Gene, 123 (1993) 33-38 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-I 119/93/$06.00

GENE 06869

33

Regulation of the expression of bacterial iron transport genes: possible role of an antisense RNA as a repressor

(Fe3 + regulation; Vibrio anguillarum; pJM1 plasmid-mediated iron transport; siderophore; anguibactin)

Patricia C. Salinas”, Lillian S. Waldbeser and Jorge H. Crosa

Department of Microbiology and Immunology, School of Medicine, Oregon Health Sciences University, Portland, OR 97201.3098, USA

Received by F. Bolivar: 20 April 1992; Revised/Accepted: 20 July/l2 August 1992; Received at publishers: 2 October 1992

SUMMARY

Expression of the iron transport genes,futB andfutA, in the bacterium Vibrio anguillarum 775 is negatively regulated

by the iron concentration in the medium. Here, we show that iron repressesfatB andfatA mRNA levels and concomi-

tantly induces the synthesis of an antisense RNA (RNAa). The presence of RNAa correlates with the inhibition of FatA

protein synthesis and thus may play a role in the iron repression off&4 expression. Since the 5’ end of RNAcl maps

125 nucleotides upstream from the start codon off&A and this RNA also extends into the coding region offat& it may

also be involved in the iron regulation off&B expression. RNAa may thus constitute a novel component of the bacterial

iron regulatory circuit.

INTRODUCTION

Environmental factors, such as iron, play an important

role in the regulation of bacterial gene expression (Cal-

derwood et al., 1987; Crosa 1990; 1984; Miller et al., 1990).

V. anguillavum 775 possesses the pJM1 plasmid-mediated

iron uptake system which is a major virulence factor of

this bacterium (Crosa, 1980). This iron uptake system

Correspondence to: Dr. J. H. Crosa, Department of Microbiology and

Immunology L220, Oregon Health Sciences University, 3181 SW Sam

Jackson Park Rd., Portland, OR 97201-3098, USA. Tel. (503) 494.7583;

Fax (503) 494-6862.

*Present address: Department of Developmental Biology, Stanford

University, Stanford, CA 94305, USA. Tel. (415)723-5083.

Abbreviations: A, absorbance (1 cm); bp, base pair(s); EDDA, ethylene-

diamine-di(o-hydroxyphenyl) acetic acid; f&l, fat& genes involved in

iron transport; kb, kilobase or 1000 bp; oligo, oligodeoxyribonucleo-

tide(s); nt, nucleotide(s); ORF, open reading frame; RNAc(, antisense

RNA, SD, Shine-Dalgarno (sequence); SDS, sodium dodecyl sulfate;

PAGE, polyacrylamide-gel electrophoresis; TcR, tetracycline resistance;

Tn, transposon; tsp, transcription start point(s); V., Vibrio; wt, wild type;

[ 1, denotes plasmid-carrier state.

includes the siderophore anguibactin, and membrane

components involved in the transport of the iron-

anguibactin complexes (Crosa, 1989). The outer mem-

brane protein FatA (M, 86000) and a 40-kDa protein,

FatB, are essential for the transport of iron into the cyto-

sol when cells are grown under iron-limiting conditions

(Actis et al., 1988; Crosa, 1989; Koster et al., 1991). The

expression of these proteins is negatively regulated by the

iron concentration of the culture medium (Actis et al.,

1988; Crosa, 1990; Tolmasky et al., 1988).

In the case of Escherichiu coli, the genes involved in

the transport of siderophore-iron complexes, as well as

those involved in the biosynthesis of the siderophore,

are negatively regulated by iron through the tran-

scriptional repressor Fur (Hantke, 1981; 1984; de

Lorenzo et al., 1987). The finding of Fur-like proteins

in different bacteria (Stoebner and Payne, 1988; Tai and

Holmes, 1988) suggests that bacterial iron-uptake sys-

tems may be controlled by a common mechanism. How-

ever, the mechanism by which iron regulation is

accomplished in V. anguillarum 775 has not yet been

established. Thus, we decided to investigate the basis of

34

iron regulation of the expression of the fatB and fatA transport genes.

RESULTS AND DISCUSSION

(a) Iron regulation of the RNA levels off&A andfatB

genes

The fatB mRNA levels were examined by an RNase

protection assay using a riboprobe made from an internal

fragment of this gene. Under iron-rich conditions thefatB

transcript was not detected (Fig. 1 b, lane A). However,

under iron limitation, a 135-ntfatB mRNA fragment was

protected (Fig. lb, lane B). Similarly, an RNase protec-

tion assay using a second riboprobe, designed to detect

fatA mRNA (Fig. Ic), identified two fragments (85 and 78

nt). These fragments were only detected with RNA iso-

lated from strains grown under iron limitation (Fig. Ic,

lane B). Since this probe is internal tofatA, the protected

85-nt fragment must represent a segment of thefatA tran-

script. The origin of the protected 78-nt RNA has not

been identified, and this RNA may be part of a processed

fatA mRNA. These results showed that iron represses the

expression of the fatB and fatA genes at the transcrip-

tional level. For fatA, our results are in accordance to

a fam

AUG Sa H AUG

those previously reported using Northern blot analysis

(Actis et al., 1988).

(b) Coordinated expression of RNAcl and the inhibition of

the synthesis of the FatA outer membrane protein

To dissect the pJMl iron transport components, Tol-

masky et al. (1988) generated transposon insertion mu-

tants defective in the iron uptake ability. Genetic and

biochemical analysis showed that mutant 17, one of these

insertion derivatives that carries the Tn3-HoHoI inser-

tion 1813 bp upstream from the fatA gene (Koster et al.,

1991) was unable to transport iron but produced FatA,

though at reduced levels detectable by immunoblot (Actis

et al., 1988). Fig. 2a (lanes A and B) shows that although

low levels of FatA are detected in mutant 17, the expres-

sion of this outer membrane protein is still regulated by

the iron concentration of the medium.

In the process of determining whether the iron trans-

port deficiency of mutant 17 could be complemented by

various recombinant plasmids, we used a high-copy-

number recombinant clone (pJHC-A122) containing a

2.3-kb Hind111 DNA fragment that encompassed the

fatB-jatA intergenic region. This plasmid could not com-

plement the iron-uptake deficient phenotype of mutant

17. However, the examination of FatA levels in mutant

faa

H St J I I I I I

- 135 nt [pJHC-S400) - 89 nt (pJHC-SSOO)

b AB M bp

C AB M bp

._ : :. i: j -154

fatB mRNA fatA mRNA

Fig. I. Regulation of the RNA levels of@!3 and fatA genes. (a) Restriction map of the clones used to prepare riboprobes. AUG, start codon; H:

NindIII; Sa, SalI; St, StuI. (Panel b) RNase protection assays offatB transcripts, with riboprobe made from pJHCS400 plasmid linearized with Sal1

and labeled with T3 polymerase. (Panel c) RNase protection assay off&4 transcripts with riboprobe made from pJHCS300 plasmid linearized with

Hind111 and labeled with T3 polymerase. Lanes A, RNA extracted from V. anguillarum strains grown in 10 PM FeCl,. Lanes B, RNA obtained from

strains grown under iron limitations. Lanes A and B: strain carrying the angR and Taf determinants essential for full expression of the pJM1 iron-

uptake system (Salinas et al.; 1989; Tolmasky et al., 1988). Lane M, end-labeled Hi&digested pBR322 used as size marker (Bolivar et al., 1977;

Maniatis et al., 1982). Methods: V. anguillarum strains were grown in M9 minimal medium with or without 10 PM FeCl, until 0.4 A6,,0, and the RNAs

were isolated by phenol extraction (von Gabain et al., 1983). pJHC-S300 or pJHC-S400 DNAs were used as templates for in vitro transcription.

Riboprobes were prepared using [w~‘P]UTP and T7 or T3 RNA polymerases according to manufacturer (Stratagene, La Jolla, CA). RNase protection

assays were performed as described by Krieg and Melton (1987). The protected RNAs were analyzed by electrophoresis on a 7 M urea/6% poly-

acrylamide gel.

a A B C D E

b MABCDE

nt

220

154

kDa

- 97.4

- 66.2

Fig. 2. Coordinated expression of RNAc( and inhibition offatA synthe-

sis. (Panel a) Immunoblot analysis of total membrane proteins of V. anguillarum 775 and of strains harboring mutation 17, either in the

presence or the absence of the recombinant clone pJHC-A122. Mutant

17 under iron-limiting (lane A) and under iron-rich conditions (lane B).

Mutant 17 carrying pJHC-A122 under iron-limiting (lane C) and under

iron-rich conditions (lane D). The wt V. anguillarum 775 under iron-

limiting conditions (lane E). The total membrane proteins were sub-

jected to 12.5% SDS-PAGE and immunoblot as previously described

(Actis et al., 1988). (Panel b) RNase protection asssay of RNAc( in mu-

tant 17 (lanes B and C) and in mutant 17 carrying pJHC-A122 (lanes

D and E). Lanes B and D, cells grown under iron-rich conditions (50

pg/ml ferric ammonium citrate). Lanes C and E, cells grown under iron-

limiting conditions (5 PM EDDA). Lane A, 167-nt riboprobe made from

Hind111 linearized pJHC-S400 DNA and labeled with [a-32P]LJTP

using T7 RNA polymerase. Lane M, Molecular weight markers consist

of 3’ end-labeled Hi&-digested pBR322 fragments. The protected

RNAs were analyzed by electrophoresis as in Fig. 1.

17 cells containing pJHC-A122 showed that a greatly reduced FatA level could be detected by immunoblot under iron-limiting conditions (Fig. 2a, lanes C and D). We hypothesized that this inhibitory effect on the expres- sion of FatA under iron limitation could be due to the

35

presence of c&acting elements that may titrate out

factors necessary for the expression off&l. Alternatively, a trans-acting factor such as an RNAa, could be encoded within the 2.3-kb DNA fragment that could have repres- sor activity. We performed RNase protection studies to determine whether an RNAcl was indeed encoded in this region. We used pJHC-S400 (Fig. la) carrying the SalI- Hind111 fragment present in pJHC-A122 to generate a riboprobe that would detect such an RNAa. Analysis of RNA samples isolated from mutant 17 carrying the high- copy-number plasmid pJHC-A122, showed the presence of an RNAa, both under iron-limiting as well as iron-rich conditions (Fig. 2b, lanes D and E). We also tested RNA isolated from mutant 17 without pJHC-A122 with the same probe. An RNAcl (the 13.5nt protected fragment) was detected in this mutant only under iron-rich condi- tions (Fig. 2b, lanes B and C). Thus, an endogenous RNAa is expressed under iron-rich conditions but not under iron limitation. Furthermore, the repression of the fatA gene by the high-copy-number plasmid pJHC-A122 is correlated with the iron-independent expression of the RNAa. It is important to mention that in this construc- tion the native RNAa promoter region is missing and the expression of RNAo( is under the control of the TcR pro- moter encoded by the pACYC184 vector. Consequently, the expression of RNAa in pJHC-A122 is no longer regu- lated by iron (L.S.W. and J.H.C., manuscript in prepa- ration).

(c) Effect of iron in the level of RNAa andfutB mRNA

We investigated whether the RNAa, observed in mu- tant 17 was also present in wt clones, such as pJHC- T2612 (Tolmasky et al., 1984) and further analyzed the effect of iron stress on the expression of thefatB mRNA and RNAa. RNA from a V. anguillarum strain containing the cloned wt iron-uptake region was tested for the pres- ence of RNAcl and fatB mRNA under iron-rich and iron- limitation conditions, using the RNase protection approach described above together with probes capable of detecting either RNAa or fatB mRNA. The results shown in Fig. 3b and c demonstrated that with the same preparations of RNA, RNAa is detected only under iron- rich conditions (Fig. 3b) whereas fatB mRNA is only found under iron-limiting conditions (Fig. 3~). The fatB transcript appeared 30 min after the addition of EDDA, with the concomitant disappearance of RNAa (Fig, 3~). Thus, there is a correlation between the release of iron

repression of the expression of the fatB and fatA genes and a decrease of the levels of RNAa, when the cells were switched from iron-rich to iron-limiting conditions.

Northern blot analysis showed that RNAa is a tran-

36

a b

c

Fig. 3. Effect of iron limitation on the levels of RNA& and fafB tran-

scripts. (Panel a) Northern blot analysis of RNAc( transcript. V. anguil-

larum strain grown in 10 yM FeCl, (lane A) and grown under iron

limitations (lane B). Arrows indicate the migration of the 1542-nt 16s

rRNA and the 2904-nt 23s rRNA. (Panel b) RNase protection analysis

of RNAa. (Panel c) RNase protection analysis offatB transcript. Metb-

ods: V. anguillaruln strain 775::Tnl-6 [pJHC-T2612] (angR+, Taf+) was

grown in 10 PM FeCI,. When the cells reached the logarithmic stage

the iron chelator EDDA was added, and samples were taken after 0.5,

1, 2, and 6 h of incubation. The protected RNAs were analyzed by

electrophoresis as in Fig. 1.

script of 650 nt (Fig. 3a) that is present in RNA samples

isolated under iron-rich conditions but not under iron-

limiting conditions.

(d) Mapping of the tsp of RNAa

Since RNAcY possesses sequences complementary to the

fatB gene and the upstream region of fatA, we investi-

gated whether the beginning of RNAcl extended to the

region containing the fatA SD sequence. We mapped ‘the

5’ end of RNAn, using primer extension, and observed

three putative tsp (la, 1 b, lc) (Figs. 4 and 5). The putative

-10 together with the -35 sequences were underlined

in Fig. 5. However, we also noticed a perfect TATAAT

sequence at the la site with a perfect corresponding - 35

sequence TTGAAA. The longest RNAa possessed a 5’

terminus at 125 nt from the start codon for thefatA gene.

Thus, RNAa could be complementary to sequences lo-

cated at the 5’ untranslated region of fatA mRNA, not

including the fatA SD region (Fig. 5), and the un-

translated 3’ end of the fatB gene.

Antisense RNAs have been implicated in the control

of DNA replication, OmpF synthesis, lysis-lysogen state,

transposition, traJ expression in DNA transfer, and

CAMP receptor protein expression (Simons, 1988; Simons

and Kleckner, 1988; Takayama and Inouye, 1990). How-

ever, in all bacterial iron transport systems examined so

nt

la - 343

lb- 107

Ic - 93

P G A T @

Fig. 4. Primer extension of RNAcl and determination of tsp. RNA

sample from strain carrying pJHCT2612 grown under iron-rich condi-

tions (lane P). The sequencing ladders (lanes G, A, T, C) were generated

from pJHCA107 (Actis et al., 1988) which containedfatB and part of

futA genes. Methods: a 20-nt primer (5’-GAACGCTCAGAAAAAGC-

TTTA) which was complementary to the sequences of the RNAcl made

within the f&B gene was used in primer extension techniques. The

synthetic oligo was labeled with [cc-‘*P]dATP (Maniatis et al.. 1982).

50 ug of the RNAs were used in primer extension techniques (Ghosh

et al., 1978). Sequencing ladders were generated by sequencing of a

plasmid carrying the intergenic region between the_futB and fatA genes

using the same primer used in primer extension analysis. The samples

were analyzed by electrophoresis as in Fig. 1.

far, iron repression of gene expression is accomplished

through the interaction of the Fe’+-repressor protein,

Fur, and the promoter sequences of iron-regulated genes

(Bagg and Neilands, 1987; Hantke, 1981; 1984; de

Lorenzo et al., 1987). It remains to be seen whether a

mechanism involving RNAcl is the only mode of regula-

tion of iron-transport genes in V. anguillarum, or whether

RNAcl is part of a complex regulatory circuit that may

also include Fur-like factors.

37

Sal1 fatB 13

GTOXXTP.4T-A~G4T~~WTCTl’TPAT~~ C &

TCG4TG+V3ZlTCA43XXT CAGCTGTAlTAGlTTGTEP32MTP.4CTA- A AGCTACGCiTTCCPI;CTAC OS&Xi--T-T

,Sla -10 -35

Fig. 5. Localization of the tsp for RNAE in thefutBzfatA intergenic region. la, lb and lc denote the tsp determined by primer extension analysis. The

3’ end of the fatB gene, the putative leftward promoter regions for tsp + la of antisense RNAa, the SD sequence, and the start codon for the fatA- encoded protein are also indicated.

(e) Conclusions

(2) The iron regulation of the transport genesfutB and

fatA is controlled at the RNA level.

(2) A high-copy-number plasmid, carrying the fatB-

fatA intergenic region has an inhibitory effect on the ex-

pression of FatA.

(3) The inhibitory effect is correlated with the presence

of an antisense RNA (RNAa), encoded by the high-copy-

number plasmid under the control of the TcR promoter.

(4) RNAa is also present in iron-rich conditions in V.

anguillaruwl strains carrying the pJM1 iron uptake region.

The presence of RNAa correlates with the absence off&B

mRNA.

(5) RNAa, a 650-nt RNA, maps within the 5’ un-

translated region off&A and the 3’ coding region of the

futB gene.

ACKNOWLEDGEMENTS

We thank Drs. Simon M. Hughes and Luis A. Actis for

critical comments on the manuscript. This work was sup-

ported by U.S. Public Health Service Grant AI-19018

from the National Institutes of Health to J.H.C.

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