Aerobactin biosynthesis and transport genes of plasmid ColV-K30 in Escherichia coli K-12.

The iron-regulated aerobactin operon, about 8 kilobase pairs in size, of the Escherichia coli plasmid ColV-K30 was shown by deletion and subcloning analyses to consist of at least five genes for synthesis (iuc, iron uptake chelate) and transport (iut, iron uptake transport) of the siderophore. The gene order iucABCD iutA was established. The genes were mapped within restriction nuclease fragments of a cloned 16.3-kilobase-pair HindIII fragment. Stepwise deletion and subsequent minicell analysis of the resulting plasmids allowed assignment of four of the five genes to polypeptides of molecular masses 63,000, 33,000 53,000, and 74,000 daltons, respectively. The 74-kilodalton protein, the product of gene iutA, is the outer membrane receptor for ferric aerobactin, whereas the remaining three proteins are involved in biosynthesis of aerobactin. The 33-kilodalton protein, the product of gene iucB, was identified as N epsilon-hydroxylysine:acetyl coenzyme A N epsilon-transacetylase (acetylase) by comparison of enzyme activity in extracts from various deletion mutants. The 53-kilodalton protein, the product of gene iucD, is required for oxygenation of lysine. The 63-kilodalton protein, the product of gene iucA, is assigned to the first step of the aerobactin synthetase reaction. The product of gene iucC, so far unidentified, performs the second and final step in this reaction. This is based on the chemical characterization of two precursor hydroxamic acids (N epsilon-acetyl-N epsilon-hydroxylysine and N alpha-citryl-N epsilon-acetyl-N epsilon-hydroxylysine) isolated from a strain carrying a 0.3-kilobase-pair deletion in the iucC gene. The results support the existence of a biosynthetic pathway in which aerobactin arises by oxygenation of lysine, acetylation of the N epsilon-hydroxy function, and condensation of 2 mol of the resulting aminohydroxamic acid with citric acid.     

Citrate-dependent iron transport system in Escherichia coli K-12

Induction of the citrate-dependent iron transport system of Escherichia coli K-12 required 0.1 mM citrate and 0.1 micrometer iron in the growth medium. Five--ten-times more iron than citrate was taken up into the cells which suggests that citrate was largely excluded from the transport. Fluorocitrate and phosphocitrate induced the citrate-dependent iron transport system although they supported iron uptake only very poorly. An outer membrane protein (FecA), belonging to the transport system, was induced in fecB mutants which were devoid of citrate-dependent iron transport. The intracellular citrate and iron concentrations were 10--100-times higher than the external concentrations required for induction of the transport system. It is concluded that only exogenous ferric citrate induced the transport system, and that citrate did not have to enter the cytoplasm. The Tn10 transposon, conferring tetracycline resistance, was inserted near the fec gene region which controls the expression of the citrate-dependent iron transport system. The determination of the cotransduction frequencies of Tn10 with the fecA and fecB markers suggested the gene order fecA fecB Tn10.     

Characterization of iucA and iucC genes of the aerobactin system of plasmid ColV-K30 in Escherichia coli.

A cloned 8.3-kilobase-pair DNA fragment carrying all the genes (iucABCD iutA) of the aerobactin iron transport system of plasmid pColV-K30 was subjected to in vitro mutagenesis to afford mutant genes iucA, iucC, and iucA iucC. Complementation analyses and identification of aerobactin precursors accumulated by Escherichia coli cells harboring the different constructions allowed assignment of the iucA and iucC genes to discrete steps in biosynthesis of the siderophore from N epsilon-acetyl-N epsilon-hydroxylysine and citrate. Plasmid pVLN10, a derivative carrying a DNA fragment complementing an iucC mutation, expressed in a minicell system a single 62,000-dalton protein as the product of this gene.     

Genetic structure and stability of a copy number mutant of IncFI group plasmid ColV-K94 in Escherichia coli K-12

Summary Mutants pWS10, pWS11, and pWS12 were derived from an, IncFI group plasmid ColV-K94 by the insertion of a transposon Tn903 (Km^r). These plasmids were all approximately 130 kb in length. The plasmid pWS12 resembled the wild type ColV-K94 in transmissibility, incompatibility and stable maintenance. Cells harboring pWS11 were poor conjugal donors but resistant to the same level of kanamycin as pWS12 containing hosts. In contrast, pWS10 conferred a higher resistance to kanamycin and exhibited reduced incompatibility properties in comparison with pWS12. The higher drug resistance associated with pWS10 appeared to be a consequence of an increase in its copy number and the generation of miniplasmids of varying sizes.Electron microscope analysis of the copy mutant pWS10 revealed that Tn903 was located at a site adjacent to a region 32.6F to 35.3F. The latter region appears to be the primary replicon of ColV-K94 and is homologous with the secondary replicon of F. The insertional mutagenesis with Tn903 brought about an extensive DNA rearrangement including the duplication and translocation of the stems of two inverted repeat structures. The DNA alterations of pWS10 were distinguishable through comparison of its EcoRI digestion patterns with those of pWS11 and pWS12.     

Transcriptional regulation of katE in Escherichia coli K-12

Escherichia coli produces two distinct species of catalase, hydroperoxidases I and II, which differ in kinetic properties and regulation. To further examine catalase regulation, a lacZ fusion was placed into one of the genes that is involved in catalase synthesis. Transductional mapping revealed the fusion to be either allelic with or very close to katE, a locus which together with katF controls the synthesis of the aerobically inducible hydroperoxidase (hydroperoxidase II). katE was expressed under anaerobic conditions at levels that were approximately one-fourth of those found in aerobically grown cells and was found to be induced to higher levels in early-stationary-phase cells relative to levels of exponentially growing cells under both anaerobic and aerobic conditions. katE was fully expressed in air and was not further induced when the growth medium was sparged with 100% oxygen. Expression of katE was unaffected by the addition of hydrogen peroxide or by the presence of additional lesions in oxyR or sodA, indicating that it is not part of the oxyR regulon. When katF::Tn10 was introduced into a katE::lacZ strain, beta-galactosidase synthesis was largely eliminated and was no longer inducible, suggesting that katF is a positive regulator of katE expression.     

Gene lon and plasmid inheritance in Escherichia coli K-12.

Lon- mutants of Escherichia coli K-12 are deficient in the inheritance of F-plasmids by conjugation. This deficiency is distinct from the conjugation deficiency caused by overproduction of capsular polysaccharide which decreases donor-recipient pair formation.     

Synthesis of linear plasmid multimers in Escherichia coli K-12.

Linear plasmid multimers were identified in extracts of recB21 recC22 strains containing derivatives of the ColE1-type plasmids pACYC184 and pBR322. A mutation in sbcB increases the proportion of plasmid DNA as linear multimers. A model to explain this is based on proposed roles of RecBC enzyme and SbcB enzyme (DNA exonuclease I) in preventing two types of rolling-circle DNA synthesis. Support for this hypothesis was obtained by derepressing synthesis of an inhibitor of RecBC enzyme and observing a difference in control of linear multimer synthesis and monomer circle replication. Reinitiation of rolling-circle DNA synthesis was proposed to occur by recA+-dependent and recA+-independent recombination events involving linear multimers. The presence of linear plasmid multimers in recB and recC mutants sheds new light on plasmid recombination frequencies in various mutant strains.     

Genetic and physical mapping of recF in Escherichia coli K-12

Summary Two factor transductional crosses place recF at approximately 82 min on the E. coli chromosome; recF is highly cotransducible with dnaA and gyrB (cou). Transductional analysis with a series of tna specialized transducing phages carrying chromosomal DNA from the tnaA region place recF between dnaA and gyrB. This analysis also indicates that a gene lying in the same region and producing an easily detectable protein (estimated MW of 45 kD) is dnaN and not recF.     

Exogenous induction of the iron dicitrate transport system of Escherichia coli K-12.

Streptonigrin was used to select mutants impaired in the citrate-dependent iron transport system of Escherichia coli K-12. Mutants in fecA and fecB could not transport iron via citrate. fecA-lac and fecB-lac operon fusions were constructed with the aid of phage Mu dl(Ap lac). Strains deficient in ferric dicitrate transport which were mutated in fecB were as inducible as transport-active strains. They expressed the FecA outer membrane protein and beta-galactosidase of the fecB-lac operon fusions. In contrast, all fecA::lac mutants and fecA mutants induced with N-methyl-N'-nitro-N-nitrosoguanidine did not respond to ferric dicitrate supplied in the growth medium. tonB fecB mutants which were lacking all tonB-related functions were not inducible. We conclude that binding of iron in the presence of citrate to the outer membrane receptor protein is required for induction of the transport system. In addition, the tonB gene has to be active. However, iron and citrate must not be transported into the cytoplasm for the induction process. These data support our previous conclusion of an exogenous induction mechanism. Mutants in fur expressed the transport system nearly constitutively. In wild-type cells limiting the iron concentration in the medium enhanced the expression of the transport system. Thus, the citrate-dependent iron transport system shares regulatory devices with the other iron transport systems in E. coli and, in addition, requires ferric dicitrate for induction.     

D-serine transport system in Escherichia coli K-12

The d-serine transport system in Escherichia coli K-12 was studied by use of a mutant unable to form d-serine deaminase, yet resistant to d-serine. The mutant is greatly impaired in its ability to accumulate d-serine, d-alanine, and glycine. Transport of l-alanine is partially affected but transport of l-serine is unaffected. The mutant is also resistant to d-cycloserine, indicating that d-serine is transported by the system responsible for uptake of d-cycloserine. The d-serine transport system is not inducible, but appears to be formed constitutively, as are the transport systems of most amino acids. The transport mutation appears to be multistep and maps to the right of malB on the E. coli linkage map.     

Genetics and regulation of peptidase N in Escherichia coli K-12

Escherichia coli K-12 strains contain a cytoplasmic activity, peptidase N, capable of hydrolyzing alanine-p-nitroanilide. Mutations in the structural gene for the enzyme, pepN, were mapped, and the properties of mutant strains were examined. The pepN locus lay between ompF and asnS at approximately 20.8 min on the E. coli chromosome. Loss of peptidase N activity through mutation had no apparent effect on the growth rate or nutritional needs of the cell. Enzyme levels in wild-type strains were constant throughout the growth cycle and were constitutive in all of the growth media tested. Starvation for carbon, nitrogen, or phosphate also did not alter enzyme levels. Constitutive expression of peptidase N is consistent with the idea that the enzyme plays a significant role in the degradation of intracellularly generated peptides.     

Replication of the nonconjugative plasmid RSF1010 in Escherichia coli K-12.

Replicating DNA molecules of the nonconjugative R plasmid RSF1010 (Smr Sur) were cleaved with the EcoRI restriction endonuclease and examined with the electron microscope. Results of this analysis indicated that replication is initiated from an origin located at about 19% of total genome size from one of the EcoRI ends. Replication proceeded either unidirectionally or bidirectionally with equal frequency. Results of the analysis of replicative intermediates of RSF1010 containing the Apr-transposable sequence (Tn) are also presented.     

Mapping of a mutation affecting regulation of iron uptake systems in Escherichia coli K-12.

The site of a mutation resulting in constitutive derepression of iron uptake systems has been localized at 15.7 min on the genetic map of Escherichia coli K-12.