FEMS Microbiology Letters 36 (1986) 281-285 281 Published by Elsevier

FEM 02544

Biochemical genetics of aerobactin biosynthesis in Escherichia coli

(Escherichia coli; aerobactin; iron uptake; hydroxamate siderophore)

Steven Ford a, Ronald A. Cooper a and Peter H. Williams b .

Departments of a Biochemistry and b Genetics, University of Leicester, Leicester LEI 7RH, U.K.

Received 6 May 1986 Revision received 6 June 1986

Accepted 8 June 1986

1. SUMMARY

Single-gene mutants of Escherichia coli defec- tive in aerobactin biosynthesis were incubated un- der non-growing conditions for 2 h with radio- labelled lysine. Analysis of the intermediates pro- duced suggested that acetylation of lysine may be the first step in aerobactin production.

2. INTRODUCTION

Aerobactin is a hydroxamate siderophore that was first isolated and characterised from culture supernatants of Aerobacter aerogenes 62-1 [1]. It comprises 2 N6-acetylated, N6-hydroxylated lysine molecules linked by peptide bonds to a molecule of citric acid. In A. aerogenes 62-1, hydroxylation of L-lysine is the first step in the biosynthetic pathway [2,3].

Aerobactin is also synthesised by invasive strains of E. coli [4,5]. It is an important virulence determinant in these strains because it confers a significant selective advantage for bacterial growth in conditions of iron limitation [6,7] such as those that exist in the tissues and body fluids of infected

* To whom correspondence should be addressed.

animals [8]. The aerobactin system in E. coli is specified by 5 genes organised as a single tran- scriptional unit [9-11]. The promoter distal gene (iutA) encodes an outer membrane protein that is the receptor for ferric-aerobactin, and the other genes (iucA-D) code for polypeptides required in the biosynthesis of the siderophore [9]. Very re- cently these polypeptides have been identified as N 2-citryl-N6-acetyl-N6-hydroxylysine synthetase (iucA), an acetyl transferase (iucB), aerobactin synthetase (iucC) and a hydroxylase (iucD); it was assumed that the biosynthetic pathway is the same as that reported for A. aerogenes 62-1 [12-14]. The data presented in this paper confirm the assignment of functions to these 4 genes but sug- gest that acetylation may precede hydroxylation in the pathway.

3. MATERIALS AND METHODS

3.1. Bacteria, plasmids and culture conditions E. coli K-12 strain W3110 [6] was used in all

the work reported here. Plasmid pABN1, which carries the entire aerobactin system of the E. coli plasmid ColV-K30 cloned in a small multicopy vector [15], was kindly provided by J.B. Neilands and A. Bindereif. Mutants designated 3, 11, 13

0378-1097/86/$03.50 © 1986 Federation of European Microbiological Societies

282

1"111OOO

i i " 11 I I pABN 1

o , , ; ~ , , ; , , ~, - - - - E H A I , B A ,uP E

i 0 i

' -" I ' - " l - ° - ° l " " ~ K 35K 45K ~ )K 7 4 K I l k b I

L - l y s i n e I I ~ N - - - a o e l y l - ~ N ' - a o e l y l - ~ N ~ - c i t r y l - I I l ~ b A e r o b a o t i n l y s i n e N e - h y d r o x y - N s - a c e t y l -

l y s i n e N e - h y d r o x y - lys ine

Fig. 1. Proposed pathway for aerobactin biosynthesis in E. coli. The 5 genes of the aerobactin iron uptake system are aligned with part of the physical map of recombinant plasmid pABN1; complete maps of this plasmid are published elsewhere [9,12,13,15]. Restriction sites for AoaI (A), BamHI (B), EcoRI (E), HindlII (H) and PvulI (P) are indicated, as are the sites of Tnl000 insertion in plasmid mutants 3, 11, 13 and 16. Gene loci are designated according to De Lorenzo et al. [13]. The estimated molecular weights (K = 1000) of the gene products [9], as well as our proposal for the biosynthetic reactions they catalyse are shown.

and 16 (Fig. 1) are TnlO00 insertion mutants derived from pABN1 [9]. Plasmid pLG141 carries the complete iucD and receptor gene sequences cloned into the vector pACYC184 [9]. Bacteria were cultured in M9 minimal salts medium [16] supplemented with glucose (0.4%, w/v) , thiamine (10 /~g/ml) and a mixture of amino acids which contained no lysine; the presence of lysine in growth media induces lysine decarboxylase which considerably reduces the potential incorporation of radiolabelled lysine into aerobactin biosyn- thetic intermediates. The chelating agent a,a'-di- pyridyl (200 /~M) was added to induce the aero- bactin system, and ampicillin or chloramphenicol (25 # g / m l ) were used to maintain the presence of the recombinant plasmids pABN1 (and deriva- tives) and pLG141, respectively.

3.2. Single and dual isotope labelling of aerobactin and biosynthetic intermediates

Bacterial cultures (10 ml) at an .4680 of 0.6 were harvested by centrifugation. Cell pellets were washed with 1 ml of 5 mM sodium phosphate buffer (pH 7.0) containing glucose (0.2%, wt /vol )

and a,a ' -dipyridyl (500 FM), resuspended in 1 ml of the same buffer and incubated with either 1 #Ci L-[U-]aC]lysine • HC1 (348 m C i / m m o l ; Amersham International, U.K.), or 40 #Ci L-[4,5-3H]lysine • HC1 plus 2 /~Ci [1-14C]sodium acetate (85 C i / m m o l and 59 mCi /mmol , respectively; Am- ersham) for 2 h at 30°C in a shaking water bath. High concentrations of dipyridyl facilitated incor- poration of radiolabelled substrates into potential intermediates rather than into cellular material. Incorporation of label was terminated by addition of absolute ethanol (2 ml) to the cell suspension and heating at 80°C for 10 min. The cells and cell debris were removed by centrifugation.

3.3. Electrophoretic separation of radiolabelled com- pounds

Reaction mixtures were analysed by paper elec- trophoresis at either pH 11.0 (50 mM borate buffer) or at pH 7.0 (50 mM sodium phosphate buffer). Purified aerobactin and N6-acetyl-N6-hy - droxylysine for use as standards were prepared as described by Gibson and Magrath [1] and by Neilands [17], respectively. These hydroxamic

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acids were detected by spraying with acidic FeC13 (1.25%, w /v , in 1 M HCI). Standard Nr-acetyl - lysine, purchased from Sigma, was detected with ninhydrin spray. Radiolabelled compounds were separated by preparative electrophoresis at pH 7.0, detected by autoradiography, and eluted with distilled water.

Cathode

3.4. Acid hydrolysis Samples in 6 M HC1 were autoclaved at 121°C

for 20 min. HC1 was removed by evaporating to dryness under reduced pressure in the presence of NaOH. Radiolabelled material was re-analysed by electrophoresis at p H 7.0.

Origin

4. RESULTS

4.1. Assignment of gene function in the biosynthesis of aerobactin

Carbonetti and Williams [9] isolated a series of mutants of plasmid pABN1 in which each individ- ual gene involved in aerobactin biosynthesis was inactivated by insertion of transposon TnlO00 (Fig. 1). Derivatives of strain W3110 harbouring these mutant plasmids were used to screen for possible radiolabelled intermediates of the biosyn- thetic pathway using [14C]lysine as a substrate. Although transposon insertion characteristically results in strong polar effects, it was previously noted that the expression of genes of the aerobac- tin system distal to the site of TnlO00 insertion is reduced but not abolished entirely, perhaps due to the activity of weak internal promoter sites [9]. This was confirmed by the fact that the 4 mutants used here showed different patterns of accumula- tion of intermediates. Analysis of incubation mix- tures by paper electrophoresis at pH 7.0 (Fig. 2) showed that mutant 3 (iucB), alone of the 4 Tn l000 insertion plasmids, did not produce sig- nificant amounts of any labelled materials other than those made by the plasmid-free ( I u - ) strain W3110. Moreover, a strain harbouring plasmid pLG141, which is capable of complementing iucD mutant plasmids but has no iucB activity [9,18], also showed no accumulation of radiolabelled products from [t4C]lysine (data not shown). We propose, therefore, that the iucB gene product

~m

Anode Iu-- iucB lucD iucA iucC iuc +

Fig. 2. Detection of radiolabelled intermediates of aerobactin biosynthesis. Bacteria carrying pABN1 (iuc + ) or Tnl000 mutant plasmids (iucA-D) and the plasmid-free strain W3110 (Iu-) were incubated with 14C-lysine and extracts were analysed by electrophoresis (pH 7.0, 1.5 kV, 100 min) on Whatman 3 MM paper, as described in the text. Radiolabelled compounds were located by autoradiography; this figure is a positive of the autoradiograph. Small amounts of cadaverine, a decarboxylation product of lysine, were observed in some samples. The identity of 'neutral material' in extracts of iucA, iucC and iucD mutants is discussed in the text. Radiolabelled aerobactin was identified by precise comigration with the standard compound. By this criterion, the small amount of negatively charged material seen with the iucD mutant was concluded not to be aerobactin. N2-citryl-N6-acetyl-Nr-hy- droxylysine carries the same charge as aerobactin but has a lower molecular weight and therefore migrates slightly faster than the complete siderophore.

mediates the first specific step in the biosynthesis of aerobactin. The other three mutants all pro- duced radiolabelled material that was neutral at p H 7.0 (Fig. 2). When this material was eluted and electrophoresed at pH 11.0 (Fig. 3), mutants 13 (iucA) and 16 (iucC), but not 11 (iucD), showed a labelled intermediate that comigrated precisely with added standard N6-acetyl-Nr-hydroxylysine.

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Origin

Ne-acety l - lysine

Ne-acety l - Ne-hydroxy- lysine

Anode

• Fig. 3. Electrophoresis at pH 11.0 of material carrying no net charge at pH 7.0. Preparative electrophoresis at pH 7.0 was used to isolate 'neutral material ' from iucA, iucC and iucD mutants as decribed in Fig. 2. Samples were re-analysed by electrophoresis at pH 11.0 (1.5 kV, 120 min) on Whatman 3 MM paper as described in the text. Radiolabelled compounds were located by autoradiography; this figure is a positive of the autoradiograph. The identity of each spot was confirmed by the inclusion of appropriate standard compounds in the ap- plied sample. From left to right: iucD; iucA; iucC

Mutant 16 (iucC) also produced a second radiolabelled compound, which migrated a little faster than the aerobactin standard at pH 7.0 (Fig. 2) and which we assume is N2-citryl-N6-acetyl - N6-hydroxylysine. Thus, our data confirm previ- ous reports [12,13] that the iucA and iucC gene products catalyse the third and fourth reactions, respectively, in the biosynthetic pathway for aerobactin (Fig. 1).

We propose that the remaining step, the second in the pathway, is catalysed by the iucD gene product. The uncharged compound (at pH 7.0) accumulated by mutant 11, and indeed other mutants which have TnlO00 inserted within the iucD coding region, is therefore the first biosyn- thetic intermediate. Potential first products in the aerobactin biosynthetic sequence are N6-hydroxy- lysine and Nr-acetyl-lysine both of which would be expected to be neutral at pH 7.0. If the com- pound accumulated by mutant 11 (iucD) is N6-hy - droxylysine, then the iucD gene cannot encode the hydroxylase, as has been suggested independently

by Gross et al. [12] and De Lorenzo et al. [13]; if the compound accumulated by mutant 11 is N 6-

acetyl-lysine, the first step in the pathway is not the same as that reported to occur in A. aerogenes 62-1 [2,3].

4.2. Identification of the accumulation product in mutant 11 (iucD)

Three pieces of evidence indicate that the com- pound accumulated by mutant 11 is N6-acetyl- lysine and not N6-hydroxylysine. First, although N6-acetyl-lysine and N6-hydroxylysine have rather similar electrophoretic mobilities at pH 11.0, the radiolabelled intermediate produced by mutant 11 comigrated precisely with unlabelled N6-acetyl - lysine added to the sample. Second, the material was susceptible to acid hydrolysis to yield lysine, consistent with its being acetylated rather than hydroxylated [1,11].

These results were further comfirmed by a dual labelling experiment in which W3110 strains con- taining pABN1 or mutant 11 (iucD) were in- cubated with 14C-labelled sodium acetate and tri- tiated lysine (Table 1). As expected [17], the strain carrying pABN1 produced aerobactin and a small amount of N6-acetyl-N6-hydroxylysine, each label- led with both 3H and 14C. If mutant 11 made N6-hydroxylysine, material carrying no charge at pH 7.0 should be labelled with trititum only; the fact that such material also contained 14C from acetate again indicates that mutation of the iucD gene results in accumulation of N6-acetyl-lysine. Mutant 11 also accumulates small amounts of two

Table 1

Evidence for the accumulation of acetylated lysine by a strain carrying pABNI : :Tn I000 mutant 11 (iucD)

Strains were incubated with [14C]acetate and [3H]lysine as described in the text.

Compound 14 C cpm

3H cpm × 10 -4

L-[4,5- 3 H]lysine 0 Aerobactin a 143.7 N 6_acetyl.N 6.hydroxylysine a 116.3 Mutant 11 neutral material 91.0

a Made by strain carrying pABN1.

negatively charged compounds derived from lysine; these have not yet been identified.

5. DISCUSSION

The work presented in this paper confirms the functional assignments published recently for the 4 aerobactin biosynthesis genes of ColV plasmids of E. coli [12-14]. The products of the iueB and iucD genes are, respectively, an acetylase and a hydroxylase involved in the formation of N 6-

acetyl-N6-hydroxylysine, the iucA gene product condenses one molecule of this hydroxamate with citrate to form NZ-citryl-N6-acetyl-N6-hydroxy- lysine, and the iucC gene product catalyses the attachment of a second molecule to complete the synthesis of aerobactin. However, our data suggest that acetylation precedes hydroxylation in the bio- synthetic pathway, in contrast to what was re- ported for A. aerogenes 62-1 [2], and appears to be assumed for E. coli [12-14]. Radioactive precursor lysine remains unmodified in the iucB mutant, but a labelled intermediate identified as N6-acetyl - lysine accumulates in the iucD mutant. Acylation precedes hydroxylation in the biosynthesis of other hydroxamates such as pulcherriminic acid [19].

Neither Gross et al. [12] nor De Lorenzo et al. [13] reported finding N6-acetyl-lysine in the cul- ture media of strains defective in known aerobac- tin biosynthesis functions. Moreover, although the acetylase encoded by iucB was observed to acetylate N6-hydroxylysine [13], its activity with L-lysine was apparently not considered. Both groups, however, reported accumulation of N6-hy - droxylysine in stationary phase culture super- natants of mutants defective in acetylase activity [12-14]. In contrast, we have monitored the accu- mulation of radiolabelled aerobactin precursors in TnlO00 mutants over a period of 2 h and no N6-hydroxylysine was detected. Studies of the substrate specificity of the purified individual enzymes should confirm that lysine is acetylated

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rather than hydroxylated as the first step in aero- bactin biosynthesis (Fig. 1).

ACKNOWLEDGEMENTS

We thank the Wellcome Trust for financial support (grant no. 12289/1.5).

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