The profile made using known E.coli Fur-binding sites (see Materials and Methods) was applied to detect new candidate FUR-boxes in the genomes of E.coli, S.typhi, Y.pestis and V.cholerae.
The profile search in the E.coli K12 genome identified 73 genes having candidate FUR-boxes in the region (–300 … +20) relative to the start of translation. A complete table of orthologous genes was constructed for the genomes of S.typhi, Y.pestis, V.cholerae, H.influenzae, P.aeruginosa and S.putrefaciens. For those genes that had no orthologs or close homologs in the above genomes, homologs in distant species were identified using BLAST (http://www.ncbi.nlm.nih.gov/blast/). Then the E.coli K12 profile was used to identify the candidate FUR-boxes upstream of orthologous and homologous genes in the other genomes. As a result, six new candidate Fur-regulated genes were found.
The same procedure was applied to the genomes of E.coli O157:H7 (–300 … +50), S.typhi (–400 … +100 relative to the start codons), Y.pestis and V.cholerae (–500 … +100 relative to the start codons). The candidate sites within coding regions of upstream genes (deeper than 50 nt) were excluded. The number of genes with candidate FUR-boxes in the four genomes was 89, 75, 88 and 94, respectively.
In all five genomes, the profile search selected a number of experimentally confirmed FUR-boxes. They are not discussed below unless these genes have been used for the verification of candidate sites.
Below we carefully describe the predicted Fur-regulons from the genomic perspective. The functional overviews are given in the Discussion. The evolutionary perspective is presented in the Conclusion.
Candidate FUR-boxes in the E.coli K12 genome
Table shows E.coli genes with new candidate FUR-boxes: fptAybiX, ydiE, b1995, b3070, yhhX.
The predicted FUR-boxes upstream of the iron-acquisition genes of E.coli and the FUR-boxes upstream of the homologous genes in other genomes
The genes fptAybiX
are likely to form an operon, as the length of the intergenetic region is only 6 nt. We have found a strong candidate FUR-box upstream of fptA
(Table and Fig. A). In the study by Oshima et al.
was annotated as a Fe(III)–pyochelin receptor due to its similarity to the P.aeruginosa
with the same function. The corresponding locus in the P.aeruginosa
genome is shown in Figure B. It also includes piuC
, the ortholog of ybiX
that encodes an iron-uptake factor, and a strong FUR-box (Table ). The arrangement of genes in P.aeruginosa
is different, as piuA
are divergently transcribed (Fig. B). This system was shown to be regulated by the P.aeruginosa
analog of the Fur (33
). A locus similar to that of P.aeruginosa
was found in the genome of V.cholerae
(Fig. C). It also has a strong FUR-box (Table ), although nothing is known about its regulation by Fur protein.
Figure 2 (A) The chemotaxis system of E.coli (taken from KEGG; 61). (B) One of the three loci containing che genes. The homologs of cheW, cheB, cheR, cheY and cheA are shown by the yellow arrows, genes encoding the MCPs are shown by the light green arrows. ( (more ...)
genes are located separately in the genome of E.coli
. Each of them has a strong upstream FUR-box (Table ). BLAST revealed their similarity to the Yersinia enterocolitica
haemin uptake proteins HemP (haemin-uptake factor) and HemR (haemin receptor), respectively (with E
-values 9e-04 and 3e-07). In the genome of Y.enterocolitica
form an operon. A FUR-box was described in its operator region and the iron-dependent transcription of hemP
was shown (34
). In the genome of S.typhi
there is a locus similar to that described in Y.enterocolitica
. It contains strong homologs of hemP
, and a strong FUR-box (Table ).
The next E.coli
gene with a candidate FUR-box, b3070
(Table ), is orthologous to viuB
-value 4e-15). This gene encodes an iron-chelator utilization protein. In V.cholerae
belongs to the iron-acquisition locus vuiAiuBviuF
shown to be regulated by Fur (35
). Transcription of viuB
is iron dependent and it has a candidate FUR-box in the promoter region (Table ) (36
). The ortholog of viuB
also has an upstream candidate FUR-box (Table ).
The gene yhhX
is a putative oxidoreductase. The possible role of oxidoreductases in the iron uptake is discussed below (see Discussion). We have found a candidate FUR-box upstream of this gene and upstream of its homolog yvaA
in the genome of Bacillus subtilis
(Table ). The E.coli
genome contains a gene b1624
with stronger similarity to yvaA
(44% identity compared with 30%) and no FUR-box. The S.typhi
ortholog of yhhX
also has a candidate FUR-box (Table ). After this study was completed, the FUR-box upstream of yhhX
was experimentally confirmed (37
Candidate FUR-boxes in the E.coli O157:H7 genome
Table shows E.coli O157:H7 genes with new candidate FUR-boxes: Z2267, Z2268, chuTWXYUV*, z4912chuAS, Z1026ybiX, Z3159, Z1178, Z1617, ydiE, yqjH, Z4385-Z4382, Z4386, yhhX.
The predicted FUR-boxes upstream of the iron-acquisition genes of E.coli O157:H7 and the FUR-boxes upstream of the homologous genes in other genomes
Some of these genes have already been described in the above paragraph. Z1026 and ybiX from E.coli O157:H7 are orthologs of the fptA and ybiX from E.coli K12, respectively; Z3159 and ydiE are orthologs of b1995 and ydiE; yqjH is orthologous to b3070; yhhX has the same name in both genomes.
A new gene iha
encoding an adhesin was recently discovered in the pathogenic strain of E.coli
). Actually, in the genome of E.coli
O157:H7 there are two identical genes for the Iha adhesin, Z1178
. We have found strong FUR-boxes upstream of both genes (Table ), and the FUR-box is conserved upstream of the orthologous gene in the genome of Y.pestis
Z2267 and Z2268 form a divergon. Z2267 encodes a putative receptor with a candidate role in enterotoxin production, determined by homology to the S.typhi gene with the same function. Z2268 encodes a putative receptor for iron transport (as annotated in GenBank). A candidate FUR-box was observed in the intergenetic region of Z2267 and Z2268 (Table ). It is conserved in the genomes of S.typhi and Y.pestis. In the latter case there are no orthologs for Z2267 and the signal was found only upstream of fyuA, a Z2268 ortholog with 24% identity.
The genes Z4385–Z4382 encode an iron ABC transporter, whereas the divergent gene Z4386 encodes an iron-compound receptor. We have found a candidate FUR-box in the Z4385–Z4386 intergenic region (Table ). The Z4385–Z4382 operon is similar to the iron ABC transporter of B.subtilis yfmCDEF and to the ferric vibriobactin ABC transporter of V.cholerae VC0776–VC0779 (although the arrangement of genes in both cases is different). Candidate FUR-boxes precede both yfmC and VC0776.
Escherichia coli O157:H7 also has a large locus of genes involved in iron acquisition with no orthologs in E.coli K12 and S.typhi. This locus includes: chuTWXYUV* and divergent Z4912-chuAS. The first gene, chuT, encodes a putative periplasmic-binding protein, chuW encodes a putative oxygen independent coproporphyrinogen III oxidase and chuX and chuY are unknown open reading frames. Further, chuU encodes a putative permease of iron-compound ABC transport system, and chuV* (Z4919) encodes a hypothetical protein highly similar to ATPases of iron ABC transporters. Finally, Z4912 is an unknown open reading frame, chuA encodes an outer membrane heme/hemoglobin receptor, and chuS encodes a putative heme/hemoglobin transport protein. A strong candidate FUR-box was found in the Z4912-chuT intergenic region. This system was also found in the genomes of Y.pestis and V.cholerae and in a reduced form in the genomes of S.putrefaciens and P.aeruginosa. The arrangement of genes in each case is unique, but a candidate FUR-box is always present in the regulatory region. There is a large intergenic space of 216 nt between Z4912 and chuA. We have found another strong candidate FUR-box upstream of chuA, although it overlaps with Z4912.
Candidate FUR-boxes in the S.typhi genome
The S.typhi genes with new candidate FUR-boxes are feoAB, sodA, hmsT*, foxR* (Table ).
The predicted FUR-boxes upstream of the iron-acquisition genes of S.typhi and the FUR-boxes upstream of the homologous genes in other genomes
Two systems, feoAB
for Fe(II) transport and sodA
encoding the superoxide dismutase, are known to be regulated by Fur in E.coli
). There are candidate FUR-boxes upstream of the sodA
orthologs in the genomes of S.typhi
and V. cholerae
(Table ). Candidate FUR-boxes upstream of the orthologs of feoAB
were found in the genomes of S.typhi
The gene hmsT*
is an ortholog of hmsT
that encodes a haemin storage protein. Both genes have candidate FUR-boxes (Table ). In Y.pestis
, the hms
-phenotype was shown to be regulated by Fur (39
). In the genome of S.typhi
this gene is followed by an ortholog of fhuF
, the Fur-regulated gene from E.coli
FoxR* of S.typhi
is an AraC/XylS-type regulator. This gene has a candidate FUR-box. There are candidate FUR-boxes upstream of closely related regulators in other genomes (alcR
; Table ). The gene alcR
from Bordetella pertussis
was shown to be regulated by Fur (40
belongs to the yersiniabactin biosynthesis locus and and its expression is Fur dependent (41
is likely to be regulated by Fur (42
). Besides, all three proteins regulate transcription of Fur-regulated iron-acquisition systems. AlcR and YbtA act in cis
, the target of PchR is located not further than 14 kb from the pchR
gene. Given these observations, we propose the foxA
gene as a candidate target for the regulation by FoxR*. Indeed, it is located immediately downstream of foxR
, it has a strong FUR-box (43
) and the product of foxA
functions as a ferrisiderophore receptor.
Candidate FUR-boxes in the Y.pestis genome
Table shows Y.pestis genes with new candidate FUR-boxes: itpPTS*, itsPTUS*, itrAS*, irp2, iucABCDiutA*, alcABC*, aclY*, psaE, iha*, hemN*, fhuE*, omrA*, hasR* and yebN*.
The predicted FUR-boxes upstream of the iron-acquisition genes of Y.pestis and the FUR-boxes upstream of the homologous genes in other genomes
We propose the name Itp* (iron transport) for a new candidate Fe(III) ABC transporter, which was found in the Y.pestis genome. The Itp system consists of a periplasmic-binding protein (ItpP*), a transmembrane protein (ItpT*) homologous to the members of the FecC/D iron permease family (BLAST E-value 3e-47) and an ATPase (ItpS*) homologous to the ATP-binding protein VC0779 of the V.cholerae ferric vibriobactin ABC transporter (BLAST E-value 3e-28). Besides, the gene located immediately downstream of itpS* has a strong similarity to the iron-regulated outer membrane ferrisiderophore receptor irgA from V.cholerae. There is a candidate FUR-box upstream of itpP* (Table ).
One more candidate Fe(III) ABC transporter found in the genome of Y.pestis
was named Its* (iron transport system). It includes a periplasmic protein (ItsP*), two transmembrane proteins (ItsT*, ItsU*) and an ATPase (ItsS*). There is a candidate FUR-box upstream of itsP*
(Table ). Iron transporter systems similar to Its exist in the genomes of V.cholerae
and Vibrio angulliarrium
). The gene fatB
is the ortholog of itsP
. The Fur-dependent regulation of fatBA
was shown in Actis et al
). Although Fur does not bind to the fatB
promoter, it regulates the expression of the latter indirectly by binding to the promoter of the antisense RNA. (45
). We have found a candidate FUR-box upstream of the itsP
ortholog in the genome of V.cholerae
(Table ). Nothing is known about the antisense RNA regulation of this transporter in V.cholerae
. Thus, the antisense RNA regulation might be unique for the V.anguillarium
system. Although the absence of a Fur-binding site upstream of fatB
does not allow us to invoke the general rule (Table ), we still predict the Fur-repressible transcription of the itsPTUS
One more iron transport locus in the genome of Y.pestis includes a candidate outer membrane siderophore receptor itrA* and a gene itrS* (iron transport). We have assigned the function of ItrS* as an iron transport ATPase by sequence similarity to the ItpS*/vibriobactin protein family. A strong candidate FUR-box was observed upstream of itrA* (Table ).
genes encoding siderophore-biosynthesis proteins, iucA*
, also have candidate FUR-boxes in their upstream regions (Table ). The former gene is a homolog of the Fur-regulated gene iucA
-value, 4e-29) and Shigella flexneri
-value, 5e-30). In E.coli
, the operon iucABCD
encodes proteins for aerobactin biosynthesis. In Y.pestis
also belongs to a siderophore-biosynthesis cluster that includes homologs of iucB
(Fig. D). The product of irp2
participates in yersiniabactin biosynthesis (46
) and it lies within a large yersiniabactin biosynthesis and uptake locus with one known and two candidate FUR-boxes (Fig. E).
One more siderophore-biosynthesis gene cluster alcABC*
is probably regulated by Fur. The alcABC*
genes lie downstream of genes fhuF*
*, and most likely form an operon with the latter (Fig. F). FhuF* is 37% identical to the FhuF protein from E.coli
. The latter is involved in the reduction of ferric iron in cytoplasmic ferrioxamine B (47
). RhsB* is homologous to the siderophore-biosynthesis protein RhsB from B.subtilis
. A strong FUR-box was found upstream of the fhuF*
gene in Y.pestis
(Table ). In the genome of E.coli
is also preceded by a strong FUR-box. The genes alcABC*
are homologous to the alcaligin-biosynthesis genes alcABC
-value, e-156). The FUR-box was observed upstream of the alcABC
operon in the genome of B.pertussis
and the transcription of alcABC
is iron dependent (48
). Moreover, in the genome of Y.pestis
, the fhuF*rhsB*alcABC*
operon lies within the locus containing other genes involved in the biosynthesis and transport of siderophores (Fig. F). One of them is alcY*
, which is 50% identical to alcC*
. It is also preceded by a candidate FUR-box (Table ). The downstream gene of alcY* fauA*
, similar to a gene for ferric alcaligin siderophore receptor from B.pertussis
A strong candidate FUR-box was found upstream of psaE
, the transcriptional regulator of biosynthesis of the Y.pestis
pH6 antigen (Table ). This antigen plays an essential role in the pathogenesis of Y.pestis
) and the psa
genes are co-localized with some iron-uptake systems (Fig. G). The role of Fur in the regulation of virulence systems is discussed below.
One more candidate Fur-regulated operon with a function in the pathogenesis is ihaAB* (Table ). The gene ihaA* encodes an adhesin similar to the one produced by the pathogenic strain of E.coli O157:H7 (iha). The latter also has an upstream candidate FUR-box (Table ; see above). The gene ihaB* encodes an exogenous ferric siderophore receptor.
The genes encoding coproporphyrinogen oxidase in Y.pestis (chuW*), E.coli O157:H7 (chuW) and in V.cholerae (VCA0909) all have candidate FUR-boxes (Table ). They probably participate in the Fe(III) reduction after it is delivered to the cytoplasm.
Two new Y.pestis
outer membrane receptors with candidate FUR-boxes are omrA*
. OmrA* is homologous to the members of the ferrisiderophore receptor family including FyuA and FhuA from E.coli
. The closest homolog of omrA*
was found in the genome of P.aeruginosa
, where it also has a candidate FUR-box (Table ). The second gene, hasR*
, is an ortholog of hasR
, encoding a heme receptor. Although the candidate FUR-box of hasR
is weak (Table ), the Fur-regulated transcription of this gene was shown experimentally (50
). In Y.pestis
lies in a locus containing other iron-acquisition genes (Fig. H).
The gene yebN* encoding a hypothetical membrane protein has a very strong candidate FUR-box. The orthologous gene yebN of E.coli also has a candidate upstream FUR-box (Table ). In the Y.pestis genome a gene for a ferrichrome receptor follows this gene.
Candidate FUR-boxes in the V.cholerae genome
Tables and show V.cholerae genes with new candidate FUR-boxes: VC1573, VCA0625, irpA, VCA0070, VCA0824, VCA0232, VCA0227, VCA0284, VCA0068, VC1643, VCA0988, VCA0923, VC1403, VC1405, tcpI, tcpP, cheY, fliLM.
The predicted FUR-boxes upstream of the iron-acquisition genes of V.cholerae and the FUR-boxes upstream of the homologous genes in other genomes
has a strong candidate FUR-box. It is similar to the fumarate hydratase genes fumC
(46% identity), P.aeruginosa
(65% identity) and other Enterobacteraceae. The transcription of fumC
is regulated by Fur and there is a known Fur-binding site upstream of the fumC
-containing operon in P.aeruginosa
). However, no FUR-boxes were found upstream of fumC
, and only weak candidate FUR-boxes were observed upstream of fumC
The product of VCA0625 was annotated as a TonB receptor-related protein (similar to the heme receptor HasR from P.aeruginosa and Serratia marcescens). A candidate FUR-box was observed upstream of this gene and upstream of the orthologous gene from P.aeruginosa (Table ).
There is a candidate FUR-box upstream of VC1264
(Table ). This gene is similar to irpA
. In Synechococcus
is expressed only under iron-deficient conditions and this gene also has a candidate Fur-binding site in the upstream region (Table ) (52
VCA0824 encodes a protein similar to diaminobutyrate aminotransferases. This gene has a candidate FUR-box site in the upstream region (Table ), whereas its product is similar to RhsA, a rhizobactin siderophore-biosynthesis protein from Sinorhizobium meliloti.
A new iron-acquisition locus with two candidate FUR-boxes consists of the ABC-transporter operon VCA0227–VCA0230
and the gene VCA0232
encoding an outer membrane protein (Fig. I). The product of VCA0232
is similar to FrpB (iron-regulated TonB-dependent outer membrane protein) from Neisseria meningitis
and ChuA (heme utilization outer membrane protein) from the pathogenic strain of E.coli
O157:H7. The genes of all three outer membrane receptors have upstream candidate FUR-boxes (Table ). The Fur-dependent regulation of chuA
has already been shown (53
). The operon frpPTUS
homologous to the ABC-transport operon VCA0227–VCA0230
is present in the genome of N.meningitis
) where it lies immediately downstream of frpB
(Fig. J). In both genomes, V.cholerae
, there are candidate FUR-boxes upstream of the transporter operons (Table ).
One more iron-acquisition system (VC0284 and ybiX*), which is likely to be regulated by Fur, consists of an outer membrane TonB-dependent receptor and an iron-uptake factor (Fig. C). This system is similar to the E.coli and P.aeruginosa iron-acquisition systems (see above).
The profile we used pulled out several V.cholerae
genes that had already been described as having candidate FUR-box in the upstream regions, namely vibF
) and vibA
Unexpectedly, a large set of genes involved in chemotaxis were found to have candidate FUR-boxes in the upstream regions. These genes encode eight MCPs (VCA0068
); homologs of the E.coli
chemotactic signal transducing proteins, VC1397
) and VC1398
); a flagellar motor switch protein VC2126
); a homolog of fliL
. All mentioned E.coli
genes are involved in the sequential steps of chemotaxis (Fig. A) (26
). Escherichia coli
has only five MCPs: tar
and only one copy of each che
. On the contrary, more than 40 MCPs were found in the genome of V.cholerae
and there are three large loci with che
-like genes. A candidate FUR-box was observed upstream of the locus containing genes least similar to the E.coli che
-genes (Fig. B). Table shows candidate FUR-boxes upstream of V.cholerae
-like genes and fliLM
homologs. The chemotaxis plays an important part in the V.cholerae
). Indeed, two of the MCPs with candidate FUR-boxes (tcpI
) are the members of the toxin co-regulated pilus-biosynthesis cluster (Fig. C). The role of Fur in the pathogenesis is discussed below (see Discussion).
The candidate FUR-boxes upstream of genes encoding MCPs in the V.cholerae genome
The profiles and comparison-based filters produced three more genes with conserved Fur-box-like sequences in the upstream regions, but the functions of these genes were not linked to the iron metabolism (Table ). Thus, they seem to be false positives. These genes are: gpmA (phosphoglycerate mutase 1 involved in glycolysis), nupC (nucleoside permease) and b2392 (NRAMP manganese transport protein) in the genome of E.coli (both strains) and their orthologs in the genomes of S.typhi and Y.pestis.
Sequences similar to FUR-boxes upstream of genes with no direct function in iron acquisition