Article Figures & Data
Figures
- Fig 1
CLUSTAL W alignment of the deduced amino acid sequence of Fur from NTHi strain 86-028NP, E. coli K-12, P. aeruginosa, H. pylori, and V. cholerae. Amino acids determined to be important in zinc coordination are marked. Open triangles indicate aspartate, arginine (P. aeruginosa), and tyrosine (E. coli) residues. Stars indicate cysteines (H. pylori and E. coli). Open squares show glutamic acids (P. aeruginosa, H. pylori, and V. cholerae). The open circle shows a zinc-coordinating histidine (P. aeruginosa, H. pylori, and V. cholerae). The zinc coordinating HHDH motif is identified using a square bracket (P. aeruginosa, H. pylori, and V. cholerae).
- Fig 2
The expression of hemoglobin-haptoglobin binding protein B is regulated by both Fur and phase variation. (A) The coding sequence for HgpB (red) contains 12 tetranucleotide repeats (TR) at the 5′ end. In the fur mutant, hgpB contains an extra tetranucleotide repeat, which generated a frameshift. These data are derived from three independent sequencing experiments. (B) qRT-PCR analyses using primers that hybridized upstream of the tetranucleotide repeats (5′) showed that expression of hgpB is classically Fur repressed. Conversely, qRT-PCR analyses using primers that hybridized downstream of the tetranucleotide repeats (3′) showed that the frameshift generated by phase variation reduced the transcription of hgpB in the fur mutant, relative to the parent. **, P < 0.05, while the P value for the 3′ data was 0.08. The data was generated using a Student paired t test, with a two-tailed distribution.
- Fig 3
Regulation of protein expression by Fur as demonstrated by PAGE analyses. Dysregulation of protein expression in sarcosyl-insoluble membrane fractions of strain 86-028NP (P) and a strain 86-028NP fur mutant (M) when grown in either sBHI (A) or DIS medium supplemented with human hemoglobin (B) was demonstrated.
- Fig 4
Characterization of strain 86-028NP-specific consensus Fur DNA binding site. (A) The model shows the frequencies of bases at each position (relative heights of the letters) and the degree of sequence conservation (total height of the letter stack). (B) The strain 86-028NP predicted Fur box is highly homologous to that derived for E. coli by Escolar et al. (63). Like the E. coli Fur box, the strain 86-028NP predicted Fur box can be considered to be two 9-bp inverted repeats (B, upper panel) or three repeats of 6 bp (two directed and one inverted) of the invariable sequence (B, lower panel). (C) The promoter regions of Fur-regulated genes bound Fur. Biotin-labeled DNA that contained predicted Fur boxes were mixed with cytoplasmic enriched extracts from either 86-028NPΔfur::Tn903 (−) or 86-Δfur::Tn903(pTS-fur) in which Fur expression was induced (+). Biotinylated DNA was subsequently isolated with streptavidin beads and resolved by PAGE. Fur that copurified with DNA was identified by Western blotting with an anti-Pseudomonas Fur antibody. Three replicate experiments were carried out, and a representative example is shown. An asterisk (*) indicates a nonspecific band that was used as a loading control. (D) To confirm that the binding of Fur to promoter regions was dependent on a predicted Fur box, the Fur binding assay was repeated with the hfeA promoter region with its predicted Fur box, as well as the hfeA promoter region in which the predicted Fur box had been scrambled.
- Fig 5
Fur is required for the persistence of NTHi strain 86-028NP when in competition with its parent in a chinchilla model of otitis media. (A) Five chinchillas were infected with a 1:1 mix of NTHi strain 86-028NP and NTHi strain 86-028NPΔfur::Tn903. Middle ear fluids were recovered by epitympanic tap on days 4, 7, and 10 postinfection, and the CFU were quantified by plating on selective media. By day 7, the fur mutant could not be recovered from any animal, while the parent persisted for the course of the experiment. (B) Ten days after infection, the fur mutant could not be recovered from middle ear mucosae. The data are expressed as the number of CFU/mg of homogenized mucosa. Two asterisks (**) denote P < 0.05, while one asterisk (*) denotes P < 0.001 as determined by a two-tailed Mann-Whitney U test.
Tables
- Table 1
Bacterial strains and plasmids
Strain or plasmid Relevant genotype Source or reference Strains H. influenzae 86-028NP NTHi strain from a child with chronic OM 20 H. influenzae 86-028NPΔfur::Tn903 Derivative of 86-028NP with an insertion/deletion mutation of fur This study H. influenzae 86-028NPΔfur::Tn903(pTS-fur) 86-028NPΔfur::Tn903 transformed with pTS-fur This study H. influenzae strain Rd A rough derivative of H. influenzae serotype d strain KW20 21 E. coli strain DY380 DH10B derivative containing a defective λ prophage; red, bet, and gam genes are controlled by the temperature-sensitive λcI857 repressor 95 E. cloni Strain used for general cloning Lucigen E. coli strain DH10B Strain used for general cloning Invitrogen Plasmids pKD3 Template plasmid used for mutant construction in E. coli; contains R6Kγ origin of replication 30 pKD3kan pKD3 derivative in which the chloramphenicol resistance gene was replaced with the kanamycin resistance gene from Tn903 This study pSPECR pCR-Blunt containing a spectinomycin resistance gene 37 pGZRS-39A Haemophilus-Actinobacillus pleuropneumoniae shuttle vector that contains the kanamycin resistance gene from Tn903 32 pSPEC1 pGZRS-39A derivative in which the kanamycin resistance gene was replaced with the spectinomycin resistance gene from pSPECR 28 pLS88 Broad-host-range plasmid isolated from Haemophilus ducreyi 35 pRSM2947 Haemophilus-E. coli shuttle vector with a temperature-sensitive origin of replication from pLS88 and an Flp recombinase gene 34 pET-30b Protein expression vector with f1 origin of replication EMD Millipore pSMART-LCKan Low-copy-number cloning vector Lucigen pT E. coli-Haemophilus shuttle vector which contains the tet repressor/promoter from pRSM2947 This study pT-fur pT containing the coding sequence for fur clones downstream of the tet repressor/promoter This study pTS-fur A derivative of pT-fur in which the kanamycin resistance gene was replaced with the spectinomycin resistance gene from pSPECR This study - Table 2
Fur-regulated genes in NTHi strain 86-028NP
NTHI no. Gene Function Fold changea Presence of Fur boxb M/P NTHi Type b Rd NTHI0002 Long-chain fatty acid coenzyme A ligase –2.2 2.42 1.42 NS N NTHI0007 fdxG Formate dehydrogenase major subunit –4.2 8.36 7.58 NO N NTHI0010 fdxH Formate dehydrogenase, iron-sulfur subunit –3.9 7.49 6.00 3.40 N NTHI0011 fdxI Formate dehydrogenase, cytochrome b556 subunit –2.7 7.47 5.23 3.20 N NTHI0012 fdhE Formate dehydrogenase accessory protein –2.4 3.35 3.43 1.88 N NTHI0088 nrdD Anaerobic ribonucleoside triphosphate reductase 9.9 –2.95 –2.54 –4.03 N NTHI0173 Hypothetical protein 3.1 Y NTHI0175 Hypothetical protein 4.0 –12.56 –3.93 –10.21 Y NTHI0177 hitA Iron-utilization periplasmic protein hFbpA 7.7 –14.27 –4.52 –8.15 Y NTHI0179 hitB Iron(III)-transport system permease protein hFbpB 8.2 NS –2.70 –3.55 Y NTHI0180 hitC Iron-utilization ATP-binding protein hFbpC 6.9 NS –2.16 –3.56 Y NTHI0202 hemR Hemin receptor 3.6 –4.72 –2.28 –2.36 Y NTHI0209 NTHI0209 Hypothetical protein –2.2 N NTHI0285 fldA Flavodoxin FldA 2.1 N NTHI0358 tonB TonB 3.0 –1.99 –1.69 –2.75 Y NTHI0359 exbD Biopolymer transport protein 3.3 –2.45 –1.87 –3.02 Y NTHI0360 exbB Transport protein ExbB 3.0 –3.07 –2.26 –2.69 Y NTHI0364 NTHI0364 Hypothetical protein –2.2 –2.27 –1.52 –1.61 Y NTHI0369 hxuC Heme/hemopexin utilization protein C 6.0 –24.69 –4.03 –10.30 Y NTHI0370 hxuB Heme/hemopexin-binding protein B 5.5 –29.31 –3.29 –9.20 Y NTHI0390 Metabolite transport protein 2.7 N NTHI0477 hfeD Putative ABC-type chelated iron transport system, permease component 3.3 –2.92 1.21 –4.35 Y NTHI0478 hfeC Putative ABC-type chelated iron transport system, permease component 3.0 –3.54 NS –6.90 Y NTHI0479 hfeB Putative ABC-type chelated iron transport system, ATPase component 2.5 –7.89 NS –8.91 Y NTHI0782 hgpB Hemoglobin-haptoglobin binding protein B –8.0 –6.52 –4.14 –1.20 Y NTHI0783 ABC transporter ATP-binding protein 4.4 NS –2.11 –1.61 N NTHI0785 ABC transporter ATP-binding protein 3.8 NS –1.87 –1.61 N NTHI0805 glpC sn-Glycerol-3-phosphate dehydrogenase subunit C –2.2 –1.35 –1.63 NS Y NTHI0806 glpB Anaerobic glycerol-3-phosphate dehydrogenase subunit B –2.8 NS –1.72 –1.24 Y NTHI0808 glpA sn-Glycerol-3-phosphate dehydrogenase subunit A –2.8 Y NTHI0813 glpK Glycerol kinase –2.1 –5.71 –3.40 –1.61 Y NTHI0831 tnaA Tryptophanase 2.1 NH N NTHI0998 frdD Fumarate reductase subunit D –2.4 NS –1.95 NS N NTHI0999 frdC Fumarate reductase subunit C –2.4 NS –1.33 NS N NTHI1000 frdB Fumarate reductase iron-sulfur subunit –2.1 N NTHI1021 hbpA Heme-binding protein A –2.2 Y NTHI1164 iga1 IgA-specific serine endopeptidase 2.1 –1.82 –1.45 –1.52 Y NTHI1168 tbp1 Transferrin-binding protein 1 17.4 –9.56 –8.16 –12.40 Y NTHI1169 tbp2 Transferrin-binding protein 2 precursor 35.2 –19.45 –16.53 –13.94 Y NTHI1171 Hypothetical protein 16.6 –15.68 –14.61 NO Y NTHI1203 Ferredoxin –2.8 N NTHI1205 dmsC Anaerobic dimethyl sulfoxide reductase chain C –4.3 NS –1.98 –1.06 Y NTHI1206 dmsB Anaerobic dimethyl sulfoxide reductase chain B –4.4 NS –2.43 NS Y NTHI1207 dmsA Anaerobic dimethyl sulfoxide reductase chain A precursor –5.0 NS –4.99 –1.10 Y NTHI1226 nrfD NrfD, formate-dependent nitrite reductase, membrane component –3.4 6.34 2.24 1.88 Y NTHI1227 nrfC NrfC, Fe-S-cluster-containing hydrogenase component 1 –3.1 6.99 1.70 1.52 Y NTHI1229 nrfB Cytochrome c nitrite reductase pentaheme subunit –3.8 9.49 NS 2.00 Y NTHI1230 nrfA Cytochrome c552 –3.8 8.33 2.53 2.13 Y NTHI1321 hpt Hypoxanthine-guanine phosphoribosyltransferase 3.3 –1.90 1.15 –1.20 N NTHI1344 NTHI1344 Hypothetical protein –2.7 N NTHI1345 artM Arginine transporter permease subunit ArtM –4.7 –4.54 –2.24 NS N NTHI1346 artQ Arginine transporter permease subunit ArtQ –4.8 –7.73 –2.77 NS N NTHI1347 artI Arginine-binding periplasmic protein –4.5 –5.87 –2.83 –1.40 N NTHI1348 artP Arginine transporter ATP-binding subunit –2.7 –5.68 –2.47 –1.20 N NTHI1449 hmw2B HMW2B, OMP-85-like protein required for HMW1A and HMW2A secretion –2.3 NH N NTHI1649 fabA 3-Hydroxydecanoyl-(acyl carrier protein) dehydratase –2.3 1.66 1.77 NS N NTHI1707 ABC transporter periplasmic protein 2.3 –6.60 –2.32 –6.12 Y NTHI1712 Hypothetical protein 3.4 N NTHI1716 Hypothetical protein 2.7 NH N NTHI1717 Hypothetical protein 2.2 NH Y NTHI1721 Hypothetical protein 2.9 NH N NTHI1722 Hypothetical protein 3.2 NH N NTHI1723 Hypothetical protein 3.4 NH N NTHI1724 Hypothetical protein 3.0 NH N NTHI1726 Hypothetical protein 2.8 NH N NTHI1727 ninB Putative recombination protein NinB 2.4 NH N NTHI1772 ftnB Ferritin –2.4 4.13 2.24 2.94 Y NTHI1773 ftnA Ferritin-like protein 1 –2.7 3.21 2.01 2.60 Y NTHI1796 pqqL Putative zinc protease 2.3 NS NS –1.86 N NTHI1961 nrdA Ribonucleotide-diphosphate reductase subunit alpha 2.6 N NTHI1962 nrdB Ribonucleotide-diphosphate reductase subunit beta 2.9 NS –1.56 –1.54 N NTHI1979 Regulator of cell morphogenesis and NO signaling –2.6 Y NTHI1984 hmw1B HMW1B, OMP-85-like protein required for secretion of HMW1A and HMW2A –2.2 NH N ↵a M, 86-028NPΔfur::Tn903; P, 86-028NP. Data for NTHi, type b, and Rd were from Whitby et al. (53) following transition from iron-heme restricted to iron-heme replete medium. NS, not significant; NH, no homolog of 86-028NP gene in strain Rd; NO, not on, i.e., there was no homologue of NTHi or type b gene in strain Rd.
↵b Y, yes; N, no.
- Table 3
Fur-regulated genes that encode proteins with hypothetical functions in NTHi strain 86-028NP
NTHI no. Predicted functiona Predicted motif(s)b NTHI0173 Nucleoside-diphosphate-sugar epimerase Permease involved in gluconate uptake NTHI0175 Methylase SAM-dependent methyltransferase NTHI0209 NTHI0364 Ribosome-binding protein Sigma 54 modulation protein NTHI1171 Type III secretion chaperone Tetratricopeptide repeats NTHI1344 YadA-like C-terminal YadA-like C-terminal NTHI1712 DNA binding excisionase NTHI1716 DNA binding Winged-helix-like domain NTHI1717 Hydrolase NTHI1721 Transcription factor DNA-binding motif NTHI1722 Transcription factor NTHI1723 Transcription factor DNA-binding motif NTHI1724 Primosomal protein DNA-binding motif NTHI1726 Metal-binding protein - Table 4
Confirmation of Fur-regulated genes in NTHi strain 86-028NPa
NTHI no. Gene Array data (M/P) qRT-PCR M/P C/P Fe(–)/Fe(+) NTHI0007 fdxG –4.2 –5.3 –1.4 –6.0 NTHI0177 hitA 5.7 11.4 –6.6 94.6 NTHI0360 exbB 3.0 5.8 2.0 5.5 NTHI0369 hxuC 6.0 9.4 –1.5 7.4 NTHI0371 hxuA 1.5 2.4 –2.1 2.3 NTHI0479 hfeB 2.5 3.7 –14.1 9.5 NTHI0481 hfeA 1.3 4.3 –3.8 9.7 NTHI0998 frdD –2.4 –1.4 1.4 –2.1 NTHI1169 tbp2 35.2 18.0 –2.9 79.0 NTHI1207 dmsA –5.0 –1.1 1.8 2.4 NTHI1230 nrfA –3.8 –7.2 –3.1 –17.1 NTHI1347 artI –4.5 –2.7b –1.2 –5.4 NTHI1348 artP –2.7 1.6 1.4 –5.0 NTHI1721 Hypothetical 2.9 1.3 –1.4 1.4 NTHI1773 ftnA –2.7 –4.8 –4.3 –1.7 NTHI1961 nrdA 2.6 2.1 –3.4 –3.5
