHSC_00219	cytoplasmic	9.67
SAOUHSC_00238	cytoplasmic membrane	9.55
SAOUHSC_00303	extracellular	8.91
SAOUHSC_00307	cytoplasmic	7.5
SAOUHSC_00308	cytoplasmic	9.97
SAOUHSC_00328	cytoplasmic membrane	10
SAOUHSC_00423	cytoplasmic membrane	8.78
SAOUHSC_00455	cytoplasmic	7.5
SAOUHSC_00548	cytoplasmic	7.5
SAOUHSC_00751	cytoplasmic	7.5
SAOUHSC_00766	cytoplasmic	7.5
SAOUHSC_00837	cytoplasmic membrane	9.55
SAOUHSC_00972	cytoplasmic	7.5
SAOUHSC_01291	cytoplasmic membrane	9.55
SAOUHSC_01402	cytoplasmic membrane	10
SAOUHSC_01931	cytoplasmic	7.5
SAOUHSC_01937	cytoplasmic membrane	9.55
SAOUHSC_02471	cytoplasmic	7.5
SAOUHSC_02889	cytoplasmic	7.5
SAOUHSC_02901	cytoplasmic	7.5
SAOUHSC_02911	cytoplasmic	7.5
SAOUHSC_02934	extracellular	8.91

SOSUI calculates the average hydrophobicity and determines if the protein is soluble from it. If hydrophobicity exists, that portion of the protein is labeled as a transmembrane region. Table 11 shows the transmembrane regions of the eight proteins. SOSUI deemed all other proteins soluble.

Table 11

PSortB cellular location of hypothetical proteins

Locus Tag	N terminal	Transmembrane Region	C terminal	Type	Length
SAOUHSC_00010	12	QECIPTLLGYAGVGISFGIVASS	34	SECONDARY	23
	40	LEIVLLCLVIYAGAAQFIMCALF	62	PRIMARY	23
	70	AIVLTVFIVNSRMFLLSMSLAPN	92	PRIMARY	23
	132	HGLNITAYLFWAISCVAGALFGE	154	PRIMARY	23
	161	TLGLDFAITAMFIFLAIAQFESI	183	SECONDARY	23
	197	AVIVMMLSLSMFMPSYLAILIAA	219	PRIMARY	23
SAOUHSC_00085	15	YFQIAYIVLMAITLCGFVICYGL	37	PRIMARY	23
	56	TIVISAIISIFVIILSIVPVIVL	78	PRIMARY	23
	93	LIVLAIIALVLCNFVSAILWFVS	115	PRIMARY	23
	8	KLLTLLLIGLAVFIQQSSVIAGV	30	PRIMARY	23
	33	SIADFITLLILVYLLFFANHLLK	55	PRIMARY	23
SAOUHSC_00091	63	FIILYTYRMIITLCLLFFDDLIF	85	PRIMARY	23
	94	STVKYAFVVIYFYLGMIIFKLGN	116	PRIMARY	23
	120	VIVTSYIISSVTIGLFCIIAGLN	142	PRIMARY	23
	167	YFAMTQIITLVLAYKYIHNYIFK	189	SECONDARY	23
	197	LWSLTTTGSKTAFIILIVLAIYF	219	PRIMARY	23
	228	NAVSVVSMSVIMLILLCFTFYNI	250	PRIMARY	23
	288	SVVWINAISVIKYTLGFGVGLVD	310	SECONDARY	23
	333	FAEWGILFGALFIIFMLYLLFEL	355	PRIMARY	23
	358	FNISGKNVTAIVVMLTMLIYFLT	380	PRIMARY	23
	382	SFNNSRYVAFILGIIVFIVQY	402	SECONDARY	21
SAOUHSC_00238	5	IINIAYLYAIIWKLKRLQKIVTS	27	PRIMARY	23
	2	SFVITVIVVYVSSFWWMTPFITY	24	SECONDARY	23
	42	QIYVMIIFFIAFCFISPVMFYQL	64	PRIMARY	23
	85	FFSVLLFCAGVAFAFYVGFPMII	107	PRIMARY	23
	126	KAYLIELIRWLFTFGLLFQLPIL	148	PRIMARY	23
SAOUHSC_00328	180	IIAPPDLTLNILLTLPLILLFEF	202	PRIMARY	23
SAOUHSC_01291	15	KIEFLIGTFIIILVILGFKIMK	36	PRIMARY	22
	24	NINILAAMMIVLVIPIMISGILF	46	PRIMARY	23
SAOUHSC_01402	50	NIDKTYIFFNIIFIDFYYYIYNV	72	SECONDARY	23
	103	FGFDEILFYTLYLLLILIVLYYL	125	PRIMARY	23
SAOUHSC_01851	9	KYIVRYHLAFVFISSFSLNFS	29	PRIMARY	21

Despite 24 proteins having cysteine resides that could form disulfide bonds; DISULFIND was unable to find potential disulfide bonding in any hypothetical protein evaluated here.

Conclusion:

Annotation of a genome does not stop after the sequence is published. We must update genomic annotations as new information on protein homology and structures are discovered. Since the annotation of over half of the S. aureus NCTC 8325 genome is as hypothetical, this study characterized 35 hypothetical proteins using bioinformatics tools and various databases for homology similarity comparisons, physiochemical characterization, domain identification, active site characterization, predicted protein-protein interactions, cellular location and stability. The examination revealed some hypothetical proteins with potentially virulent domains and protein-protein interactions including Oantigen, superoxide dismutase, siderophore synthesis, and bacterial ferric iron reductase. Other hypothetical proteins appear to metabolic or transport proteins including major facilitator superfamily, ABC transporters, GTPases, and Sadenosylmethionine decarboxylase. While this contributes to the current understanding of S. aureus, there is more work to do. More homology and structural information is needed in public repositories to be able to fully evaluate some hypothetical proteins, especially the smaller ones. This process will have to be repeated at regular intervals until the entire genome is properly annotated and should be done with all genomes as part of regular maintenance. Automation of this process would help ensure up-to-date databases. Until then, these data describing what is currently available for these 35 hypothetical proteins will contribute to the scientific understanding of S. aureus, aiding in the discovery of therapeutic targets.