JOURNAL OF BACTERIOLOGY, Mar. 2006, p. 1899–1910 0021-9193/06/$08.00⫹0 doi:10.1128/JB.188.5.1899–1910.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 188, No. 5
Transcription Profiling of the mgrA Regulon in Staphylococcus aureus Thanh T. Luong,1 Paul M. Dunman,2 Ellen Murphy,3 Steven J. Projan,4 and Chia Y. Lee1* Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 722051; Department of Microbiology and Pathology, University of Nebraska Medical Center, Omaha, Nebraska 681982; Wyeth Research, Pearl River, New York3; and Wyeth Research, Cambridge, Massachusetts4 Received 26 August 2005/Accepted 8 December 2005
MgrA has been shown to affect multiple Staphylococcus aureus genes involved in virulence and antibiotic resistance. To comprehensively identify the target genes regulated by mgrA, we employed a microarray method to analyze the transcription profiles of S. aureus Newman, its isogeneic mgrA mutant, and an MgrA-overproducing derivative. We compared genes that were differentially expressed at exponential or early stationary growth phases. Our results showed that MgrA affected an impressive number of genes, 175 of which were positively regulated and 180 of which were negatively regulated in an mgrA-specific manner. The target genes included all functional categories. The microarray results were validated by real-time reverse transcriptionPCR quantitation of a set of selected genes from different functional categories. Our data also indicate that mgrA regulates virulence factors in a fashion analogous to that of the accessory gene regulatory locus (agr). Accordingly, exoproteins are upregulated and surface proteins are downregulated by the regulator, suggesting that mgrA may function in concert with agr. The fact that a large number of genes are regulated by mgrA implies that MgrA is a major global regulator in S. aureus. The Staphylococcus accessory gene regulator, sarA, is the best-characterized small cytoplasmic transcriptional regulator. SarA regulates its target genes either directly or through the agr system by binding to the agr promoters. Several SarA homologs have been identified, and some have been molecularly characterized (7). Recently, we and others have identified a novel global regulator, MgrA (15, 24, 39). Mutation or overexpression of mgrA affects the production of several virulence factors including capsules, protein A, and alpha-toxin (24). In addition, MgrA has been shown to affect antibiotic resistance and autolysis (15, 39). MgrA, which contains a DNA-binding helix-turn-helix motif, is a small transcriptional regulator related to the SarA family of regulators. MgrA regulates certain target genes by directly binding their promoter region (15, 16, 39). Previously, we showed by gel electrophoresis that MgrA profoundly affects the expression of extracellular proteins (24). However, the number of genes or the biological processes that are regulated by MgrA are not known. Here, we employ microarray methodology to determine the range of genes regulated by MgrA. Our data suggest that in addition to the already-known virulence factors, MgrA can act as a repressor or activator of a large number of genes, including genes involved in metabolic functions.
Staphylococcus aureus can cause a diverse range of diseases, from superficial skin infections to serious infections such as osteomyelitis, septic arthritis, pneumonia, infected implant failure, and toxic shock syndrome (23). This bacterium is the prominent cause of nosocomial infections. A large number of virulence factors including secreted proteins, cell wall-tethered proteins, and cytoplasmic and integrated membrane proteins are believed to be involved in the infection processes. These virulence factors are coordinately regulated by a network of regulatory genes, which can be grouped into two major classes, two-component systems (TCSs) and small transcription regulators. The sensors of the 16 TCSs found in the S. aureus genome are likely to be responsible for sensing various environmental cues and transmitting the information to the responders, which either alone or in conjunction with other small transcription regulators regulate “downstream” genes (1, 7, 31). Among the TCSs, the accessory gene regulator (agr) system is the best-characterized system. agr is a quorum-sensing system that is activated by responding to the accumulation of an autoinducing peptide during cell growth (reviewed in reference 31). The autoinducing peptide is processed from AgrD by AgrB (40), which is also believed to export the peptide. The signal is then sensed by AgrC and transmitted to responder AgrA, which binds to the agr promoters (18). Activation results in the upregulation of the agrDCBA operon and RNAIII, a small RNA effector molecule of the agr system. RNAIII activates the production of many exoproteins and represses several cell surface proteins at the transcriptional level. Interestingly, RNAIII has also been shown to regulate alpha-toxin and protein A production by an antisense mechanism at the translational level (14, 30).
MATERIALS AND METHODS Bacterial strains and culture conditions. S. aureus Newman and its derivatives were used for the sources of RNA. Bacteria were cultivated in Trypticase soy broth (Difco Laboratories, Detroit, Mich.) with shaking. Chromosomal transduction using phage 52A was carried out as previously described (21). The mgrA deletion mutant strain CYL1050 was constructed by transduction of ⌬mgrA::cat from CYL1040 (24) to the Newman strain. The MgrA-overproducing strain CYL907 was constructed by cotransduction of Tn917 and the mgrA5614 allele (a promoter-up mutation linked by Tn917 insertion) from CYL183 (24) to the Newman strain. Mutant strain CYL1050 and the overproducing strain CYL907 were grown in media supplemented with chloramphenicol at 5 g/ml and erythromycin at 10 g/ml, respectively.
* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot 511, Little Rock, AR 72205. Phone: (501) 526-7687. Fax: (501) 686-5359. E-mail:
[email protected]. 1899
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LUONG ET AL.
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TABLE 1. Primers used in real-time RT-PCR with SYBR green probes Primer
N315 ORF a
Sequence
SG16S1 SG16S2 SGhu1 SGhu2 SGsarA1 SGsarA2 arcA3 arcA4 betB3 betB4 lacA5 lacA6 marR3 marR4 lukD3 lukD4 lytN3 lytN4 splA3 splA4 tcaR3 tcaR4 wcaG3 wcaG4
SArRNA01 SArRNA01 SA1305 SA1305 SA0573 SA0573 SA2428 SA2428 SA2406 SA2406 SA1997 SA1997 SA0904 SA0904 SA1637 SA1637 SA1090 SA1090 SA1631 Sa1631 SA2147 SA2147 SA0123 SA0123
CGTGCTACAATGGACAATAC AAA ATCTACGATTACTAGCGATTCCA GCTGGGATATCAATTTCTTTACC ATCCAAAACTCACTTGCTAAAGG CTGTATTGACATACATCAGCGAA GTTCTTTCATCATGCTCATTACG ATGTCAGGGGTACGTAAGGAA GAGGCTTGTGGATCTCTAGTA ATCGCGAAGCGTTAGCACGA CGTCTTTATCTGCTAATCCAGC TCAGATGAAGCTGGCAAACGA GCTACAGCCAAAGTTGCATCA GAAGATTTGGACAACGCACGG GTTCCCTGCTAATCTTCATCG ACTTAAGGCAGCCGGAAACAT CATTTGATTCTGAACTAACCGAA TGAAGGTGCTGTTAAAACATCG CTTGGCTTGAGTTTTTCGGAGT CATTTGTGGGTGGTACTGGTG CTCGAATGATGTGCTGATACTC GTAAATAAGGCCGCAGTAAGC GCTCTACCTTTGTCAGTTAAGG TGTATTTGGGCCAAGACAGGA CTAGTTTGCAGTCCGTCACC
a
ORF, open reading frame.
RNA isolation. Cultures of S. aureus grown overnight were diluted 1:100 into fresh Trypticase soy broth medium with appropriate antibiotics and grown at 37°C for 2 to 5 h (optical densities at 660 nm of 0.43 to 0.46 and 3.90 to 4.13, respectively), at which time samples were collected for RNA isolation. The culture samples were centrifuged at 10,000 ⫻ g at 4°C for 5 min and washed once with Tris-EDTA (TE) buffer (10 mM Tris, 1 mM EDTA, pH 7.6). The cell pellets were resuspended in TE buffer and kept frozen at ⫺80°C after the addition of an equal volume of a 1:1 ice-cold alcohol-acetone mixture. After centrifugation at 10,000 ⫻ g at 4°C for 5 min, cell pellets were air dried and resuspended on ice in 500 l TE buffer. The cell suspensions were transferred to lysing matrix B tubes and processed twice in an FP120 FastPrep cell disruptor (MP Biomedicals, Irvine, Calif.) for 40 s at setting 6.0 each time. The tubes were centrifuged at maximum speed in a tabletop centrifuge for 15 min at 4°C, and the upper-phase samples were transferred to a 1.5-ml microtube. The RNA samples were further purified and treated with DNase I using the RNeasy kit from QIAGEN, Inc. (Valencia, Calif.), according to the instructions of the manufacturer. The RNA was quantified spectrophotometrically, and the absence of DNA was verified by PCR using primers listed in Table 1. Microarray profiling. RNA was converted to cDNA, and microarray analysis was performed according to the manufacturer’s instructions (Affymetrix Expression Analysis Technical Manual; Affymetrix, Inc., Santa Clara, Calif.) for antisense prokaryotic arrays essentially as described previously by Beenken et al. (2). To ensure reproducibility, two RNA samples from each strain were prepared at each growth phase from two separate experiments. Each RNA sample was hybridized to two separate GeneChips. Genes with at least a twofold difference (t test; P ⱕ 0.05) in RNA titer between the wild-type strain and the mgrA deletion mutant or between the wild-type strain and the MgrA-overproducing strain were considered differentially expressed in an mgrA-dependent manner. Real-time RT-PCR. To confirm the microarray data, we selected genes from different functional categories to assay their relative expression levels by realtime reverse transcription (RT)-PCR. Briefly, one-step quantitative RT-PCR was performed by incubating DNase I-treated RNA with SuperScript III platinum SYBR Green One-Step qRT-PCR master mix (Invitrogen Corporation, Carlsbad, Calif.) using the ABI Prism 7300 detection system (Applied Biosystems, Foster City, Calif.). The cDNA was subjected to real-time PCR using the primer pairs listed in Table 1. Cycling conditions were 48°C for 30 min and 95°C for 15 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min and a dissociation step at 95°C for 15 s, 60°C for 30 s, and 95°C for 15 s. Relative expression levels were determined by the comparative threshold cycle (⌬⌬Ct) method (Applied Biosystems User Bulletin no. 2).
RESULTS AND DISCUSSION Identification of mgrA-regulated genes by microarray. MgrA has been shown to regulate several virulence factors in S. aureus (15, 24, 39). In an effort to further characterize the role of MgrA in virulence gene regulation, DNA microarray studies were performed with the RNA isolated from strain Newman and its isogenic mgrA deletion mutant (CYL1050). We also included an MgrA-overproducing strain (CYL907) in our studies, since we previously found that MgrA overexpression affected the expression of many genes (24). However, strain CYL907 contains a Tn917 insertion (at the hypothetical gene SA0636 of N315) that is not present in strain Newman and CYL1050. To ensure that this insertion does not affect mgrA expression, we compared the mgrA promoter activity of strain CYL908, which contains Tn917 at the same insertion site as CYL907 but possesses a wild-type allele of mgrA, to that of strain Newman. No difference in promoter activity was observed between the two strains (results not shown) by using a plasmid-based promoter–xylE reporter gene fusion method, indicating that the insertion does not affect mgrA expression. To take into account the possibility of growth phase-dependent regulation, we isolated the RNA at exponential (2 h) and early stationary (5 h) growth phases. The levels of gene expression between the wild type and the mgrA mutant and between the wild type and the overproducing strain were compared. A change of at least twofold was considered significant. Genes with decreased expression in the mgrA deletion mutant compared to the wild type and genes with decreased expression in the wild type compared to the Mgr-overproducing strain were grouped as mgrA-upregulated genes. Genes that were oppositely affected were grouped as downregulated by mgrA. A total of 350 genes were found to be regulated by mgrA at either 2 h or 5 h or at both sampling times. More specifically, 175 genes were found to be upregulated by mgrA (Table 2), and 180 genes were found to be downregulated by mgrA (Table 3). These genes were found in various functional groups and were grouped according to the classification described previously by Kunst et al. (19). The number of genes affected by mgrA was much greater at 2 h than at 5 h (about 2.5-fold), suggesting that mgrA is an early-growth-phase regulator. There were only 52 genes (14.8% of the total affected) that exhibited MgrA effects at both the log phase and stationary phase, suggesting that most of the genes respond to a limited range of MgrA concentrations. It should be noted here that we found that the mgrA transcript in the overproducing strain was 3.2-fold and 1.8-fold higher than that of the wild-type strain at 2 h and 5 h, respectively. Our microarray results confirm previous reports that mgrA is a pleiotropic regulator that can act either as an activator or as a repressor (15, 24, 39). Unexpectedly, we found that five genes (opuD, pyrG, pyrP, scrA, and stpC) were positively regulated by mgrA at one time point but negatively regulated at the other. For example, scrA was found to be upregulated 3.1-fold at 2 h when the wild-type strain and the deletion mutant were compared but was downregulated 5.5-fold at 5 h when the wild-type strain and the overproducing strain were compared. The simplest explanation for these seemingly conflicting results is that such target genes are regulated by mgrA through more than one pathway (such as through other regulators) in which the
TRANSCRIPTION PROFILING OF THE S. AUREUS mgrA REGULON
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TABLE 2. Genes upregulated by mgr Fold change ORF
wt vs mgr⌬ 2h
Cell envelope and cellular processes N315-SA1269 COL-SA0002 N315-SA2261 N315-SA2233 N315-SA2302 N315-SA1655 N315-SA1674 N315-SA0682 N315-SA0250 N315-SA1987 N315-SA0531 N315-SA2408 N315-SA1718 N315-SA1270 N315-SA2239 N315-SA0541 N315-SA1675 N315-SA0871 N315-SA0368 N315-SA0810 N315-SA0809 N315-SA0808 N315-SA0807 N315-SA0374 N315-SA1042 N315-SA2167 N315-SA2316 N315-SA2303 N315-SA0211 N315-SA0522 N315-SA1182 N315-SA0201 Intermediary metabolism N315-SA1272 N315-SA0342 N315-SA1510 N315-SA1959 N315-SA0376 N315-SA0375 N315-SA0202 N315-SA0430 N315-SA2334 N315-SA0686 N315-SA0687 N315-SA2409 N315-SA1724 N315-SA1929 N315-SA1460 N315-SA1921 N315-SA0373 N315-SA2397 N315-SA0642 N315-SA0643 COL-SA2141 N315-SA0119 Information pathways N315-SA1678 N315-SA1041 N315-SA1870 N315-SA1869 N315-SA0573 N315-SA2147 N315-SA2379 N315-SA2495 N315-SA1956 N315-SA0461 N315-SA0014 N315-SA0577
5h
a
mgr⫹⫹ vs wt 2h
Gene
5h
norB tetK
2.1 24.8 2.8 2.0
stpC
4.2 2.1 5.2 2.1 7.1 2.5 3.4
3.6 2.6
2.0
lytS opuD prop cudT putP
3.2 2.0 2.3 7.3 2.1 3.4 mnhD mnhE mnhF mnhG pbuX pyrP scrA srtA smpC
2.2 2.4 2.4 2.5 4.5 19.0 3.1 2.1 5.3 2.0 2.8
mscL rlp
2.8 3.2
3.3 2.5 2.1 4.6 3.9 2.9 2.6 2.7 2.1 2.3 3.0 2.2 2.2 3.2 2.0 2.1 3.2 5.0 3.3 2.3 2.6
Alanine dehydrogenase Acetyl-CoA C-acetyltransferase homolog Glyceraldehyde-3-phosphate dehydrogenase 2 Glucosamine-fructose-6-phosphate aminotransferase GMP synthase Inositol-monophosphate dehydrogenase HP, similar to gamma-glutamyltranspeptidase precursor Glutamate synthase large subunit 3-Hydroxy-3-methylglutaryl-CoA synthase Ribonuceloside diphosphate reductase major subunit Ribonucleoside diphosphate reductase minor subunit Anaerobic ribonucleotide reductase activator protein Adenylosuccinate lyase CTP synthase GTP pyrophosphokinase Thymidine kinase Xanthine phosphoribosyltransferase HP, similar to pyridoxal phosphate-dependent aminotransferase HP, similar to cobalamin synthesis-related protein HP, similar to aryl alcohol dehydrogenase Site-specific recombinase family protein, degenerate HP, similar to diaminopimelate decarboxylase
furB pyrR rsbW sigB sarA tcaR
Transcription regulator Fur family homolog Pyrimidine operon repressor chain A anti-B factor B factor Staphylococcal accessory regulator A TcaR transcription regulator HP, similar to transcriptional regulator tetR family HP, putative transcriptional regulator Lytic regulatory protein truncated with Tn554 Transcription-repair-coupling factor 50S ribosomal protein L9 HP, similar to FimE recombinase
2.3
2.0 9.7 2.1 2.1 2.4 2.1 5.8 2.9 2.1 2.1 2.3
Blt-like protein, efflux pump Tetracycline resistance protein HP, similar to efflux pump HP, similar to integral membrane efflux protein HP, similar to ABC transporter ABC transporter ecsA homolog Glutamate ABC transporter ATP-binding protein HP, similar to di-tripepride ABC transporter Two-component sensor histidine kinase Glycine betaine transporter homolog Proline/betaine transporter homolog Choline transporter High-affinity proline permease HP, similar to amino acid permease HP, similar to amino acid transporter HP, similar to cationic amino acid transporter HP, similar to glutamine-binding periplasmic protein HP, similar to Na⫹/H⫹-dependent alanine carrier protein HP, similar to proton/sodium-glutamate symport protein Na⫹/H⫹ antiporter subunit Na⫹/H⫹ antiporter subunit Na⫹/H⫹ antiporter subunit Na⫹/H⫹ antiporter subunit Xanthine permease Uracil permease PTS system, sucrose-specific IIBC component Sortase Similar to membrane-spanning protein HP, similar to NADH-dependent dehydrogenase HP, similar to poly(glycerol phosphate) ␣-glucosyltransferase Large-conductance mechanosensitive channel RGD-containing lipoprotein
ald atoB gapB glmS guaA guaB ggt gltB mvaS nrdE nrdF nrdG purB pyrG relA tdk xprT
5.3
5.2
Descriptionb
mfd rplI
Continued on following page
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LUONG ET AL. TABLE 2—Continued Fold change ORF
wt vs mgr⌬ 2h
Other functions N315-SA0144 N315-SA0145 N315-SA0146 N315-SA0147 N315-SA0148 N315-SA0149 N315-SA0150 N315-SA0151 N315-SA0152 N315-SA0153 N315-SA0154 N315-SA0155 N315-SA0156 N315-SA0157 N315-SA0158 N315-SA0159 N315-SA0252 N315-SA2406 N315-SA2207 N315-SA1637 N315-SA1638 N315-SA1813 N315-SA1812 N315-SA0388 N315-SA0390 COL-SA0472 N315-SA2463 N315-SA0746 N315-SA1758 N315-SA0128 N315-SA1631 N315-SA1630 N315-SA1629 N315-SA1628 COL-SA1865 N315-SA1549 N315-SA0744 N315-SA0462 N315-SA0754 COL-SA0321 N315-SA0102 N315-SA0521 Similar to unknown proteins N315-SA2210 N315-SA2158 N315-SA1900 N315-SA2133 N315-SA1327 N315-SA2298 N315-SA0701 N315-SA0863 N315-SA0943 N315-SA0961 N315-SA1976 COL-SA0645 COL-SA0651 COL-SA0650 COL-SA0647 N315-SA1849 COL-SA2631 N315-SA0463 COL-SA0654 N315-SA0753 N315-SA0648 N315-SA0464 N315-SA0212 N315-SA0427 N315-SA1459
a
mgr⫹⫹ vs wt
5h
2h
5h
3.1 2.9 3.1 3.6
9.7 9.1 10 8.6 8.2 6.6 6.5 6.1 5.8 5 4.7 4.1 4.1 3.7 3.3 3.0
6.2 5.6 5.5 5.3 5.0 5.3 5.3 5.3 5.9 5.3 4.7 4.5 4.3 3.8
14.0 4.2 5.8 15.0 13.1
4.8 4.2
4.0 5.9 2.8 3.2 2.2 4.4 2.4
2.1 11.2 13.5 9.8 6.1 8.1
cap5A cap5B cap5C cap5D cap5E cap5F cap5G cap5H cap5I cap5J cap5K cap5L cap5M cap5N cap5O cap5P lrgA gbsA hlgA lukD lukS lukF lukM set12 set14 lip nuc sak sodd splA splB splC splD splE htrA ssp
3.1 13.7
Gene
2.0 2.7
2.2 11.1 2.1 2.3 2.2 2.3 2.9
sdrE
bioX
2.6 2.4
ywpF
3.4 2.4 2.3 2.4 2.7 2.7 3.1 2.0 3.8 2.8 2.5 2.4 2.3 2.2 2.1 2.1 2.1 2.0 2.0 2.0 2.0 2.0 2.0
Descriptionb
Capsular polysaccharide synthesis enzyme Capsular polysaccharide synthesis enzyme Capsular polysaccharide synthesis enzyme Capsular polysaccharide synthesis enzyme Capsular polysaccharide synthesis enzyme Capsular polysaccharide synthesis enzyme Capsular polysaccharide synthesis enzyme Capsular polysaccharide synthesis enzyme Capsular polysaccharide synthesis enzyme Capsular polysaccharide synthesis enzyme Capsular polysaccharide synthesis enzyme Capsular polysaccharide synthesis enzyme Capsular polysaccharide synthesis enzyme Capsular polysaccharide synthesis enzyme Capsular polysaccharide synthesis enzyme Capsular polysaccharide synthesis enzyme Antiholin-like protein LrgA Glycine betaine aldehyde dehydrogenase GbsA Gamma-hemolysin chain II precursor Leukotoxin, LukD Synergohymenotropic toxin LukS Synergohymenotropic toxin precursor Leukocidin chain LukM Exotoxin 12 Exotoxin 14 Exotoxin 2 Triacylglycerol lipase precursor Staphylococcal nuclease Staphylokinase precursor Superoxide dismutase Serine protease SplA Serine protease SplB Serine protease SplC Serine protease SplD Serine protease SplE Serine proteinase htrA Extracellular ECM and plasma binding protein HP, similar to low-temperature-requirement B protein HP, similar to lactococcal prophage ps3 protein 05 Bacteriophage L54a, repressor protein 67-kDa myosin-cross-reactive streptococcal antigen homolog SD-rich fibrinogen-binding, bone sialoprotein-binding protein
HP, similar to BioX protein HP, similar to TpgX protein Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP Conserved HP
Continued on following page
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TABLE 2—Continued Fold change ORF
wt vs mgr⌬ 2h
No similarity COL-SA2420 N315-SA0292 N315-SA0633 N315-SA0644 N315-SA1001 N315-SAS084 N315-SA2343 N315-SA2224 N315-SA1477 N315-SA1755 N315-SAS014 N315-SAS025 N315-SA1168 COL-SA0859 COL-SA2480 COL-SA1528 COL-SA1529 COL-SA1532 COL-SA2677 N315-SA2280 N315-SAS083 N315-SA0120 N315-SA2272 N315-SA1321 N315-SA0090 N315-SA0279 N315-SA0647 N315-SA0291 N315-SA2076 COL-SA2629 COL-SA2693 COL-SA1533 N315-SAS008
5h
a
mgr⫹⫹ vs wt 2h
Gene
5h
4.8 2.3 2.2 2.1 10.2 2.0 5.3 2.0 2.1 14.9 2.0 2.0 2.1 30.0 4.4 2.3 2.2 3.0 3.8 2.8 2.5 2.3 2.2 2.2 2.1 2.1 2.0 2.0 2.0
Descriptionb
2.7
4.1 3.7 2.5 2.9 2.6 2.4 2.6 3.4 splF
3.5 2.7
HP HP HP HP HP HP HP HP HP HP (bacteriophage phiN315) HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP 100% protein ID to PTS system sucrose-specific IIBC component ScrA, S. aureus MW2 91.4% protein ID to HP SA1320, S. aureus N315 8.1% protein ID to conserved HP SA0553, S. aureus N315 85.7% protein ID to MW2624, S. aureus MW2 98.9% protein ID to serine Protease SplF, S. aureus MW2 91.4% protein ID to SA1320 HP, S. aureus N315
a Values in boldface type indicate an undetected level in one strain such that the severalfold change could not be accurately calculated. “wt vs mgr⌬” indicates an increased expression level in the wild type compared to the mgrA deletion mutant. “mgr⫹⫹ vs wt” indicates an increased expression level in the overproducing strain compared to the wild type. b Abbreviations: PTS, phosphotransferase; HP, hypothetical protein; ID, identity; CoA, coenzyme A; ECM, extracellular matrix.
target genes are regulated oppositely in different pathways depending on the level of MgrA and, perhaps, the growth conditions. Confirmation of microarray results by real-time RT-PCR. To confirm the results obtained by microarray, we employed real-time RT-PCR to estimate the amount of transcripts after 5 h of incubation for eight selected mgrA target genes in different functional categories. The expression of either 16S rRNA or hu (N315-SA1305) was used as the control for estimating the severalfold changes. The expression of the hu gene was not affected by mgrA (data not shown). The results shown in Table 4 were comparable with what was observed by DNA microarray results except that the severalfold changes were much more profound in real-time RT-PCR, indicating that real-time RT-PCR is more sensitive than GeneChips, as previously suggested (2). Our data were also validated by the findings that mgrA transcript was not found in mutant CYL1050 and that the transposon Tn917-associated genes were only found in overproducing strain CYL907 containing Tn917.
mgrA regulation of genes involved in polysaccharide synthesis. We have previously shown that mgrA activates the expression of capsular polysaccharide genes encoded in the cap5(8) operon (24). Our profiling results showed that all 16 genes in this locus were upregulated by mgrA at 2 h and that all but two genes (cap5K and cap5N) were upregulated at 5 h when the overproducer was compared to the wild type (Table 2). When the deletion mutant was compared to the wild-type strain, only cap5ABCD was found to be upregulated at 5 h. These results are in good agreement with our previous Northern blotting results, promoter fusion assay, and capsule measurement. Our profiling results also showed that mgrA regulated other putative polysaccharide synthesis genes. We found that five genes with high similarity to polysaccharide synthesis genes encoded in the SA0123-SA0127 (N315) locus were affected by mgrA. However, contrary to the cap5(8) genes, these genes were downregulated by mgrA (Table 3). Like the cap5(8) genes, these genes were strongly regulated at both 2 h and 5 h, indicating that MgrA is a major regulator for these genes. This
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LUONG ET AL. TABLE 3. Genes downregulated by mgrA Fold changea ORF
mgr⌬ vs wt 2h
Cell envelope and cellular processes N315-SA0650 N315-SA0099 N315-SA0599 N315-SA2302 N315-SA2314 N315-SA0297 N315-SA0229 N315-SA1992 N315-SA1993 N315-SA1960 N315-SA2167 N315-SA0233 N315-SA0214 N315-SA1090 N315-SA1025 N315-SA1091 N315-SA1987 N315-SA0304 N315-SA1042 N315-SA2446 N315-SA0443 N315-SA0849 N315-SA2081 N315-SA2239 N315-SA2489 N315-SA2073 N315-SA1374 N315-SA1373 N315-SA0578 N315-SA2311 N315-SA0581 N315-SA0579 N315-SA0580 N315-SA2227 Intermediary metabolism N315-SA2428 N315-SA2427 N315-SA2008 N315-SA1045 N315-SA1046 N315-SA2312 N315-SA1493 N315-SA2465 N315-SA2464 N315-SA1997 N315-SA1996 N315-SA1995 N315-SA1994 N315-SA1991 N315-SA0344 N315-SA2063 N315-SA2068 N315-SA1963 N315-SA0016 N315-SA1043 N315-SA1044 N315-SA1048 N315-SA1047 N315-SA1929 N315-SA2318 N315-SA2319 N315-SA2082 N315-SA2083 N315-SA2084 N315-SA2088 N315-SA2085 N315-SA2086 N315-SA2087
5h
wt vs mgr⫹⫹ 2h
Gene
5h
2.9 4.6 3.0 3.3
norA sbtA abcA stpC
2.5 2.4 3.3 14.8 15.4 2.6 5.5 2.7
2.7 30.7 2.3 22.6 2.9
2.2 31.6 19.7 2.0 2.7
4.3 2.0 2.5 4.6 3.5 2.7 2.2 3.0 2.4 4.0 4.3 3.7 3.9 4.0
lacE lacF mtlF scrA uhpT lytN mraY eprH opuD nanA pyrP secY
modB gspE 7.1
5.8
17.6 16.4 6.8 27.8 2.4 3.2 3.1
2.0
6.4
2.5 2.3 2.6
10.8 15.7 17.8 15.1 13.9
2.4 2.3 2.4 2.9 4.2 2.4 2.5 2.5 2.4 2.3 14.9 26.7 14.9 11.9 16.4 15.8 10.2
3.2 3.1 11.2 13.1
Descriptionb
arcA arcB budB carA carB ddh hemD hisF hisI lacA lacB lacC lacD lacG metE moaA moeA mtlD purA pyrB pyrC pyrE pyrF pyrG sdhA sdhB ureA ureB ureC ureD ureE ureF ureG
Quinolone resistance protein HP, similar to transmembrane efflux pump protein ATP-binding cassette transporter A HP, similar to ABC transporter HP, similar to ABC transporter (ATP-binding protein) HP, similar to ABC transporter (ATP-binding protein) HP, similar to nickel ABC transporter nickel-binding protein PTS system, lactose-specific IIBC component PTS system, lactose-specific IIA component PTS system, mannitol-specific IIBC component PTS system, sucrose-specific IIBC component PTS enzyme maltose- and glucose-specific factor II homolog Hexose phosphate transport protein Cell wall hydrolase Phospho-N-muramic acid-pentapeptide translocase Endopeptidase resistance gene Glycine betaine transporter N-Acetylneuraminate lyase subunit Uracil permease HP, similar to preprotein translocase HP, similar to signal peptidase II homolog HP, similar to peptide binding protein OppA HP, similar to urea transporter HP, similar to amino acid transporter HP, similar to high-affinity nickel transport protein Probable molybdenum transport permease HP, similar to late competence protein ComGA HP, similar to DNA transport machinery protein ComGB HP, similar to NADH dehydrogenase HP, similar to NAD(P)H-flavin oxidoreductase MnhD homolog, similar to Na⫹/H⫹ antiporter subunit HP, similar to Na⫹/H⫹ antiporter HP, similar to Na⫹/H⫹ antiporter Truncated HP, similar to D-serine/D-alanine/glycine transporter
Arginine deiminase Ornithine transcarbamoylase Alpha-acetolactate synthase Carbamoyl-phosphate synthase small chain Carbamoyl-phosphate synthase large chain D-Specific D-2-hydroxyacid dehydrogenase Uroporphyrinogen III synthase Cyclase-like protein Histidine biosynthesis bifunctional protein Galactose-6-phosphate isomerase LacA subunit Galactose-6-phosphate isomerase LacB subunit Tagatose-6-phosphate kinase Tagatose-1,6-diphosphate aldolase 6-Phospho-beta-galactosidase 5-Methyltetrahydropteroyl-triglutamate-homocysteine methyltransferase Molybdenum cofactor biosynthesis protein A Molybdopterin biosynthesis protein Mannitol-1-phosphate-5-dehydrogenase Adenylosuccinate synthase Aspartate transcarbamoylase chain A Dihydroorotase Orotate phosphoribosyltransferase Orotidine-5-phosphate decarboxylase CTP synthase HP, similar to L-serine dehydratase HP, similar to beta subunit of L-serine dehydratase Urease gamma subunit Urease beta subunit Urease alpha subunit Urease accessory protein Urease accessory protein Urease accessory protein Urease accessory protein
Continued on following page
TRANSCRIPTION PROFILING OF THE S. AUREUS mgrA REGULON
VOL. 188, 2006
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TABLE 3—Continued Fold change ORF
mgr⌬ vs wt 2h
N315-SA1814 N315-SA0234 N315-SA0098 N315-SA2007 Information pathways N315-SA0108 N315-SA2092 N315-SA0904 N315-SA1961 N315-SA2320 N315-SA0627 N315-SA1485 N315-SA0897 N315-SA0565 N315-SA1092 Other functions N315-SA2456 N315-SA2455 N315-SA0123 N315-SA0124 N315-SA0125 N315-SA0126 N315-SA0127 N315-SA2329 COL-SA0095 COL-SA2676 COL-SA1806 COL-SA2150 N315-SA1004 N315-SA2431 N315-SA2447 COL-SA1472 N315-SA1267 N315-SA2285 N315-SA2284 COL-SA0479 N315-SA2414 N315-SA1147 COL-SA0478 N315-SA0777 N315-SA0102 COL-SA1186 COL-SA1187 N315-SA1617
Similar to unknown proteins N315-SA1618 N315-SA0335 N315-SA2448 N315-SA2315 N315-SA2378 N315-SA0296 N315-SA0332 N315-SA0333 N315-SA1154 N315-SA0447 N315-SA1432 N315-SA1433 N315-SA0710 N315-SA0778 N315-SA1451 N315-SA2371 N315-SA1618 N315-SA0625 N315-SA0635 N315-SA0624
5h
a
wt vs mgr⫹⫹ 2h
5h
3.0 3.0 5.2 7.5
HP, HP, HP, HP,
sarS
2.1 2.6 12.2
19.6 3.3 2.6 2.2 2.4
radC
2.2 2.5 4.5
3.4 ⬎4.6 10.1 11.0 7.4 10.5 8.9 2.5
capB capC 33.5 14.3 6.1 5.6 2.0 16.4
8.8 4.7 28.7
3.8
3.0 6.9
cidA spa mrp fmtB fib isaB
2.8 10.7 2.1
3.6 9.2 72.6 64.1 4.7 4.0
ebh ebhA 2.3 gpxA hflX
11.7 2.9 3.1 2.1 2.9 2.8 3.1 3.7
2.2 2.5 2.1 2.7 3.2 2.6 2.5 2.8 3.8 2.1 5.3 5.9 2.9 2.2
Descriptionb
Gene
6.0
4.8 6.2 3.7 6.1 11.9
6.1 18.4 2.8
similar similar similar similar
to to to to
succinyl-diaminopimelate desuccinylase inosine-uridine-preferring nucleoside hydrolase aminoacylase alpha-acetolactate decarboxylase
Staphylococcal accessory regulator A homolog HP, similar to transcription regulator HP, probable ATL autolysin transcription regulator HP, similar to transcription antiterminator BglG family HP, similar to regulatory protein PfoR HP, similar to LysR family transcriptional regulator DNA repair protein RadC homolog (Tn554 inserted) HP, similar to prolyl aminopeptidase HP, similar to endonuclease III HP, similar to DNA-processing Smf protein
Capsular polysaccharide biosynthesis Capsular polysaccharide biosynthesis HP, similar to UDP-glucose-4-epimerase (galE-1) HP, similar to glycosyltransferase TuaA HP, similar to EpsG (exopolysaccharide G) HP, similar to capsular polysaccharide synthesis protein 14H HP, similar to capsular polysaccharide synthesis protein 14L Conserved HP Immunoglobulin G binding protein A precursor LPXTG motif cell wall anchor domain protein LPXTG motif cell wall anchor domain protein FmtB (Mrp) protein HP, similar to fibrinogen-binding protein Immunodominant antigen B HP, similar to streptococcal hemagglutinin protein Pathogenicity protein, putative HP, similar to streptococcal adhesin emb HP, similar to accumulation-associated protein HP, similar to accumulation-associated protein Surface protein, putative HP, similar to glutathione peroxidase HP, similar to GTP-binding protein proteinase modulator homolog ynbA Exotoxin 3 HP, similar to nitrogen fixation protein NifU 67-kDa myosin-cross-reactive streptococcal antigen homolog Antibacterial protein Antibacterial protein HP, similar to latent nuclear antigen (Kaposi’s sarcoma- associated herpesvirus)
Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved Conserved
HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP
Continued on following page
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LUONG ET AL.
J. BACTERIOL. TABLE 3—Continued Fold change
ORF
mgr⌬ vs wt 2h
No similarity N315-SA2091 COL-SA1473 COL-SA1474 COL-SA2433 COL-SA2027 N315-SA2443 N315-SA2444 N315-SA2445 N315-SA2473 N315-SA1001 COL-SA1372 N315-SA1514 COL-SA2069 N315-SA0748 N315-SA0749 N315-SA1008 COL-SA1345 COL-SA1346 COL-SA2187 N315-SA2372 N315-SA2373 N315-SA2218 COL-SA2510 COL-SA2420 N315-SA0221 COL-SA0859 N315-SAS028 COL-SA1042 N315-SA1015 N315-SA1619 N315-SAS014 COL-SA1174 COL-SA2559 COL-SA0492 COL-SA2558 N315-SA0644 N315-SA1017 N315-SA1620
2.9 13.8 14.3 8.6 3.3 8.0 6.8 7.0 4.7 3.1 2.3 2.2 2.7
5h
a
wt vs mgr⫹⫹ 2h
Gene
5h
5.1 7.9
3.5 3.2 2.6 2.2 2.2 10.0 4.6 4.9 3.3
2.4
2.0 2.1 2.2 2.4 2.4 2.4 2.7 2.9 2.9 3.3 3.8 4.1 4.1 2.3 2.3 3.2 24.3 4.8 3.1 135.2 21.0 3.7 99.4 7.1 19.0
Descriptionb
20.3
4.1 2.6 2.3 3.5
HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP HP 100% protein ID to truncated FmtB, S. aureus MW2 30.1% protein ID to fibrinogen-binding protein A ClfA 100% protein ID to hemagglutinin, S. aureus MW2575 83.5% protein ID to Ebh, S. aureus MW2 77.5% protein ID EbhA, S. aureus N315 27.8% protein ID to S. aureus surface protein Pls 65.8% protein ID to Ebh, S. aureus MW2 63.2% protein ID to Ebh, S. aureus MW2 98.1% protein ID to NasE assimilatory nitrite reductase, S. aureus N315 34.1% protein ID to Fb1 receptin precursor, Staphylococcus lugdunensis strain 2342 94.5% protein ID to SA1003, S. aureus N315 89.5% protein ID to SA2243, S. aureus N315 100% protein ID to PTS system sucrose-specific IIBC component ScrA, S. aureus MW2
a Values in boldface type indicate an undetected level in one strain such that the severalfold change could not be accurately calculated. “mgr⌬ vs wt” indicates an increased expression level in the mgrA deletion mutant compared to the wild type. “wt vs mgr⫹⫹” indicates an increased expression level in the wild type compared to the overproducing strain. b Abbreviations: PTS, phosphotransferase; HP, hypothetical protein; ID, identity; ATL, autolysin.
is in contrast to the majority of the genes identified in this study, which showed regulation at only one time point but not both. To confirm that these genes are repressed by mgrA, we compared the transcript of the first gene of this cluster of the wild-type strain to those of the mutant strains. As shown in Table 4, MgrA strongly repressed the SA0123 gene (nearly 2,000-fold repression). These results are in agreement with the microarray data. Our microarray study also found that two genes (N315-SA2455 and N315-SA2456) with high similarity to
cap5(8)BC were downregulated in an mgrA-specific manner. These genes are clustered with the third gene, SA2457, with high similarity to cap5(8)A. However, mutations within this locus did not affect the type 5 or type 8 capsule (unpublished data). The cluster is adjacent to the ica locus, which encodes genes required for poly-N-acetylglucosamine synthesis involved in biofilm formation. However, these capABC-like genes have not been implicated in poly-N-acetylglucosamine synthesis (9). None of the ica genes were regulated by mgrA in
TRANSCRIPTION PROFILING OF THE S. AUREUS mgrA REGULON
VOL. 188, 2006
TABLE 4. Relative quantification of gene expression regulated by MgrA by real-time RT-PCRa ORF
N315-SA2428 N315-SA1637 N315-SA1090 N315-SA0904 N315-SA0573 N315-SA1631 N315-SA2147 N315-SA0123
Fold change ⫾ SE
Gene
arcA lukD lytN sarA splA tcaR
CYL907
CYL1050
⫺1.1 ⫾ 0.1 ⫺21.1 ⫾ 2.9 ⫹2.1 ⫾ 0.3 ⫹1.5 ⫾ 0.03 ⫺3.4 ⫾ 0.5 ⫹1.6 ⫾ 0.1 ⫺3.4 ⫾ 0.8 ⫺1.8 ⫾ 0.4
⫺27.9 ⫾ 9.1 ⫹83.9 ⫾ 9.9 ⫺2,190.0 ⫾ 152.1 ⫺564.4 ⫾ 19.8 ⫺1.4 ⫾ 0.2 ⫹22.8 ⫾ 2.7 ⫹6.5 ⫾ 0.7 ⫺1,923.5 ⫾ 398.1
a The severalfold changes are expressed in comparison to wild-type S. aureus Newman; ⫺ indicates reduction, and ⫹ indicates increase. Standard errors were calculated based on at least two independent experiments.
our microarray study. Taken together, our results showed that mgrA not only regulated all the polysaccharide genes encoded by the cap5(8) locus but also controlled two other putative polysaccharide loci, suggesting that MgrA is a major regulator involved in polysaccharide production in S. aureus. MgrA is a major autolytic regulator. In S. aureus, autolysis is a very complex process. Three major autolysin genes, atl, lytM, and lytN, have been identified (32, 34, 36). In addition, the cidAB operon, which encodes a holin-like protein, has been shown to promote autolysis by functioning to facilitate either the transport of murein hydrolases or their activity (34). The lrgAB operon, which encodes an antiholin component, has been shown to counteract cidAB and thus inhibits extracellular murein hydrolase activity (13). The expression of lrgAB is in turn positively regulated by lytSR, a two-component regulatory system (5). Recently, ArlRS, another two-component regulatory system, has been shown to negatively regulate autolysis (11, 12). Ingavale et al. (15) showed that mgrA was a major regulator of autolysis, affecting the expression of lytSR, lrgAB, arlRS, lytM, and lytN. Indeed, we found that mgrA upregulated lytS and lrgA and downregulated lytN and cidA. Our results are therefore generally in agreement with the results reported previously by Ingavale et al. (15). In addition, our data also showed that N315-SA1956, a putative lytic regulator (which is truncated by Tn554 in N315), was upregulated by mgrA and that SA0904, a putative regulator of autolysin Atl, was downregulated. Taken together, our results indicate that mgrA is a major controlling factor for autolysis, negatively regulating autolytic genes but positively regulating antiautolytic factors. Our microarray studies also revealed that mgrA regulated several genes involved in cell wall synthesis and the osmotic stress response. These genes include mraY (N315-SA1025, encoding muramic acid pentapetide translocase), eprH (N315-SA1091, an endopeptidase resistance gene), opuD (N315-SA1987, encoding the glycine betaine transporter), proP (N315-SA0531, encoding a proline/betaine transport homolog), gbsA (SA2406, encoding glycine betaine aldehyde dehydrogenase), cudT (N315-SA2408, encoding the choline transporter), and putP (N315-SA1718, encoding high-affinity proline permease). Perturbation of genes involved in cell wall synthesis is likely to weaken the cell wall, whereas an alteration of the gene involved in osmoprotectant synthesis or transport may lead to cell lysis under osmotic stress. Thus, these genes are likely to be indirectly involved in autolysis. These data
1907
suggest that mgrA also regulates autolysis at a different level, further supporting the notion that mgrA is a major autolytic regulator. Among the genes related to autolysis, lytN, eprH, and N315SA0904 were most profoundly regulated by mgrA at both 2 h and 5 h with high degrees of severalfold changes. To confirm these results, the effect on the expression of lytN and SA0904 by mgrA was measured by quantitative RT-PCR. As shown in Table 4, both genes were highly expressed in the mgrA deletion mutant and slightly repressed in the MgrA-overproducing strain, suggesting that lytN and SA0904 are highly repressed by mgrA in the wild-type strain and that a further increase of MgrA in the overproducing strain therefore does not decrease the level of repression. Regulation of transporters and transmembrane proteins by mgrA. MgrA has been found to affect several efflux pumps including norA (N315-SA0650), norB (N315-SA1269), and tet38 (N315-SA0132) (38, 39). Efflux pumps are involved in antibiotic resistance by effectively extruding antimicrobial agents from bacterial cells. NorA and NorB are multidrug resistance pumps, whereas tet38 confers only tetracycline resistance. In this study, we found that mgrA affected the expression of norA and norB but not tet38. However, our results showed that mgrA downregulated norA and upregulated norB, which is the opposite of results previously reported (38, 39). Since these previous studies used strain ISP794, a derivative of 8325-4 known to contain a mutation affecting sigB expression, it is likely that the discrepancy can be due to a sigB difference between strains ISP794 and Newman. Furthermore, we also found that mgrA affected sigB expression in the studies reported here (see below). However, our results on norA regulation by mgrA are consistent with those reported in a recent study by Kaatz et al. (17), who showed that mgrA downregulated norA in three different strains, ISP794, SH1000 (a sigBpositive 8325-4 strain), and Newman. It should be noted here that methodological differences between laboratories exist, and thus, one cannot exclude the possibility that the discrepancy between our results and those reported previously (38, 39) is due to strain differences. In addition to norA and norB, the tetK efflux gene (COL SA0002) was found to be upregulated at 2 h. Furthermore, we found that two additional putative efflux pump genes (N315SA2233 and SA2261) were upregulated and one (N315SA0099) was downregulated by mgrA. Besides the efflux pump proteins, mgrA appears to regulate a plethora of transporter proteins and transmembrane proteins. These include sortase A (SrtA), preprotein transporter SecY, Na⫹/H⫹ antiporters, ABC transporters, amino acid transporters, ion transporters, a pyrimidine transporter, and sugar transporters. A total of about 50 genes in this group were either upregulated or downregulated by mgrA, suggesting that mgrA plays an important role in controlling various transport systems. Secreted and cell wall-associated proteins are oppositely regulated by mgrA. Mutation of mgrA has a profound effect on extracellular protein production (15, 24). In the current work, we found that mgrA positively affected 19 genes encoding secreted toxins, enzymes, and proteases (Table 2). Most noticeable among this group are genes in three loci encoding leukocidal toxins, which are bicomponent toxins composed of two components designated class S and class F. At least six S
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LUONG ET AL.
proteins and five F proteins have been identified in S. aureus (reviewed in reference 27). Four leukotoxin operons have been characterized to date: ␥-hemolysin (hlgABC), pvl (lukSF-PV), lukED, and lukMF-PV. The genes are encoded in the chromosome. The pvl (Panton-Valentine leukocidin [PVL]) operon is carried by lysogenic phages and is associated with 2 to 3% of clinical isolates, whereas other genes are more frequently present (27). However, PVL has been linked to severe necrotizing infections such as pneumonia and necrotizing faciitis (reference 28 and references therein). Strain N315 contains hlg, lukFM, and lukDE in its chromosome in which lukDE is located within the pathogenicity island SaPIn3 (20). The lukFM and hlgA (separately transcribed with hlgBC) genes are upregulated by mgrA at 2 h, whereas the lukDE genes are profoundly upregulated at 5 h. In addition, lukDE is upregulated by the MgrA-overproducing strain at 2 h. Thus, MgrA can be considered to be a major regulator for leukotoxins. It would be of interest to know whether mgrA also regulates pvl in PVL-positive strains. Another notable cluster of secreted genes that is upregulated by mgrA is the spl operon encoding serine proteases SplABCDEF. Like the lukDE genes, spl genes are substantially upregulated at 5 h. Interestingly, both lukDE and splABCDF are located closely within SaPIn3 (20). To confirm the microarray results, we performed real-time RT-PCR to compare the transcription of lukD and splA genes in the wild-type strain, the mgrA mutant strain, and the MgrA-overproducing strain. Our results showed that splA and lukD were expressed about 54-fold and 30-fold less, respectively, in the mgrA deletion mutant than in the wild type (Table 4). Our results showed that a total of 13 cell wall-anchored or cell surface-associated proteins were regulated by mgrA. These include protein A (Spa) and several putative cell wall proteins containing an LPXTG anchoring domain. In particular, Spa was highly regulated by mgrA at both 2 h and 5 h, a finding consistent with previous reports (15, 24). The cell wall proteins with an LPXTG domain are anchored to the cell wall by sortase A protein (encoded by srtA) (25). However, srtA was upregulated by mgrA (Table 2). Interestingly, our results also revealed that these cell wall-associated or cell surface-associated proteins, except SdrE, were repressed by mgrA (Table 3). Such regulation of cell surface proteins is in contrast to the mgrA regulation of secreted proteins in which all but one (exotoxin 3) was repressed by mgrA (see above). This mode of regulation is similar to that of agr regulation of these two groups of genes in which secreted exoproteins are activated and cell surface proteins are repressed by agr (31). The mechanism by which the agr locus transcriptionally regulates target genes is unknown. Based on the parallel between agr and mgrA, it is tempting to speculate that mgrA may interact with agr to regulate exoproteins and cell surface proteins. Indeed, it has been shown that mgrA mutations significantly reduced RNAIII, the effector of agr, and that mutations of agr slightly decreased mgrA expression (15, 16). However, these studies were conducted using derivatives of strain 8325-4, and therefore, whether the results can be extrapolated to strain Newman requires further studies. Control of metabolic proteins by mgrA. A number of proteins involved in metabolism are regulated by mgrA: 31 genes are upregulated, while 21 are downregulated. Several clusters within this group are highly repressed by mgrA, including the lac operon
J. BACTERIOL.
involved in the utilization of lactose, the urease genes encoded in the ure operon (contains eight genes, ureABCEFGD, and the putative urease transporter SA2081), and the arc genes (Table 3). Utilization of urea by urease produces ammonia, thereby enhancing bacterial acid resistance (29). The arc genes are involved in arginine degradation leading to energy generation as well as ammonia production that could render bacteria acid tolerant under anaerobic conditions (6). Although only the arcAB genes are listed in Table 3, the other three arc genes (arcDCR) in the same cluster were also repressed dramatically, but they did not meet the statistical cutoff used in this study. Both the ure and arc gene clusters have been shown to be induced in biofilms in which the bacteria are likely to be in an acidic environment (2, 33). Thus, in this context, it would be of interest to test whether mgrA affects biofilm formation. The expression of arcA was confirmed by quantitative RT-PCR (Table 4). MgrA also highly repressed the ddh gene encoding NAD⫹-dependent D-lactate dehydrogenase, which may be involved in low-level vancomycin resistance (4), suggesting that mgrA may affect vancomycin resistance. Other genes involved in biological processes that were regulated to a lesser extent by mgrA include genes involved in nucleotide synthesis: pyr genes (pyrRBC, pyrEF, and pyrG) required for pyrimidine synthesis, carAB for carbamoyl phosphate synthesis, purAB for purine synthesis, and guaAB for GMP synthesis. Interestingly, purB and guaAB are upregulated by mgrA, whereas all others genes involved in nucleotide metabolism are repressed by mgrA. It is noteworthy that the pyr gene cluster and the carAB genes were also induced in biofilm in the study by Beenken et al. (2) but not in the study by Resch et al. (33). Regulators affected by mgrA. One means by which mgrA achieves its global regulatory role is through the controlling of other regulators. We found that mgrA marginally upregulated sarA transcription (2.4-fold) when MgrA was overproduced after 5 h of incubation. SarA has been shown to control more than 100 genes (10). To verify these results, we performed real-time RT-PCR and confirmed that sarA transcription was elevated 3.4-fold in overproducing strain CYL907 but was not affected by mutation of mgrA (Table 4), consistent with the microarray results. Ingavale et al. (16) reported recently that sarA was not affected by mgrA deletion; however, no MgrAoverproducing strain was used in that study. SigB is an alternative sigma factor affecting well over 250 genes (3). We found that sigB and the antisigma factor (rsbW) were activated by mgrA to about the same extent when MgrA was overexpressed at 2 h but not at 5 h. Since both sarA and sigB are broadspectrum regulators, some of the effects of mgrA on target genes could be indirect, via effects on these regulators. Two other sarA homologs, sarS and tcaR, with narrow target ranges were also regulated by mgrA. The sarS gene is an activator of spa, whereas the tcaR gene positively controls sarS (and thus spa) and sasF, a cell wall-anchored protein (8, 26, 37). Ingavale et al. have shown that MgrA binds to the sarS promoter and proposed that mgrA represses spa by repressing sarS (16). This is in agreement with our microarray data showing that mgrA repressed sarS at 2 h under conditions of MgrA overexpression (Table 3). However, our data also showed that tcaR was activated by mgrA at 5 h in the mgrA-deleted mutant compared to the wild type (Table 2). Since mgrA strongly downregulated spa at both 2 h and 5 h (Table 3), activation of tcaR by mgrA as
TRANSCRIPTION PROFILING OF THE S. AUREUS mgrA REGULON
VOL. 188, 2006
shown by our microarray results is contradictory, suggesting that the regulation of spa by mgrA is more complicated than the model proposed previously by Ingavale et al. (16). Besides the regulators discussed above, mgrA also potentially regulated 10 additional putative transcriptional regulators (Tables 2 and 3). Thus, many of the genes found in this study could be mediated through these regulators and could therefore be indirectly regulated by mgrA. The expression of the tcaR gene was also verified by the real-time RT-PCR method (Table 4). Conclusions. The multigene regulator MgrA has previously been shown to influence the expression of a diverse set of target genes (15, 24, 39). In this report, we employed microarray technology to identify the genes and better characterize the biological processes that are modulated by mgrA. We found that, as expected, mgrA regulated a wide range of genes including metabolic genes that have not previously been determined to be regulated in an mgrA-dependent manner. In addition, we found 134 genes of unknown function that were regulated by mgrA. The total number of genes regulated by MgrA was remarkably large, making it the regulator with widest target gene spectrum among all staphylococcal global regulators characterized to date. Most of the mgrA target genes reported previously were confirmed in this study; however, some were not detected. Most notably, the hla gene encoding ␣-hemolysin was not detected. The discrepancy could be explained by strain differences. Indeed, we have shown that hla in strain Newman was slightly upregulated by mgrA but was repressed in strain Becker (16, 24). Previously, Ingavale et al. showed that hla was activated in the strain RN6390 background (16). Gene profiling of several staphylococcal global regulators has been reported. These include agr, sarA, rot, sigB, and arl (3, 10, 22, 35). We now provide the profiling results of one more global regulatory gene, mgrA. A comparison of these profiling results is likely to provide insights into the regulatory network governing virulence gene regulation at the genomic level. Already, a pattern has emerged, namely, that agr and mgrA have similar effects on virulence gene regulation, whereas rot and sigB have the opposite effects. These analyses will undoubtedly lead to a greater understanding of the pathogenesis of this major human pathogen. ACKNOWLEDGMENT This work was supported by grant AI54607 from the National Institute of Allergy and Infectious Diseases. REFERENCES 1. Arvidson, S., and K. Tegmark. 2001. Regulation of virulence determinants in Staphylococcus aureus. Int. J. Med. Microbiol. 291:159–170. 2. Beenken, K. E., P. M. Dunman, F. McAleese, D. Macapagal, E. Murphy, S. J. Projan, J. S. Blevins, and M. S. Smeltzer. 2004. Global gene expression in Staphylococcus aureus biofilms. J. Bacteriol. 186:4665–4684. 3. Bischoff, M., P. Dunman, J. Kormanec, D. Macapagal, E. Murphy, W. Mounts, B. Berger-Ba ¨chi, and S. Projan. 2004. Microarray-based analysis of the Staphylococcus aureus B regulon. J. Bacteriol. 186:4085–4099. 4. Boyle-Vavra, S., B. L. de Jonge, C. C. Ebert, and R. S. Daum. 1997. Cloning of the Staphylococcus aureus ddh gene encoding NAD⫹-dependent D-lactate dehydrogenase and insertional inactivation in a glycopeptide-resistant isolate. J. Bacteriol. 179:6756–6763. 5. Brunskill, E. W., and K. W. Bayles. 1996. Identification and characterization of a putative regulatory locus that affects autolysis in Staphylococcus aureus. J. Bacteriol. 178:611–618. 6. Casiano-Colon, A., and R. E. Marquis. 1988. Role of the arginine deiminase system in protecting oral bacteria and an enzymatic basis for acid tolerance. Appl. Environ. Microbiol. 54:1318–1324.
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