Iro Gene Research Articles

Characteristics of iron cycle and its driving mechanism during the development of biological soil crusts associated with desert revegetation

Iron (Fe) is an essential element for almost all living organisms. In addition, microbial Fe cycling drives the biogeochemical cycles of other elements, although little is known about characteristics of Fe cycling and its driving mechanism in desert ecosystems. Here, we investigated the key microbial functional genes involved in Fe(II) oxidation and Fe(III) reduction and the associated factors affecting the soil during the development of biological soil crusts (BSCs), which are among the most important landscapes in desert ecosystems. We also analyzed the relationships among the genes involved in Fe, carbon (C), and nitrogen (N) cycling. The available Fe content and diversity of genes associated with microbial Fe cycling increased after 61 years of BSC development. Our results suggested that Fe(II) oxidation in BSCs is mainly driven by the iro gene from microaerophilic Fe oxidizing microorganisms (FeOMs), whereas Fe(III) reduction is driven mainly by c-type cytochromes through regulation by the OmcS gene. The presence of both genes significantly correlated with that of genes involved in labile organic C degradation, denitrification, and N reduction. Fungal abundance, soil nutrient content (mainly total N), and the Fe-manganese (Mn) oxide-bound fraction (RED-Fe) were the main soil factors affecting the gene profile associated with microbial Fe cycling in BSCs (explaining 74.8% of the variation). These results indicated that the main Fe cycling process in desert ecosystems can adapt to the local environment. Furthermore, the interaction between Fe and the C or N cycles, as well as the synergistic effects of soil quality improvement, actively promotes effective and dynamic Fe cycling in BSCs.

Comparative genomics of Salmonella enterica subsp. diarizonae serovar 61:k:1,5,(7) reveals lineage-specific host adaptation of ST432.

Unlike most Salmonella enterica subsp. diarizonae , which are predominantly associated with cold-blooded animals such as reptiles, the serovar IIIb 61:k:1,5,(7) (termed SASd) is regarded as host-adapted to sheep. The bacterium is rarely associated with disease in humans but, nevertheless, SASd isolates are sporadically obtained from human clinical samples. It is unclear whether these transmissions are directly linked to sheep or whether transmissions may, for example, occur through other domestic animals also carrying SASd. For this reason, we utilized whole-genome sequencing to investigate a set of 119 diverse SASd isolates, including sheep-associated and human-associated isolates, as well as isolates obtained from other matrices. We discovered that serovar IIIb 61:k:1,5,(7) is composed of two distinct lineages defined by their sequence types ST432 and ST439. These two lineages are distinguished by a number of genetic features, as well as their prevalence and reservoir. ST432 appears to be the more prevalent sequence type, with the majority of isolates investigated in this study belonging to ST432. In contrast, only a small number of isolates were attributed to ST439. While ST432 isolates were of sheep, human or other origin, all ST439 isolates with source information available, were obtained from human clinical samples. Regarding their genetic features, lineage ST432 shows increased pseudogenization, harbours a virB/D4 plasmid that encodes a type IV secretion system (T4SS) and does not possess the iro gene cluster, which encodes a salmochelin siderophore for iron acquisition. These characteristics likely contribute to the ability of ST432 to persistently colonize the intestines of sheep. Furthermore, we found isolates of the lineage ST432 to be highly clonal, with little variation over the sampling period of almost 20 years. We conclude from the genomic comparisons that SASd underlies a microevolutionary process and that it is specifically lineage ST432 that should be considered as host-adapted to sheep.

Biochemical characterization of wild-type and mutant (Q9F and S21Y/V22D)iron oxidases isolated from Acidithiobacillus ferrooxidans M1

Iron oxidase, a member of the high potential iron-sulfur protein (HiPIP) family within the iron-sulfur cluster, was thought to be involved in the iron respiratory electron transport chain in Acidithiobacillus ferrooxidans. A. ferrooxidans M1 strain was isolated from Murgul copper mine. The iro gene of this bacterium encoding iron oxidase was cloned, and the complete nucleotide sequence was disclosed. The gene was cloned and overexpressed successfully. The highly conserved amino acid residues within the iron oxidase enzyme sequence were determined, and their Q9F and S21Y/V22D recombinants were created through site-directed mutagenesis. Wild-type and recombinant iron oxidase enzymes were purified and further characterized. The biochemical properties and kinetic parameters of wild-type and mutant enzymes were determined and compared. The optimal temperature of the wild-type enzyme was 25 °C, and maximal activity was observed at pH 4.0. The Km and Vmax values of wild-type enzyme were 0.27 ± 0.09 mM and 0.083 ± 0.01 μmol/min/mg protein, respectively. Although the mutant enzymes were almost comparable to wild-type enzyme, their maximal activities moved from pH 4.0 to pH 3.5, and pH stability of S21Y/V22D mutant was improved compared to wild type.

Insights into the pathways of iron- and sulfur-oxidation, and biofilm formation from the chemolithotrophic acidophile Acidithiobacillus ferrivorans CF27

The iron-oxidizing acidithiobacilli cluster into at least four groups, three of which (Acidithiobacillus ferrooxidans, Acidithiobacillus ferridurans and Acidithiobacillus ferrivorans) have been designated as separate species. While these have many physiological traits in common, they differ in some phenotypic characteristics including motility, and pH and temperature minima. In contrast to At. ferrooxidans and At. ferridurans, all At. ferrivorans strains analysed to date possess the iro gene (encoding an iron oxidase) and, with the exception of strain CF27, the rusB gene encoding an iso-rusticyanin whose exact function is uncertain. Strain CF27 differs from other acidithiobacilli by its marked propensity to form macroscopic biofilms in liquid media. To identify the genetic determinants responsible for the oxidation of ferrous iron and sulfur and for the formation of extracellular polymeric substances, the genome of At. ferrivorans CF27 strain was sequenced and comparative genomic studies carried out with other Acidithiobacillus spp.. Genetic disparities were detected that indicate possible differences in ferrous iron and reduced inorganic sulfur compounds oxidation pathways among iron-oxidizing acidithiobacilli. In addition, strain CF27 is the only sequenced Acidithiobacillus spp. to possess genes involved in the biosynthesis of fucose, a sugar known to confer high thickening and flocculating properties to extracellular polymeric substances.

Phenotypic and genetic characterization of a novel strain of Acidithiobacillus ferrooxidans (AF2)

A comparative study on the phenotypic and genetic characteristics among Acidithiobacillus ferrooxidans (AF2), a typic strain ATCC23270 and a previously isolated strain AF3 was performed. AF2 can use ferrous ion (Fe2+) or elemental sulfur (S0) as sole energy source, but oxidizes S0 more effectively than Fe2+, which is different from ATCC23270 and AF3. The G+C content of AF2 is 51.8% (molar fraction), however, ATCC23270 and AF3 strains have G+C content of 63.7% and 64.8% (molar fraction), respectively. The DNA-DNA hybridization results show that AF2 has 41.53% and 52.38% genome similarity to ATCC 23270 and AF3, respectively, but AF3 has a high genome similarity of 89.86% to ATCC 23270 strain. Rusticyanin (rus) and subunit III of aa3-type cytochrome oxidase (coxC) genes are not detected in AF2, but Fe2+ oxidase (iro) gene can be detected. To understand the genomic organization of iro gene, a cosmid library of AF2 genome was constructed and iro gene-containing clone was screened. The sequencing result shows that although the nucleotide sequence of iro gene in AF2 is completely identical to that of ATCC 23270 strain, its genomic organization is different from that of ATCC 23270. In AF2, iro is located at downstream of purA gene, while it is located at downstream of petC-2 gene in ATCC 23270 strain. These results indicate that AF2 is a novel strain of A. ferrooxidans, and that phenotypic differences among the strains of A. ferrooxidans are closely correlated with their genetic polymorphisms.

Isolation and characterization of acidophilic bacterium from Dongxiangshan Mine in Xinjiang Province, China

One bioleaching bacterium, named as strain DXS, was isolated from acid mine drainages (AMDs) of Dongxiangshan Mine of Hami, Xinjiang Province, China. The strain DXS is gram-negative and rod-shaped with a size of (0.40±0.05) μm×(1.3±0.5) μm. The optimal temperature and pH for growth are 30 °C and pH 2.0, respectively. It can grow autotrophically by using ferrous iron, elemental sulfur and NaS2O3 as sole energy sources. In the phylogenetic tree, strain DXS has similarity with Acidithiobacillus ferrooxidans type strain ATCC 23270 with 99.57% sequence similarity. The cloning and sequencing of Iro protein gene (iro) and tetrathionate hydrolase gene (tth) reveal that strain DXS is completely identical in iro gene sequence to A. ferrooxidans LY (DQ166841), and almost identical in tth gene sequene to A. ferrooxidans (AB259312) (only two nucleotides change). The bioleaching experiments of marmatite and pyrite reveal that the leached zinc and iron concentrations reach 3.01 g/L and 2.75 g/L, respectively. The strain has a well potential application in industry bioleaching.

Isolation and characterization of acidophilic bacterium from Gaofeng Mine in China

An acidophilic, chemolithotrophic and ferrous oxidizing bacterium strain GF was isolated from the acid mine drainage (AMD) of Gaofeng Mine, Guangxi Province, China using 9K enrichment medium, and then purified on solid ferrous-agarose medium. The physiological experiments show that it can use ferrous or sulfur as sole energy and a low level (0.1%, w/v) of peptone can accelerate the growth of the isolated strain. The optimum pH and temperature for growth are 2.0 and 30 °C, respectively. The isolated strain shares 99.64% identities of 16S rRNA gene with the type strain Acidthiobacillus ferrooxidans ATCC 23270 and 100% identities of iro gene (CDS) with A. ferrooxidans strain Fe-1. These results show that the strain can be considered as Acidthiobacillus ferrooxidans. Because of the high activity of oxidizing ferrous and sulfide mineral, strain GF was used in bioleaching of marmatite. The Zn concentration is 0.273 g/L under the steriled control and 7.30 g/L with adapted GF strain incubated after 29 d in leaching marmatite. The isolated strain GF can be used to leach marmatite in industry application.

Identification and characterization of four strains of Acidithiobacillus ferrooxidans isolated from different sites in China

Four strains of Acidithiobacillus ferrooxidans (A. ferrooxidans), AF1, AF2, AF3 and AFc, were isolated from samples with different geological sources using a 9K medium. These four isolates were identified as A. ferrooxidans by phenotypic and 16S rDNA sequence analyses. All four isolates were able to use ferrous ion (Fe(2+)), elemental sulfur (S0) or pyrite as a sole energy source, but they showed differences in pH optima and range of activity, optimum temperature of activity, resistance to chloride (KCl) and heavy metal ions, and oxidation rates of Fe(2+), S0 and pyrite. AF3 was the most active strain when using Fe(2+) as the energy source, while AFc grew best using pyrite as the energy source. AF2 appeared to differ from the other three strains in substrate utilization, as it oxidizes S0 and pyrite more effectively than Fe(2+). RAPD analysis of genomic DNA from these isolates showed that banding profiles of their genomic DNA exhibited some differences, and the genomic banding profile of AF2 was significantly different from that of others. To obtain an insight into the molecular biology of the process of the energy production of these strains, several genes involved in the iron respiratory chain were cloned and sequenced, including Fe(2+) oxidase (iro), rusticyanin (rus) and subunit III of aa3-type cytochrome oxidase (cox C) genes. The results revealed that the iro gene can be cloned from all of the four strains and the nucleotide sequences were shown to be completely identical in each. However, rus and coxC genes could be amplified only from AF1, AF3 and AFc, not from AF2. These results suggested that the phenotypic differences of the four strains of A. ferrooxidans from different sites correlated with their genetic polymorphism, which may result from the different environments in which they lived, and that the strain AF2 was phenotypically and genetically significantly different from the other three strains.

The colicin G, H and X determinants encode microcins M and H47, which might utilize the catecholate siderophore receptors FepA, Cir, Fiu and IroN.

The colicin G producer Escherichia coli CA46, the colicin H producer E. coli CA58 and E. coli Nissle 1917 (DSM 6601) were shown to produce microcin H47 and the newly described microcin M. Both microcins were exported like colicin V by an RND-type export system, including TolC. The gene cluster encoding microcins H47 and M in strains CA46 and CA58 is nearly identical to that in strain DSM 6601, except that two additional genes are included. A Fur box identified in front of the microcin-encoding genes explained the observed iron regulation of microcin production. The catecholate siderophore receptors Fiu, Cir and FepA from E. coli and IroN, Cir and FepA from Salmonella were identified as receptors for microcins M, H47 and E492. IroN takes up the glucose-containing catecholate siderophore salmochelin, whose synthesis is encoded in the iro gene cluster found in Salmonella and certain, often uropathogenic, E. coli strains. A gene in this iro cluster, iroB, which encodes a putative glycosyltransferase, was also found in the microcin H47/M and microcin E492 gene clusters. These microcins could aid the producing strain in competing against enterobacteria that utilize catecholate siderophores.

Characterization of an iroBCDEN gene cluster on a transmissible plasmid of uropathogenic Escherichia coli: evidence for horizontal transfer of a chromosomal virulence factor.

The chromosomal iroBCDEN gene cluster first described for Salmonella enterica is involved in the uptake of catecholate-type siderophore compounds. An orthologous gene cluster has recently been detected in Escherichia coli strains which cause extraintestinal disease. This E. coli iroBCDEN gene cluster has an impact on virulence and has been reported to be located in a pathogenicity island on the chromosome. In this study we characterized an iro gene cluster of a uropathogenic E. coli isolate which is located on a transmissible plasmid related to the R64 plasmid of S. enterica. This cluster is highly homologous to the chromosomal iro cluster of E. coli. When introduced into an E. coli fepA cir fiu aroB mutant, IroN, but not IroBCDE, mediated the utilization of structurally related catecholate siderophores, including 2,3-dihydroxybenzoyl-L-serine, 2,3-dihydroxybenzoyl-D-ornithine, 2,3-dihydroxybenzoic acid, and enterochelin. This study supports the idea of an ongoing horizontal transfer of putative virulence factors and the mobilization of single virulence gene clusters, which lead to a modular assembly of virulence determinants such as pathogenicity islands.

Biochemical and EPR characterization of a high potential iron-sulfur protein inThiobacillus ferrooxidans

A soluble acid-stable high potential iron-sulfur protein (HiPIP) was purified from Thiobacillus ferrooxidans using the periplasmic extraction method. It was isolated in the form of a tetramer consisting of four subunits with a molecular mass of 5582 Da, and its biochemical and biophysical properties were characterized. The N-terminal amino acid sequence (15 residues) was compared with the nucleotide sequence of the iro gene isolated from another strain and the two sequences were found to be identical. The iron content measurement together with optical and EPR spectroscopic studies of the purified protein were consistent with the presence of one [4Fe-4S] cluster per subunit. The EPR spectrum recorded in the oxidized state was attributed to a [4Fe-4S]3+ cluster and the redox potential has been determined to be +380 mV.

Biochemical and EPR characterization of a high potential iron-sulfur protein in Thiobacillus ferrooxidans

A soluble acid-stable high potential iron-sulfur protein (HiPIP) was purified from Thiobacillus ferrooxidans using the periplasmic extraction method. It was isolated in the form of a tetramer consisting of four subunits with a molecular mass of 5582 Da, and its biochemical and biophysical properties were characterized. The N-terminal amino acid sequence (15 residues) was compared with the nucleotide sequence of the iro gene isolated from another strain and the two sequences were found to be identical. The iron content measurement together with optical and EPR spectroscopic studies of the purified protein were consistent with the presence of one [4Fe-4S] cluster per subunit. The EPR spectrum recorded in the oxidized state was attributed to a [4Fe-4S] 3+ cluster and the redox potential has been determined to be +380 mV.

Identification of thepurA gene encoding adenylosuccinate synthetase inThiobacillus ferrooxidans

The purA gene of Thiobacillus ferrooxidans encoding adenylosuccinate synthetase [EC 6.3.4.4] was identified in the upstream region of the iro gene encoding Fe(II)-oxidase (J. Biol. Chem 267:11242-11247, 1992). The purA gene consisted of 1290 base-pairs, which translated into a 29-amino-acid protein. The gene is functionally active, because it is able to complement an Escherichia coli purA-deficient strain. The deduced gene product has a high degree (60.9%) of sequence identity with that (432 aa) of E. coli purA gene, and both the products share GDEGKGK-DETG-TKLD sequences which are supposed to be GTP-binding domain. The downstream region of the iro gene contained another open-reading frame (ORF) of 1218 bp, and this showed high homology (56.6% over 249 bp) with E. coli ORF-II, which is found as a second ORF and truncated form in the downstream region of the purA gene. Comparison of the gene organization in the flanking region of purA gene between T. ferrooxidans and E. coli is also described.

Molecular cloning of the gene encoding Thiobacillus ferrooxidans Fe(II) oxidase. High homology of the gene product with HiPIP.

The amino-terminal sequence of Thiobacillus ferrooxidans Fe(II) oxidase (linked to cytochrome c552) was determined, and the iro gene that encodes this enzyme was cloned using degenerate oligonucleotides as a probe. The DNA sequence of a region (856 base pairs) which encompasses the iro gene revealed that the enzyme was encoded by a 273-base pair open reading frame and consists of 90 amino acids, including a possible 37-residue signal sequence. The iro gene seems to be transcribed independently of any other gene because the transcriptional products are 0.45 and 0.6 kilobases in size. Current protein databases revealed that the iro gene product is a new member of the high redox potential iron sulfur proteins, which generally function in electron transport but do not show enzymatic activity.