Iron-binding compounds from Agrobacterium spp.: biological control strain Agrobacterium rhizogenes K84 produces a hydroxamate siderophore.
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Iron-binding compounds were produced in various amounts in response to iron starvation by a collection of Agrobacterium strains belonging to the species A. tumefaciens, A. rhizogenes, and A. vitis. The crown gall biocontrol agent A. rhizogenes strain K84 produced a hydroxamate iron chelator in large amounts. Production of this compound, and also of a previously described antibiotic-like substance called ALS84, occurred only in cultures of strain K84 grown in iron-deficient medium. Similarly, sensitivity to ALS84 was expressed only when susceptible cells were tested in low-iron media. Five independent Tn5-induced mutants of strain K84 affected in the production of the hydroxamate iron chelator showed a similar reduction in the production of ALS84. One of these mutants, M8-10, was completely deficient in the production of both agents and grew poorly compared to the wild type under iron-limiting conditions. Thus, the hydroxamate compound has siderophore activity. A 9.1-kb fragment of chromosomal DNA containing the Tn5 insertion from this mutant was cloned and marker exchanged into wild-type strain K84. The homogenote lost the ability to produce the hydroxamate siderophore and also ALS84. A cosmid clone was isolated from a genomic library of strain K84 that restored to strain M8-10 the ability to produce of the siderophore and ALS84, as well as growth in iron-deficient medium. This cosmid clone contained the region in which Tn5 was located in the mutant. Sequence analysis showed that the Tn5 insert in this mutant was located in an open reading frame coding for a protein that has similarity to those of the gramicidin S synthetase repeat superfamily. Some such proteins are required for synthesis of hydroxamate siderophores by other bacteria. Southern analysis revealed that the biosynthetic gene from strain K84 is present only in isolates of A. rhizogenes that produce hydroxamate-type compounds under low-iron conditions. Based on physiological and genetic analyses showing a correlation between production of a hydroxamate siderophore and ALS84 by strain K84, we conclude that the two activities share a biosynthetic route and may be the same compound. (+info)
Siderophore uptake and use by the yeast Saccharomyces cerevisiae.
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The non-reductive uptake of several siderophores (ferrioxamine B, ferrichrome, triacetylfusarinine C and ferricrocin) by various strains of Saccharomyces cerevisiae was studied. Several aspects of siderophore transport were examined, including specificity of transport, regulation of transport and intracellular localization of the ferri-siderophores. Ferrioxamine B was taken up preferentially via the products of the SIT1 gene and triacetylfusarinine C by the TAF1 gene product, but the specificity was not absolute. Ferrichrome and ferricrocin uptake was not dependent on a single major facilitator superfamily (MFS) gene product. The apparent specificity of transport was strongly dependent on the genetic background of the cells. Non-reductive uptake of siderophores was induced under more stringent conditions (of iron deprivation) than was the reductive uptake of ferric citrate. Regulation of transport depended on the transcriptional factors Aft1 and Tup1/Ssn6. Cells disrupted for the TUP1 or SSN6 genes were constitutively derepressed for the uptake of ferrichrome, ferricrocin or ferrioxamine B, but not for the uptake of triacetylfusarinine C. Cells bearing the AFT1(up) mutation accumulated large amounts of ferric siderophores. Intracellular decomplexation of the siderophores occurred when transcription of the AFT1(up) gene was repressed. Ferrioxamine B and ferrichrome seemed to accumulate in an endosomal compartment, as shown by biochemical studies and by confocal microscopy study of cells loaded with a fluorescent derivative of ferrichrome. Endocytosis was, however, not involved in the non-reductive uptake of siderophores. (+info)
Essential PchG-dependent reduction in pyochelin biosynthesis of Pseudomonas aeruginosa.
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The biosynthetic genes pchDCBA and pchEF, which are known to be required for the formation of the siderophore pyochelin and its precursors salicylate and dihydroaeruginoate (Dha), are clustered with the pchR regulatory gene on the chromosome of Pseudomonas aeruginosa. The 4.6-kb region located downstream of the pchEF genes was found to contain three additional, contiguous genes, pchG, pchH, and pchI, probably forming a pchEFGHI operon. The deduced amino acid sequences of PchH and PchI are similar to those of ATP binding cassette transport proteins with an export function. PchG is a homolog of the Yersinia pestis and Y. enterocolitica proteins YbtU and Irp3, which are involved in the biosynthesis of yersiniabactin. A null mutation in pchG abolished pyochelin formation, whereas mutations in pchH and pchI did not affect the amounts of salicylate, Dha, and pyochelin produced. The pyochelin biosynthetic genes were expressed from a vector promoter, uncoupling them from Fur-mediated repression by iron and PchR-dependent induction by pyochelin. In a P. aeruginosa mutant lacking the entire pyochelin biosynthetic gene cluster, the expressed pchDCBA and pchEFG genes were sufficient for salicylate, Dha, and pyochelin production. Pyochelin formation was also obtained in the heterologous host Escherichia coli expressing pchDCBA and pchEFG together with the E. coli entD gene, which provides a phosphopantetheinyl transferase necessary for PchE and PchF activation. The PchG protein was purified and used in combination with PchD and phosphopantetheinylated PchE and PchF in vitro to produce pyochelin from salicylate, L-cysteine, ATP, NADPH, and S-adenosylmethionine. Based on this assay, a reductase function was attributed to PchG. In summary, this study completes the identification of the biosynthetic genes required for pyochelin formation from chorismate in P. aeruginosa. (+info)
Analysis of the pmsCEAB gene cluster involved in biosynthesis of salicylic acid and the siderophore pseudomonine in the biocontrol strain Pseudomonas fluorescens WCS374.
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Mutants of Pseudomonas fluorescens WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylic acid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb EcoRI fragment of pMB374-07. The SA and pseudomonine biosynthesis region was identified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of P. putida. Sequence analysis of the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs (pmsC and pmsB) showed homologies with chorismate-utilizing enzymes; a third ORF (pmsE) encoded a protein with strong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. The region also contained a putative histidine decarboxylase gene (pmsA). A putative promoter region and two predicted iron boxes were localized upstream of pmsC. We determined by reverse transcriptase-mediated PCR that the pmsCEAB genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genes was achieved in P. putida and Escherichia coli cells. In E. coli, deletions affecting the first ORF (pmsC) diminished SA production, whereas deletion of pmsB abolished it completely. The pmsB gene induced low levels of SA production in E. coli when expressed under control of the lacZ promoter. Several lines of evidence indicate that SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn5 mutant (374-05) that is simultaneously impaired in SA and pseudomonine production. (+info)
Characterisation of Hafnia alvei isolates from human clinical extra-intestinal specimens: haemagglutinins, serum resistance and siderophore synthesis.
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Extra-intestinal Hafnia alvei isolates are rarely considered to be pathogenic. To investigate whether such strains are able to produce virulence factors, a total of 70 clinical H. alvei isolates was compared with clinical extra-intestinal isolates of other members of the enterobacterial tribe Klebsiellae (Kiebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens). Whereas mannose-sensitive haemagglutination (MSHA) was less common in H. alvei (59%) than in K. pneumoniae (86%) and E. cloacae (89%) isolates, the incidences of mannose-resistant haemagglutination indicative of type 3 pili (MR/K-HA) and of serum resistance properties were not lower. All H. alvei strains secreted siderophores but, unlike the other enterobacterial species examined, the siderophore type was neither enterobactin nor aerobactin. Although the low pathogenicity of H. alvei isolates could not be attributed to any of the factors investigated, the mean number of factors expressed by each H. alvei isolate was significantly lower than that expressed by K. pneumoniae and E. cloacae isolates but did not differ significantly from that of S. marcescens. Based on these findings, the low pathogenicity of H. alvei appears to be due to its low frequency of expression of virulence factors as compared with clinically significant species such as K. pneumoniae and E. cloacae. (+info)
Regulation of ornibactin biosynthesis and N-acyl-L-homoserine lactone production by CepR in Burkholderia cepacia.
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The CepR-CepI quorum-sensing system has been shown to regulate production of the siderophore ornibactin, extracellular proteases, and N-octanoyl-homoserine-L-lactone (OHL) in Burkholderia cepacia strain K56-2. To examine the effect of cepIR on production of other siderophores, cepR mutants were constructed in strains that produce pyochelin in addition to salicylic acid and ornibactins. Pc715j-R1 (cepR::tp) hyperproduced ornibactin but produced parental levels of pyochelin and salicylic acid, suggesting that CepR is a negative regulator of ornibactin synthesis but not pyochelin or salicylic acid. Pc715j-R1 was also protease deficient and OHL negative. The effects of cepR on ornibactin biosynthetic genes were examined by constructing cepR pvdA-lacZ and cepR pvdD-lacZ mutants and monitoring beta-galactosidase activity. There was an increase in expression of pvdA in the cepR mutant compared to the level in its parent strain in both low- and high-iron media during stationary phase. When the outer membrane protein profiles of a cepR mutant and the wild-type strain were compared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, there did not appear to be any difference in levels of expression of the ornibactin receptor. Experiments with cepI-lacZ and cepR-lacZ transcriptional fusions indicated that cepI was not expressed in the cepR mutant and that cepR acts as a negative regulator of its own expression. By a thin-layer chromatography assay for N-acyl homoserine lactones, OHL and N-hexanoyl-L-homoserine lactone (HHL) were detectable in K56-2 and Pc715j, both wild-type strains. OHL was not detectable and HHL was only weakly detectable in the cepI and cepR mutants. These results suggest that CepR is both a positive and negative transcriptional regulator and that CepR may influence the expression of ornibactin biosynthetic genes in addition to the expression of the cepIR quorum-sensing system. (+info)
Reduced virulence of a Bordetella bronchiseptica siderophore mutant in neonatal swine.
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One means by which Bordetella bronchiseptica scavenges iron is through production of the siderophore alcaligin. A nonrevertible alcaligin mutant derived from the virulent strain 4609, designated DBB25, was constructed by insertion of a kanamycin resistance gene into alcA, one of the genes essential for alcaligin biosynthesis. The virulence of the alcA mutant in colostrum-deprived, caesarean-delivered piglets was compared with that of the parent strain in two experiments. At 1 week of age, piglets were inoculated with phosphate-buffered saline, 4609, or DBB25. Two piglets in each group were euthanatized on day 10 postinfection. The remainder were euthanatized at 21 days postinfection. Clinical signs, including fever, coughing, and sneezing, were present in both groups. Nasal washes performed 7, 14, and 21 days postinoculation demonstrated that strain DBB25 colonized the nasal cavity but did so at levels that were significantly less than those achieved by strain 4609. Analysis of colonization based on the number of CFU per gram of tissue recovered from the turbinate, trachea, and lung also demonstrated significant differences between DBB25 and 4609, at both day 10 and day 21 postinfection. Mild to moderate turbinate atrophy was apparent in pigs inoculated with strain 4609, while turbinates of those infected with strain DBB25 developed no or mild atrophy. We conclude from these results that siderophore production by B. bronchiseptica is not essential for colonization of swine but is required for maximal virulence. B. bronchiseptica mutants with nonrevertible defects in genes required for alcaligin synthesis may be candidates for evaluation as attenuated, live vaccine strains in conventionally reared pigs. (+info)
Genetic organization of the region encoding regulation, biosynthesis, and transport of rhizobactin 1021, a siderophore produced by Sinorhizobium meliloti.
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Eight genes have been identified that function in the regulation, biosynthesis, and transport of rhizobactin 1021, a hydroxamate siderophore produced under iron stress by Sinorhizobium meliloti. The genes were sequenced, and transposon insertion mutants were constructed for phenotypic analysis. Six of the genes, named rhbABCDEF, function in the biosynthesis of the siderophore and were shown to constitute an operon that is repressed under iron-replete conditions. Another gene in the cluster, named rhtA, encodes the outer membrane receptor protein for rhizobactin 1021. It was shown to be regulated by iron and to encode a product having 61% similarity to IutA, the outer membrane receptor for aerobactin. Transcription of both the rhbABCDEF operon and the rhtA gene was found to be positively regulated by the product of the eighth gene in the cluster, named rhrA, which has characteristics of an AraC-type transcriptional activator. The six genes in the rhbABCDEF operon have interesting gene junctions with short base overlaps existing between the genes. Similarities between the protein products of the biosynthesis genes and other proteins suggest that rhizobactin 1021 is synthesized by the formation of a novel siderophore precursor, 1,3-diaminopropane, which is then modified and attached to citrate in steps resembling those of the aerobactin biosynthetic pathway. The cluster of genes is located on the pSyma megaplasmid of S. meliloti 2011. Reverse transcription-PCR with RNA isolated from mature alfalfa nodules yielded no products for rhbF or rhtA at a time when the nifH gene was strongly expressed, indicating that siderophore biosynthesis and transport genes are not strongly expressed when nitrogenase is being formed in root nodules. Mutants having transposon insertions in the biosynthesis or transport genes induced effective nitrogen-fixing nodules on alfalfa plants. (+info)