Cryo-EM structures for the Mycobacterium tuberculosis iron-loaded siderophore transporter IrtAB

The adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter, IrtAB, plays a vital role in the replication and viability of Mycobacterium tuberculosis (Mtb), where its function is to import iron-loaded siderophores. Unusually, it adopts the canonical type IV exporter fold. Herein, we report the structure of unliganded Mtb IrtAB and its structure in complex with ATP, ADP, or ATP analogue (AMP-PNP) at resolutions ranging from 2.8 to 3.5 Å. The structure of IrtAB bound ATP-Mg2+ shows a "head-to-tail" dimer of nucleotide-binding domains (NBDs), a closed amphipathic cavity within the transmembrane domains (TMDs), and a metal ion liganded to three histidine residues of IrtA in the cavity. Cryo-electron microscopy (Cryo-EM) structures and ATP hydrolysis assays show that the NBD of IrtA has a higher affinity for nucleotides and increased ATPase activity compared with IrtB. Moreover, the metal ion located in the TM region of IrtA is critical for the stabilization of the conformation of IrtAB during the transport cycle. This study provides a structural basis to explain the ATP-driven conformational changes that occur in IrtAB.

Identification of a novel strain, Streptomyces blastmyceticus JZB130180, and evaluation of its biocontrol efficacy against Monilinia fructicola / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Peach brown rot, caused by Monilinia fructicola, is one of the most serious peach diseases. A strain belonging to the Actinomycetales, named Streptomyces blastmyceticus JZB130180, was found to have a strong inhibitory effect on M. fructicola in confrontation culture. Following the inoculation of peaches in vitro, it was revealed that the fermentation broth of S. blastmyceticus JZB130180 had a significant inhibitory effect on disease development by M. fructicola. The fermentation broth of S. blastmyceticus JZB130180 had an EC50 (concentration for 50% of maximal effect) of 38.3 µg/mL against M. fructicola, as determined in an indoor toxicity test. Analysis of the physicochemical properties of the fermentation broth revealed that it was tolerant of acid and alkaline conditions, temperature, and ultraviolet radiation. In addition, chitinase, cellulase, and protease were also found to be secreted by the strain. The results of this study suggest that S. blastmyceticus JZB130180 may be used for the biocontrol of peach brown rot.

Endophytic Bacillus strains enhance pearl millet growth and nutrient uptake under low-P

Abstract Bacterial endophytes are considered to have a beneficial effect on host plants, improving their growth by different mechanisms. The objective of this study was to investigate the capacity of four endophytic Bacillus strains to solubilize iron phosphate (Fe-P), produce siderophores and indole-acetic acid (IAA) in vitro, and to evaluate their plant growth promotion ability in greenhouse conditions by inoculation into pearl millet cultivated in a P-deficient soils without P fertilization, with Araxá rock phosphate or soluble triple superphosphate. All strains solubilized Fe-P and three of them produced carboxylate-type siderophores and high levels of IAA in the presence of tryptophan. Positive effect of inoculation of some of these strains on shoot and root dry weight and the N P K content of plants cultivated in soil with no P fertilization might result from the synergistic combination of multiple plant growth promoting (PGP) traits. Specifically, while B1923 enhanced shoot and root dry weight and root N P content of plants cultivated with no P added, B2084 and B2088 strains showed positive performance on biomass production and accumulation of N P K in the shoot, indicating that they have higher potential to be microbial biofertilizer candidates for commercial applications in the absence of fertilization.

Promotion of iron nutrition and growth on peanut by Paenibacillus illinoisensis and Bacillus sp. strains in calcareous soil

ABSTRACT This study aimed to explore the effects of two siderophore-producing bacterial strains on iron absorption and plant growth of peanut in calcareous soil. Two siderophore-producing bacterial strains, namely, YZ29 and DZ13, isolated from the rhizosphere soil of peanut, were identified as Paenibacillus illinoisensis and Bacillus sp., respectively. In potted experiments, YZ29 and DZ13 enhanced root activity, chlorophyll and active iron content in leaves, total nitrogen, phosphorus and potassium accumulation of plants and increased the quality of peanut kernels and plant biomass over control. In the field trial, the inoculated treatments performed better than the controls, and the pod yields of the three treatments inoculated with YZ29, DZ13, and YZ29 + DZ13 (1:1) increased by 37.05%, 13.80% and 13.57%, respectively, compared with the control. Based on terminal restriction fragment length polymorphism analysis, YZ29 and DZ13 improved the bacterial community richness and species diversity of soil surrounding the peanut roots. Therefore, YZ29 and DZ13 can be used as candidate bacterial strains to relieve chlorosis of peanut and promote peanut growth. The present study is the first to explore the effect of siderophores produced by P. illinoisensis on iron absorption.

Screening and characterization of endophytic Bacillus and Paenibacillus strains from medicinal plant Lonicera japonica for use as potential plant growth promoters

Abstract A total of 48 endophytic bacteria were isolated from surface-sterilized tissues of the medicinal plant Lonicera japonica, which is grown in eastern China; six strains were selected for further study based on their potential ability to promote plant growth in vitro (siderophore and indoleacetic acid production). The bacteria were characterized by phylogenetically analyzing their 16S rRNA gene similarity, by examining their effect on the mycelial development of pathogenic fungi, by testing their potential plant growth-promoting characteristics, and by measuring wheat growth parameters after inoculation. Results showed that the number of endophytic bacteria in L. japonica varied among different tissues, but it remained relatively stable in the same tissues from four different plantation locations. Among the three endophytic strains, strains 122 and 124 both had high siderophore production, with the latter showing the highest phosphate solubilization activity (45.6 mg/L) and aminocyclopropane-1-carboxylic acid deaminase activity (47.3 nmol/mg/h). Strain 170 had the highest indoleacetic acid (IAA) production (49.2 mg/L) and cellulase and pectinase activities. After inoculation, most of the six selected isolates showed a strong capacity to promote wheat growth. Compared with the controls, the increase in the shoot length, root length, fresh weight, dry weight, and chlorophyll content was most remarkable in wheat seedlings inoculated with strain 130. The positive correlation between enzyme (cellulose and pectinase) activity and inhibition rate on Fusarium oxysporum, the IAA production, and the root length of wheat seedlings inoculated with each tested endophytic strain was significant in regression analysis. Deformity of pathogenic fungal mycelia was observed under a microscope after the interaction with the endophytic isolates. Such deformity may be directly related to the production of hydrolytic bacterial enzymes (cellulose and pectinase). The six endophytic bacterial strains were identified to be Paenibacillus and Bacillus strains based on the results of 16S rRNA gene sequencing analysis and their physiological and biochemical characteristics. Results indicate the promising application of endophytic bacteria to the biological control of pathogenic fungi and the improvement of wheat crop growth.

Stress tolerant virulent strains of Cronobacter sakazakii from food

BACKGROUND: Cronobacter sakazakii is considered as an emerging foodborne pathogen. The aim of this study was to isolate and characterize virulent strains of Cronobacter sakazakii from food samples of Bangladesh. RESULT: Six (6) Cronobacter sakazakii was isolated and identified from 54 food samples on the basis of biochemical characteristics, sugar fermentation, SDS-PAGE of whole cell protein, plasmid profile and PCR of Cronobacter spp. specific genes (esak, gluA, zpx, ompA, ERIC, BOX-AIR) and sequencing. These strains were found to have moderately high antibiotic resistance against common antibiotics and some are ESBL producer. Most of the C. sakazakii isolates were capable of producing biofilm (strong biofilm producer), extracellular protease and siderophores, curli expression, haemolysin, haemagglutinin, mannose resistant haemagglutinin, had high cell surface hydrophobicity, significant resistance to human serum, can tolerate high concentration of salt, bile and DNase production. Most of them produced enterotoxins of different molecular weight. The isolates pose significant serological cross-reactivity with other gram negative pathogens such as serotypes of Salmonella spp., Shigella boydii, Shigella sonnei, Shigella flexneri and Vibrio cholerae. They had significant tolerance to high temperature, low pH, dryness and osmotic stress. CONCLUSION: Special attention should be given in ensuring hygiene in production and post-processing to prevent contamination of food with such stress-tolerant virulent Cronobacter sakazakii.

Colonization and plant growth promoting characterization of endophytic Pseudomonas chlororaphis strain Zong1 isolated from Sophora alopecuroides root nodules

The endophytic strain Zong1 isolated from root nodules of the legume Sophora alopecuroides was characterized by conducting physiological and biochemical tests employing gfp-marking, observing their plant growth promoting characteristics (PGPC) and detecting plant growth parameters of inoculation assays under greenhouse conditions. Results showed that strain Zong1 had an effective growth at 28 ºC after placed at 4-60 ºC for 15 min, had a wide range pH tolerance of 6.0-11.0 and salt tolerance up to 5% of NaCl. Zong1 was resistant to the following antibiotics (µg/mL): Phosphonomycin (100), Penicillin (100) and Ampicillin (100). It could grow in the medium supplemented with 1.2 mmol/L Cu, 0.1% (w/v) methylene blue and 0.1-0.2% (w/v) methyl red, respectively. Zong1 is closely related to Pseudomonas chlororaphis based on analysis the sequence of 16S rRNA gene. Its expression of the gfp gene indicated that strain Zong1 may colonize in root or root nodules and verified by microscopic observation. Furthermore, co-inoculation with Zong1 and SQ1 (Mesorhizobium sp.) showed significant effects compared to single inoculation for the following PGPC parameters: siderophore production, phosphate solubilization, organic acid production, IAA production and antifungal activity in vitro. These results suggest strains P. chlororaphi Zong1 and Mesorhizobium sp. SQ1 have better synergistic or addictive effect. It was noteworthy that each growth index of co-inoculated Zong1+SQ1 in growth assays under greenhouse conditions is higher than those of single inoculation, and showed a significant difference (p < 0.05) when compared to a negative control. Therefore, as an endophyte P. chlororaphis Zong1 may play important roles as a potential plantgrowth promoting agent.

Growth of Brassica juncea under chromium stress: influence of siderophores and indole 3 acetic acid producing rhizosphere bacteria.

Plant growth promoting rhizobacterial (PGPR) strains A3 and S32 have been shown to promote the growth of Brassica juncea under chromium stress which has been related to the microbial production of siderophores and indole 3 acetic acid (IAA). The aim of the present study is to evaluate the importance of siderophores and IAA producing PGPR on the growth of Brassica juncea under chromium stress. The production of IAA and siderophores were observed in the strains A3 and S32, respectively. Both PGPR strains promote the growth of Brassica juncea under chromium stress. The maximum growth was observed in plants inoculated with siderophores producing strain 32. Both the bacterial inoculum did not influence the uptake of chromium by plants. The present observation showed that PGPR isolates A3 and S32 are capable of protecting the plants against the inhibitory effects of chromium by producing the siderophores and IAA.

Chemical properties and NMR spectroscopic identification of certain fungal siderophores.

Siderophores of six fungi viz. Aspergillus sp. ABp4, Aureobacidium pullulans, Penicillium oxalicum, P. chrysosporium, Mycotypha africana and Syncephalastrum racemosum were examined for their (1) electrophoretic mobilities to determine the acidic, basic or neutral charge; (2) Fe (III) binding nature viz., mono-, di-, or trihydroxamate; (3) amino acid composition; and (4) NMR (nuclear magnetic resonance) spectroscopy to determine their structure. Electrophoretic mobilities of siderophores of 3 fungi (P. oxalicum, P. chrysosporium, and M, africana) exhibited net basic charge, siderophores of 2 fungi (Aspergillus sp. ABp4 and S. racemosum) were acidic and 1 fungus (A. pullullans) was neutral. Electrophoresis of ferrated siderophore at pH 2 and colour of the spots indicated that siderophores of Aspergillus sp. ABp4 and P. oxalicum and A. pullulans were trihydroxamates, whereas siderophore of P. chrysosporium was dihydroxamate. Amino acid composition of siderophores purified by XAD-2 column chromatography, revealed the presence of asparagine, histidine, and proline in Aspergillus sp. ABp4, serine and alanine in P. chrysosporium, and valine in M. africana. The structure of purified siderophores as revealed by NMR spectroscopy identified siderophore of AB - 2670 (A. pullulans) as asperchrome F1, and AB-513 (M. africana) as rhizoferrin. The peak obtained for siderophore AB-5 (Aspergillus sp. ABp4) did not show resemblance to any known siderophore, therefore may be an exception.

Ferric reductase, superoxide dismutase and alkaline phosphatase activities in siderophore producing fungi.

Enzymes associated with release of iron from internalized ferrated siderophore (ferrisiderophore reductase), with damage to the cell at high iron concentration (superoxide dismutase) and siderophore synthesis (alkaline phosphatase), were examined in 3 test fungi viz., Aspergillus sp. ABp4, Aureobasidium pullulans and Rhizopus sp. Extracellular ferrisiderophore reductase activity was present in all the three fungi, but Aureobasidium pullulans, that showed the highest activity (84.3 microM min(-1)), was the only one to produce intra-cellular ferric reductase (147.9 microM min(-1)). Superoxide dismutase was produced by Aureobasidium pullulans and Rhizopus sp., but not by Aspergillus sp. ABp4, that showed intra-cellular enzyme activity in case of ferric reductase and alkaline phosphatase. Maximum SOD activity was seen in Aureobasidium pullulans both extra-cellularly (93.83 ng ml(-1)) and intra-cellularly (57.14 ng ml(-1)). All the test fungi examined, produced intra-cellular alkaline phosphatase. There was no extracellular alkaline phosphatase. Among the three fungi, Aureobasidium pullulans showed highest alkaline phosphatase activity (129.9 microM min(-1)) and Aspergillus sp. ABp4 the least (76.4 microM min(-1)).

Rhizobia as a biological control agent against soil borne plant pathogenic fungi.

Rhizobia promote the growth of plants either directly through N2 fixation, supply of nutrients, synthesis of phytohormones and solubilization of minerals, or indirectly as a biocontrol agent by inhibiting the growth of pathogens. The biocontrol effect of rhizobia is due to the secretion of secondary metabolites such as antibiotics and HCN. Siderophore production in iron stress conditions provides rhizobia an added advantage, resulting in exclusion of pathogens due to iron starvation.

Growth and Siderophore Production of Staphylococci in Human Peritoneal Dialysate

Although activity of iron uptake system (IUS) was thought to play an important role in staphylococcal growth in human peritoneal dialysate (HPD) solution, siderophore production, one of the well-known IUS, was not yet detected directly in HPD solution. Therefore, we tried to detect siderophore production directly in HPD solution by using a newly developed chrome azurol S (CAS) agar diffusion assay and to investigate the effect of IUS activity on bacterial growth in HPD solution. According to the susceptibility test for streptonigrin and the productivity of siderophore in the iron-deficient (ID) medium, Staphylococcus aureus ATCC 6538 strain and Staphylococcus epidermidis clinical isolate had higher IUS activity and grew better than S. aureus ATCC 25923 strain in the ID medium. These bacteria did not grow and produce siderophore in the unused chronic ambulatory peritoneal dialysis solution. However, these bacteria grew and produced siderophore in the HPD solution. Moreover, S. aureus ATCC 25923 strain with lower activity of IUS grew poorly and produced smaller amount of siderophore in HPD compared to S. aureus ATCC 6538 strain and S. epidermidis clinical isolate with higher activity of IUS like in the ID medium. To the best of our knowledge, this is the first report that sidero-phore production is directly detected in the HPD by CAS agar diffusion assay. These results indicated that activity of IUS plays an important role in bacterial growth in the HPD solution and pathogenesis of continuous ambulatory peritoneal dialysis peritonitis.