Genetics and environmental regulation of Shigella iron transport systems

Shigella spp. have transport systems for both ferric and ferrous iron. The iron can be taken up as free iron or complexed to a variety of carriers. All Shigella species have both the Feo and Sit systems for acquisition of ferrous iron, and all have at least one siderophore-mediated system for transport of ferric iron. Several of the transport systems, including Sit, Iuc/IutA (aerobactin synthesis and transport), Fec (ferric di-citrate uptake), and Shu (heme transport) are encoded within pathogenicity islands. The presence and the genomic locations of these islands vary considerably among the Shigella species, and even between isolates of the same species. The expression of the iron transport systems is influenced by the concentration of iron and by environmental conditions including the level of oxygen. ArcA and FNR regulate iron transport gene expression as a function of oxygen tension, with the sit and iuc promoters being highly expressed in aerobic conditions, while the feo ferrous iron transporter promoter is most active under anaerobic conditions. The effects of oxygen are also seen in infection of cultured cells by Shigella flexneri; the Sit and Iuc systems support plaque formation under aerobic conditions, whereas Feo allows plaque formation anaerobically.     

IS1-mediated mobility of the aerobactin system of pColV-K30 in Escherichia coli

Summary Genes determining the high affinity iron transport system mediated by the siderophore aerobactin are flanked in the enterobacterial plasmid pColV-K30 by inverted repeats of IS1 sequences, suggesting that the aerobactin genes are part of a transposon. To study this possibility, the entire region between the two IS1 sequences was cloned as an 18 kb HindIII-BamHI restriction fragment in pUC8 giving plasmid pMO1. A number of derivatives of pMO1, in which aerobactin genes were tagged with a kanamycin resistance gene, were prepared in order to assess the ability of both IS1s to promote the formation of cointegrates with pCJ105, an F derivative devoid of insertion sequences. Mating-out assays indicated that both flanking IS1s were active in cointegrate formation at detectable frequencies. In some cases, the cointegrates could be resolved, the final result being a transposition-like event for the entire aerobactin system.     

Isolation and some properties of lysineN 6-hydroxylase fromEscherichia coli strain EN222

Summary Lysine N⁶-hydroxylase was isolated as a soluble enzyme from the supernatant after ultrasonication ofEscherichia coli strain EN222 which contained the structural gene on a multicopy plasmid (as described by Engelbrecht and Braun in 1986). The apoenzyme prepared by dialysis was purified by ammonium sulfate precipitation and fast protein liquid chromatography using Superose 12 and Mono Q columns. The molecular mass as determined by gel filtration was 200 kDa and 50 kDa by SDS/polyacrylamide gel electrophoresis. The enzyme binds 0.79 molecule FAD/50 kDa. The activity of the enzyme is strictly dependent on NADPH. Its properties are similar to other flavoprotein monooxygenases of the EC group 1.14.13.     

Assay for acetyl coenzyme A: Alkylhydroxylamine N-acetyltransferase in Klebsiella pneumoniae ATCC25304

Abstract An alkylhydroxylamine N -acetyltransferase was partially purified from extracts of Kelbsiella pneumoniae . N ⁶-Hydroxylysine, N ⁵-hydroxyornithine and N -methylhydroxylamine were acetylated with decreasing velocity. N⁶-Acetyl- l -lysine showed noncompetitive inhibition while l -ornithine, l -lysine, their α-acetyl derivatives and N ⁵-hydroxylysine were without effect at 0.1 M. A sensitive assay using ¹⁴C-labelled acetyl coenzyme A was developed.     

Identification of the siderophores from Vibrio hollisae and Vibrio mimicus as aerobactin

Abstract Hydroxamate siderophores were purified from low-iron cultures of Vibrio hollisae ATCC 33564 and Vibrio mimicus ATCC 33653. By a combination of ¹H and ¹³C NMR spectroscopy, fast atom bombardment mass spectrometry, and compositional analysis, both of the siderophores were identified as aerobactin, a citrate-based dihydroxamate siderophore, which is highly prevalent in species of the family Enterobacteriaceae . Four other clinical strains belonging to these species also produced aerobactin. In response to iron limitation, all strains expressed two high molecular mass outer membrane proteins. The protein with an apparent molecular mass of 77 kDa, which was common to all strains examined, may be the ferric aerobactin receptor.     

Aerobactin production linked to transferable antibiotic resistance in Escherichia coli strains isolated from sewage

Abstract The production of aerobactin has been suggested to be a virulence factor in Escherichia coli . We have studied the production of aerobactin in 155 E. coli strains, isolated from sewage, which carry conjugative antibiotic resistance plasmids, and 88 (57%) of these strains produced aerobactin, and 59 (38%) co-transferred production of the siderophore and antibiotic resistance. In 35 (22%) of the transconjugants, both characters seemed to be encoded into the same plasmid. Those aerobactin-producing-antibiotic resistance plasmids had different sizes and antibiotic resistance patterns. The ecological implications of these results are discussed.     

Lysine N-hydroxylase and N-acetyltransferase of the aerobactin system of pColV plasmids in Escherichia coli

The time course of formation of lysine N-hydroxylase and N-transacetylase in Escherichia coli bearing cloned genes derived from pColV-K311 was followed and the influence of iron concentration on the enzyme induction was studied. Specific activities of both enzymes were determined for 8 strains prepared by Braun and coworkers [1,2] comprising the separated genes on the vectors pBR322 or pACYC184. The assignment of genes aerA and aerB to the first two enzymes of aerobactin biosynthesis [2] was confirmed. The active enzyme encoded by aerA (N-hydroxylase) has an M_r of 52 000 and that of aerB (N-transacetylase) an M_r of 70 000, as determined by gel filtration. The M_r of the N-transacetylase was 35 000 in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).     

Expression of high affinity iron uptake systems by clinical isolates of Klebsiella

Seventeen isolates of Klebsiella aerogenes, K. pneumoniae, K. oxytocum and K. edwardsii were examined for their ability to express iron-regulated outer membrane proteins (IROMPs) and high affinity iron-chelating agents (siderophores). In response to iron deprivation, all strains induced at least 4 IROMPs in the approximate M_r range 70 000–85 000 and the phenolate siderophore enterobactin. Six strains also produced the hydroxamate siderophore aerobactin. The Klebsiella enterobactin receptor was identified as an 81 000 M_r iron-repressible outer membrane (OM) protein which appears to be highly conserved and shows considerable antigenic homology with that of Escherichia coli.     

Aeribactin synthesis in a cell-free system of Aerobacter Aerogenes 62-1

The supernatant fraction of the cell-free system of Aerobacter aerogenes 62-1 was found to contain enzyme(s) capable of synthesising aerobactin from citrate and N⁶-acetyl-N⁶-hydroxylysine. Studies with partially purified enzyme systems revealed that aerobactin production was dependent on Mg²⁺ and ATP, the latter being used for the activation of both the precursors, citrate and N⁶-acethyl-N⁶-hydroxylysine. The generation of ADP in the activation process was consistent with the formation of phosphorylated precursor intermediates in the reactions leading to the peptide bond formation.     

Aerobactin-mediated iron uptake system in intestinal Escherichia coli

Abstract Escherichia coli strains isolated from children with diarrhea were studied to determine the incidence of the components of the aerobactin system. The production of aerobactin and the presence of the aerobactin receptor were demonstrated using bioassay techniques. The presence and location of the genes for the aerobactin system was determined by colony and Southern DNA-DNA hydridization. It was found that aerobactin and the aerobactin receptor could be coded by either chromosomal or plasmid genes. A high percentage of strains expressed the receptor in the absence of aerobactin. Of 19 enterotoxin-producing strains one was found to have the complete aerobactin system, while 11 expressed only the aerobactin receptor which was most frequently coded by a plasmid.