Role of siderophore in iron uptake in cowpea Rhizobium GN1 (peanut isolate): Possible involvement of iron repressible outer membrane proteins

Abstract Siderophore produced by cowpea Rhizobium GN1 (Peanut isolate) was shown to be involved in iron uptake by this organism. Siderophore enhanced iron uptake in iron-starved cells. SDS-PAGE analysis of the outer membrane proteins showed two iron repressible outer membrane proteins with approximate molecular mass of 80 kDa and 76 kDa. A siderophore non-producing mutant, which was unable to grow on a medium containing synthetic iron chelators unless and until iron was added exogenously in the medium, could use siderophore of the wild-type for iron uptake indicating that the receptor for Fe-siderophore complex was intact in the mutant.     

Chemical and structural characterization of hydroxamate siderophore produced by marine <Emphasis Type="Italic">Vibrio harveyi</Emphasis>

In the present study, 22 different bacteria were isolated from open ocean water from the Gulf of Mannar, India. Of the 22 isolates, 4 were identified as Vibrio spp. (VM1, VM2, VM3 and VM4) and found to produce siderophores (iron-binding chelators) under iron-limited conditions. Different media were found to have an influence on siderophore production. Maximum siderophore production was observed with VM1 isolate in MM9 salts medium at 48 h of incubation. The isolate was confirmed as Vibrio harveyi based on 16S rRNA gene sequencing and phylogenetic analysis. Fourier-transform infrared (FTIR) and ¹H nuclear magnetic resonance (NMR) spectra revealed the hydroxamate nature of the siderophore produced. Further characterization of the siderophore revealed it to be of dihydroxamate nature, forming hexadentate ligands with Fe(III) ions. A narrow shift in ultraviolet (UV)–Vis spectrum was observed on photolysis due to ligand oxidation. Growth-promotion bioassay with Aeromonas hydrophila, Staphylococcus aureus and E. coli confirmed the iron-scavenging property of the siderophore produced by Vibrio harveyi.     

A comparative study of siderophore production by fungi from marine and terrestrial habitats

Siderophore producing potential of 20 fungal isolates (same 10 species from each marine and terrestrial habitat) were examined and compared. Except marine Aspergillus flavus, all isolates produced siderophores as evidenced by positive reaction in FeCl₃ test, CAS assay and CAS agar plate test. The results indicated widespread occurrence of siderophores in both the habitats. Examination of the chemical nature of siderophores revealed that mucoraceous fungi produced carboxylate, while others produced hydroxamate siderophores. Thus, the nature of siderophore was found to be independent of habitat. Among all the isolates, Cunninghamella elegans (marine form) was maximum siderophore producer (1987.5 μg/ml) followed by terrestrial form of C. elegans (1248.75 μg/ml). There was no marked variation in siderophore concentration of Penicillium funiculosum strains. Comparison of quantification of siderophore production between marine and terrestrial revealed that four terrestrial isolates (Aspergillus niger, Aspergillus ochraceous, Penicillium chrysogenum, Penicillium citrinum) were ahead in siderophore production, while, the other four marine isolates (Aspergillus versicolor, C. elegans, Rhizopus sp., Syncephalastrum racemosum) were found to be more potent siderophore producers, indicating that they were equally competent.     

Symbiotic influence of endophytic Bacillus pumilus on growth promotion and probiotic potential of the medicinal plant Ocimum sanctum

Bacillus pumilus was isolated from surface-sterilized tissues of the medicinal plant Ocimum sanctum. Scanning electron microscopic (SEM) imaging confirmed the presence of a rod shaped bacterium within the plant tissues. The bacterium was identified as B. pumilus by biochemical analyses and 16S rRNA gene sequencing. In vitro analyses indicate that the isolated strain of B. pumilus was endowed with multiple plant growth promotion (PGP) traits such as phosphate solubilization and the production of indole acetic acid (IAA), siderophore and hydrogen cyanide (HCN). Phosphate solubilization (37.3 μg ml^?1) and IAA production (36.7 μg ml^?1) by the isolate was found to reach a maximum after 60 h of incubation. Siderophore mediated iron sequestration by B. pumilus may confer a competitive advantage to the host with respect to pathogen inhibition. Siderophore produced by the isolate was found to be of a trihydroxamate type with hexadentate nature. The B. pumilus isolate also exhibited cellulolytic, proteolytic and chitinolytic activity. Cell free supernatant, culture filtrates of the isolate were found to suppress the growth of fungal phytopathogens. The culture filtrate retained its antifungal activity even after exposure to heat. In addition to PGP, the isolate exhibited probiotic properties such as acid tolerance (pH2), bile salt tolerance (2 %), auto-aggregation, antibiotic resistance and the absence of haemolytic activity. These finding suggest the possibility of utilizing this endophytic strain of B. pumilus as a bioinoculant to enhance plant growth and also as a probiotic.     

Iron repressibility of siderophore and transferrin-binding protein in Staphylococcus aureus

In order to investigate whether the iron acquisition mechanisms of Staphylococcus aureus are induced by iron restriction in vitro, we examined S. aureus ATCC 6538 for production of siderophore and expression of transferrin-binding protein (SA-tbp) in normal or deferrated brain heart infusion broth (BHI). Siderophore production was earlier and greater in the deferrated BHI. The SA-tbp, detected by ligand blot assay, was expressed only in the deferrated BHI. When human transferrin was added to the deferrated BHI, siderophore production was later and lower than when transferrin was not present. In conclusion, both iron acquisition mechanisms of S. aureus were found to be iron-repressible and via both of them, human transferrin-bound iron was utilized for growth under iron-restricted condition.     

Siderophore Production by Pathogenic Mucorales and Uptake of Deferoxamine B

Clinical reports have established that mucormycosis, mainly caused by Rhizopus spp., frequently occurs in patients treated with deferoxamine B (DFO, Desferal^®) which is misappropriated by these fungi. Siderophore production by twenty mucoralean isolates was therefore investigated using a commercial iron-depleted culture medium. Siderophore production was detected for most of the isolates. Our experiments confirmed that feroxamine B (iron chelate of DFO) promoted in vitro growth of Rhizopus arrhizus. Electrophoretic analysis of somatic extracts revealed iron-regulated proteins of 60 and 32 kDa which were lacking in iron-depleted culture conditions. Using a fluorescent derivative of deferoxamine B, we showed by fluorescence microscopy the entry of the siderophore within the fungal cells, thus suggesting a shuttle mechanism encompassing the uptake of the entire siderophore-ion complex into the cell. This useful tool renders possible a better understanding of iron metabolism in Mucorales which could lead to the development of new diagnostic method or new antifungal therapy using siderophores as imaging contrast agents or active drug vectors.     

Influence of Environmental Factors on Bacteriocin Production by Human Isolates of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> MM19 and <Emphasis Type="Italic">Pediococcus acidilactici</Emphasis> MM33

The influence of temperature, initial pH, and carbon and nitrogen sources on bacteriocin secreted by Lactococcus lactis MM19 (MM19) and Pediococcus acidilactici MM33 (MM33) was evaluated. It was found that 30 and 45 °C were the growth temperatures for higher nisin and pediocin production by MM19 and MM33, respectively. The initial pH values for higher production of nisin and pediocin were 9 and 6, respectively. Glucose and wheat peptone E430 were found as suitable carbon and nitrogen sources, respectively, for highest nisin production by MM19 at 30 °C and initial pH of 9. In these conditions, nisin production could be increased by 6.7 times as compared to the control medium (de Man, Rogosa, and Sharpe—MRS broth). Similarly, fructose and pea peptone were suitable carbon and nitrogen sources, respectively, for highest production of pediocin by MM33 at 45 °C and initial pH of 6. In these conditions, pediocin production by MM33 was increased by three times as compared to the control medium (tryptone-glucose-yeast extract—TGE broth).     

Regulation of siderophore production by iron Fe(III) in certain fungi and fluorescent pseudomonads

Regulation of siderophore production in response to iron concentration in the medium was examined. Threshold concentration was recorded for twenty fungi and three rhizobacterial pseudomonads. Organisms showed difference in threshold values at which they stopped siderophore elaboration. In nine fungi (3 aspergilli, 1 penicillium, N. crassa, F. dimerum and 3 mucors) siderophore production was repressed at 3 microM Fe(III). Siderophore production was repressed at 27 microM of Fe (III) in 3 aspergilli, 2 penicillia and 3 pseudomonads. Rest of the fungi had cut off values at 6, 9, 15, 21 microM of Fe(III) concentration.     

ε-PolyLysine production from sugar cane molasses by a new isolates of Bacillus sp. and optimization of the fermentation condition

An attempt was made to use cane molasses as a culture medium for ε-PolyLysine (ε-PL) production by a natural bacterial isolate. The bacterium was identified as Bacillus sp., as confirmed by 16S rDNA sequence analysis. A BLAST result of the sequence indicated that the closest relative of this Bacillus BHU strain was B. thuringiensis, with 97 % homology. The molasses was found to be a better culture medium compared to commonly used culture media comprised of either glucose or glycerol as a carbon source. The various physicochemical parameters were studied for culture growth and polymer production, and were further optimized using response surface methodology (RSM). The correlation coefficient of the resulting model was found to be R ²?=?0.9828. The RSM predicted optimum conditions for ε-PL production (2.46 g/l) by the Bacillus strain was achieved by using molasses, 59.7 g/l; yeast extract, 15.2 mg/l; pH, 6.8 and fermentation time, 42 h at 30 °C. This study represents the first report on the potential application of cane molasses (a byproduct of sugarcane industries) as a culture medium for ε-PL production by Bacillus species. The specific Bacillus strain used in the present study can be exploited for developing a novel technology using inexpensive renewable resources for ε-PL production, a polymer of commercial interest.     

Potential of Siderophore Production by Bacteria Isolated from Heavy Metal: Polluted and Rhizosphere Soils

Recently, heavy metals have been shown to have a stimulating effect on siderophore biosynthesis in various bacteria. In addition, several studies have found that siderophore production is greater in bacteria isolated from soil near plant roots. The aim of this study was to compare the production of siderophores by bacterial strains isolated from heavy metal-contaminated and uncontaminated soils. Chrome azurol sulphonate was used to detect siderophore secretion by several bacterial strains isolated from heavy metal-contaminated and rhizosphere-uncontaminated soils with both a qualitative disc diffusion method and a quantitative ultraviolet spectrophotometric method. Siderophore production by rhizosphere bacteria was significantly greater than by bacteria isolated from contaminated soil. The Pearson’s correlation test indicated a positive correlation between the amount of siderophore produced by bacteria isolated from the rhizosphere using the quantitative and qualitative detection methods and the amount of heavy metal in the soil. However, a significant negative correlation was observed between the amount of siderophore produced by bacteria isolated from heavy metal-contaminated soil and the amount of heavy metal (r value of ?0.775, P < 0.001).     

Hemolytic Activity and Siderophore Production in Different Aeromonas Species Isolated from Fish

The hemolytic activity and siderophore production of several strains of motile aeromonads were determined. The hemolytic activity of Aeromonas caviae and Aeromonas eucrenophila was enhanced after trypsinization of the samples. The enhancement of hemolysis was observed in strains that carried an aerolysin-like gene, detected by a PCR procedure. Siderophore production was demonstrated in all but one strain of Aeromonas jandaei. No apparent relationship was observed between the presence of plasmid DNA and hemolysis or siderophore production.     

Siderophore production by actinomycetes isolates from two soil sites in Western Australia

The actinomycetes are metabolically flexible soil micro-organisms capable of producing a range of compounds of interest, including siderophores. Siderophore production by actinomycetes sampled from two distinct and separate geographical sites in Western Australia were investigated and found to be generally similar in the total percentage of siderophore producers found. The only notable difference was the proportion of isolates producing catechol siderophores with only 3% found in site 1 (from the north-west of Western Australia and reportedly containing 40% magnetite) and 17% in site 2 (a commercial stone fruit orchard in the hills east of Perth with a soil base ranging from sandy loam to laterite). Further detailed characterization of isolates of interest identified a Streptomyces that produced extracellularly excreted enterobactin, the characteristic Enterobacteriaceae siderophore, and also revealed some of the conditions required for enterobactin production. Carriage of the entF gene, which codes for the synthetase responsible for the final assembly of the tri-cyclic structure of enterobactin, was confirmed by PCR in this isolate. Another separate Streptomyces produced a compound that matched the UV/VIS spectra of heterobactin, a siderophore previously only described in Rhodococcus and Nocardia.     

嗜水气单胞菌铁载体的提纯及特性分析

提纯了嗜水气单胞菌（Ａｈ）Ｊ１ 株的铁载体（ｓｉｄｅｒｏｐｈｏｒｅ） ,并对其特性进行了初步分析。Ａｈ Ｊ１ 株的培养上清液经聚酰胺柱层析、双蒸水洗脱、乙酸乙脂沉淀和真空冻干,获得白色粉末。用ＣＡＳ法及Ａｒｎｏｗ 法检测均为阳性,证实为铁载体,含有２ ,３二羟基苯甲酸（２ ,３ＤＨＢ） 功能团,属酚盐类铁载体。高压液相色谱分析表明,此种铁载体仅含甘氨酸、赖氨酸及色氨酸。上述纯化的铁载体,在体外培养条件下能促进产铁载体为弱阳性的Ａｈ Ｎ９ａ 株的生长,且能对抗ＥＤＤＡ 对细菌生长的抑制作用,显示铁载体能促进细菌的增殖,在细菌的感染致病过程中可能起重要作用。     

Photobactin: a Catechol Siderophore Produced by Photorhabdus luminescens, an Entomopathogen Mutually Associated with Heterorhabditis bacteriophora NC1 Nematodes

The nematode Heterorhabditis bacteriophora transmits a monoculture of Photorhabdus luminescens bacteria to insect hosts, where it requires the bacteria for efficient insect pathogenicity and as a substrate for growth and reproduction. Siderophore production was implicated as being involved in the symbiosis because an ngrA mutant inadequate for supporting nematode growth and reproduction was also deficient in producing siderophore activity and ngrA is homologous to a siderophore biosynthetic gene, entD. The role of the siderophore in the symbiosis with the nematode was determined by isolating and characterizing a mini-Tn5-induced mutant, NS414, producing no detectable siderophore activity. This mutant, being defective for growth in iron-depleted medium, was normal in supporting nematode growth and reproduction, in transmission by the dauer juvenile nematode, and in insect pathogenicity. The mini-Tn5 transposon was inserted into phbH; whose protein product is a putative peptidyl carrier protein homologous to the nonribosomal peptide synthetase VibF of Vibrio cholerae. Other putative siderophore biosynthetic and transport genes flanking phbH were characterized. The catecholate siderophore was purified, its structure was determined to be 2-(2,3-dihydroxyphenyl)-5-methyl-4,5-dihydro-oxazole-4-carboxylic acid [4-(2,3-dihydroxybenzoylamino)-butyl]-amide, and it was given the generic name photobactin. Antibiotic activity was detected with purified photobactin, indicating that the siderophore may contribute to antibiosis of the insect cadaver. These results eliminate the lack of siderophore activity as the cause for the inadequacy of the ngrA mutant in supporting nematode growth and reproduction.     

Screening siderophore producing bacteria as potential biological control agent for fungal rice pathogens in Thailand

Rice (Oryza sativa) is a staple food in Thailand and, in addition, feeds around one half of the world’s population. Therefore, diseases of rice are of special concern. Rice is destroyed by 2 main pathogens, Fusarium oxysporum and Pyricularia oryzae the causative agents of root rot and blast in rice respectively. These pathogens result in low grain yield in Thailand and other Southeast Asian countries. Soil samples were taken from paddy fields in Northern Thailand and bacteria were isolated using the soil dilution plate method on Nutrient agar. Isolation yielded 216 bacterial isolates which were subsequently tested for their siderophore production and effectiveness in inhibiting mycelial growth in vitro of the rice pathogenic fungi; Alternaria sp., Fusarium oxysporum, Pyricularia oryzae and Sclerotium sp., the causal agent of leaf spot, root rot, blast and stem rot in rice. It was found that 23% of the bacteria isolated produced siderophore on solid plating medium and liquid medium, In dual culture technique, the siderophore producing rhizobacteria showed a strong antagonistic effect against the Alternaria (35.4%), Fusarium oxysporum (37.5%), Pyricularia oryzae (31.2%) and Sclerotium sp. (10.4%) strains tested. Streptomyces sp. strain A 130 and Pseudomonas sp. strain MW 2.6 in particular showed a significant higher antagonistic effect against Alternaria sp. while Ochrobactrum anthropi D 5.2 exhibited a good antagonistic effect against F. oxysporum. Bacillus firmus D 4.1 inhibited P. oryzae and Kocuria rhizophila 4(2.1.1) strongly inhibited Sclerotium sp. P. aureofaciens AR 1 was the best siderophore producer overall and secreted hydroxamate type siderophore. This strain exhibits an in vitro antagonistic effect against Alternaria sp., F. oxysporum and P. oryzae. Siderophore production in this isolate was maximal after 15 days and at an optimal temperature of 30°C, yielding 99.96 ± 0.46 μg ml^?1 of siderophore. The most effective isolates were identified by biochemical tests and molecular techniques as members of the Genus Bacillus, Pseudomonas and Kocuria including B. firmus D 4.1, P. aureofaciens AR1 and Kocuria rhizophila 4(2.1.1). The study demonstrated antagonistic activity towards the target pathogens discussed and are thus potential agents for biocontrol of soil borne diseases of rice in Thailand and other countries.     

Response surface optimization of cellulase production from Aneurinibacillus aneurinilyticus BKT-9: An isolate of urban Himalayan freshwater

Due to their vast industrial potential, cellulases have been regarded as the potential biocatalysts by both the academicians and the industrial research groups. In the present study, culturable bacterial strains of Himalayan Urban freshwater lake were investigated for cellulose degrading activities. Initially, a total of 140 bacterial strains were isolated and only 45 isolates were found to possess cellulose degrading property. On the basis of preliminary screening involving cellulase activity assay on CMC agar (with clear zone of hydrolysis) and biosafety assessment testing, only single isolate named as BKT-9 was selected for the cellulase production studies. Strain BKT-9 was characterized at the molecular level using rRNA gene sequencing and its sequence homology analysis revealed its identity as Aneurinibacillus aneurinilyticus. Further, various physico-chemical parameters and culture conditions were optimized using one factor approach to enhance cellulase production levels in the strain BKT-9. Subsequently, RSM based statistical optimization led to formulation of cellulase production medium, wherein the bacterial strain exhibited ~60 folds increase in enzyme activity as compared to un-optimized culture medium. Further studies are being suggested to scale up cellulase production in A. aneurinilyticus strain BKT-9 so that it can be utilized for biomass saccharification at an industrial level.     

Hydroxamate-siderophore production and utilization by marine eubacteria

Siderophore (iron-binding chelator) production was examined in 30 strains of open ocean bacteria from the generaVibrio, Alteromonas, Alcaligenes, Pseudomonas, andPhotobacterium. The results showed that hydroxamate-type siderophore production was widely distributed in various marine species, except for isolates ofAlteromonas macleodii andV. nereis. In all cases, the ability to produce siderophores was under the control of iron levels in the medium and satisfied the iron requirements of the siderophore bioassay organism. On the basis of chemical assay and bacterial bioassays, none of the examined isolates produced phenolate-type siderophores. Several isolates produces siderophores that were neither hydroxamatenor phenolate-type siderophores. Some strains such asAlteromonas communis produce siderophores that could be used by many other isolates. In contrast, the siderophore produced byAlcaligenes venustus had little cross-strain utilization. These findings suggest that the ability to produce siderophores may be common to open ocean bacteria.     

Combat of iron-deprivation through a plant growth promoting fluorescent Pseudomonas strain GRP3A in mung bean (Vigna radiata L. Wilzeck)

Microorganisms and plants sustain themselves under iron-deprived conditions by releasing siderophores. Among others, fluorescent pseudomonads are known to exert extensive biocontrol action against soil and root borne phytopathogens through release of antimicrobials and siderophores. In this study, production and regulation of siderophores by fluorescent Pseudomonas strain GRP3A was studied. Among various media tested, standard succinate medium (SSM) promoted maximum siderophore production of 56.59 mg l(-1). There were low levels of siderophore in complex media like King's B medium, trypticase soya medium and nutrient medium (41.27, 29.86 and 27.63 mg l(-1)), respectively. In defferrated SSM, siderophore level was quantified to be 68.74 mg l(-1). Supplementation with iron (FeCl3) resulted in decreased siderophore levels depending on concentration. Siderophore production was promoted by Zn2+ (78.94 mg l(-1)), Cu2+ (68.80 mg l(-1)) whereas Co2+ (57.33 mg l(-1)) and Fe3+ reduced siderophore production (37.44 mg l(-1) as compared to control (55.97 mg l(-1)). Strain GRP3A showed plant growth promotion under iron limited conditions.     

Marine isolate Citricoccus sp. KMM 3890 as a source of a cyclic siderophore nocardamine with antitumor activity

A novel actinobacterium, designated KMM 3890 was isolated from a bottom sediment sample collected from the Sakhalin shallow environment. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated strain KMM 3890 affiliation to the genus Citricoccus. In addition to its hemolytic activity, this strain exhibited inhibitory activity against Gram-positive bacteria. It was found that the marine isolate Citricoccus sp. KMM 3890 produced and excreted into the culture medium a large amount of the compound, which was isolated and structurally characterized as known cyclic siderophore nocardamine on the basis of combined spectral analyses. Nocardamine showed inhibitory effects to colony formation of T-47D, SK-Mel-5, SK-Mel-28 and PRMI-7951 tumor cell lines and a weak antimicrobial against Gram-positive bacteria and no revealed cytotoxic activity. This study can be considered as the first report on marine isolate of the genus Citricoccus producing nocardamine with antitumor activity.     

Siderophore production by Fusarium venenatum A3/5

Fusarium venenatum A3/5 was grown in iron-restricted batch cultures and iron-limited chemostat cultures to determine how environmental conditions affected siderophore production. The specific growth rate in iron-restricted batch cultures was 0.22 h(-1), which was reduced to 0.12 h(-1) when no iron was added to the culture. D(crit) in iron-limited chemostat culture was 0.1 h(-1). Siderophore production was correlated with specific growth rate, with the highest siderophore production occurring at D=0.08 h(-1) and the lowest at D=0.03 h(-1). Siderophore production was greatest at pH 4.7 and was significantly reduced at pHs above 6.0. Siderophore production could be enhanced by providing insoluble iron instead of soluble iron in continuous flow cultures.