Journal of Microbiology

Journal Articles

Tn5 Transposon-based Mutagenesis for Engineering Phage-resistant Strains of Escherichia coli BL21 (DE3): Yinfeng Wang , Guanhua Xuan , Houqi Ning , Jiuna Kong , Hong Lin , Jingxue Wang; J. Microbiol. 2023;61(5):559-569. Published online May 22, 2023; DOI: https://doi.org/10.1007/s12275-023-00048-2

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Abstract: Escherichia coli is a preferred strain for recombinant protein production, however, it is often plagued by phage infection during experimental studies and industrial fermentation. While the existing methods of obtaining phage-resistant strains by natural mutation are not efficient enough and time-consuming. Herein, a high-throughput method by combining Tn5 transposon mutation and phage screening was used to produce Escherichia coli BL21 (DE3) phage-resistant strains. Mutant strains PR281-7, PR338-8, PR339-3, PR340-8, and PR347-9 were obtained, and they could effectively resist phage infection. Meanwhile, they had good growth ability, did not contain pseudolysogenic strains, and were controllable. The resultant phage-resistant strains maintained the capabilities of producing recombinant proteins since no difference in mCherry red fluorescent protein expression was found in phage-resistant strains. Comparative genomics showed that PR281-7, PR338-8, PR339-3, and PR340-8 mutated in ecpE, nohD, nrdR, and livM genes, respectively. In this work, a strategy was successfully developed to obtain phage-resistant strains with excellent protein expression characteristics by Tn5 transposon mutation. This study provides a new reference to solve the phage contamination problem.

Devosia rhizoryzae sp. nov., and Devosia oryziradicis sp. nov., novel plant growth promoting members of the genus Devosia, isolated from the rhizosphere of rice plants: Geeta Chhetri , Inhyup Kim , Minchung Kang , Jiyoun Kim , Yoonseop So , Taegun Seo; J. Microbiol. 2022;60(1):1-10. Published online November 26, 2021; DOI: https://doi.org/10.1007/s12275-022-1474-8

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Two novel Gram-negative, aerobic, asporogenous, motile, rodshaped, orange and white pigmented, designated as LEGU1T and G19T, were isolated from the roots of rice plants, collected from Goyang, South Korea. Phylogenetic analysis based on their 16S rRNA gene sequences revealed that they belonged to the genus Devosia and formed a different lineage and clusters with different members of the genus Devosia. These strains shared common chemotaxonomic features. In particular, they had Q-10 as the sole quinone, phosphatidylglycerol, diphosphatidylglycerol as the principal polar lipids and C16:0, C18:1 ω7c 11-methyl and summed feature 8 (comprising C18:1 ω7c/ C18:1 ω6c) as the main fatty acids. The draft genome sequences of strains LEGU1T and G19T were 3,524,978 and 3,495,520 bp in size, respectively. Their average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were 72.8–81.9% and 18.7–25.1%, respectively, with each other and type strains of related species belonging to the genus Devosia, suggesting that these two strains represent novel species. The G + C content of strains LEGU1T and G19T were 62.1 and 63.8%, respectively. Of the two strains, only LEGU1T produced carotenoid and flexirubin-type pigment. Both strains produced siderophore and indole acetic acid (IAA) in the presence of L-tryptophan. Siderophore biosynthesis genes, auxin responsive genes and tryptophan biosynthesis genes were present in their genomes. The present study aimed to determine the detailed taxonomic positions of the strains using the modern polyphasic approach. Based on the results of polyphasic analysis, these strains are suggested to be two novel bacterial species within the genus Devosia. The proposed names are D. rhizoryzae sp. nov., and Devosia oryziradicis sp. nov., respectively. The plant growth promoting effects of these strains suggest that they can be exploited to improve rice crop productivity. The type strain of D. rhizoryzae is LEGU1T (KCTC 82712T = NBRC 114485T) and D. oryziradicis is G19T (KCTC 82688T = NBRC 114842T).

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Review

[MINIREVIEW] Interdependence between iron acquisition and biofilm formation in Pseudomonas aeruginosa: Donghoon Kang , Natalia V. Kirienko; J. Microbiol. 2018;56(7):449-457. Published online June 14, 2018; DOI: https://doi.org/10.1007/s12275-018-8114-3

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Abstract

Bacterial biofilms remain a persistent threat to human healthcare due to their role in the development of antimicrobial resistance. To combat multi-drug resistant pathogens, it is crucial to enhance our understanding of not only the regulation of biofilm formation, but also its contribution to bacterial virulence. Iron acquisition lies at the crux of these two subjects. In this review, we discuss the role of iron acquisition in biofilm formation and how hosts impede this mechanism to defend against pathogens. We also discuss recent findings that suggest that biofilm formation can also have the reciprocal effect, influencing siderophore production and iron sequestration.

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Journal Articles

Characterization of siderophore produced by Pseudomonas syringae BAF.1 and its inhibitory effects on spore germination and mycelium morphology of Fusarium oxysporum: Sumei Yu , Chunying Teng , Jinsong Liang , Tao Song , Liying Dong , Xin Bai , Yu Jin , Juanjuan Qu; J. Microbiol. 2017;55(11):877-884. Published online October 27, 2017; DOI: https://doi.org/10.1007/s12275-017-7191-z

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Abstract

In this study, an antagonistic bacterium against Fusarium oxysporum was identified and designated as Pseudomonas syringae strain BAF.1 on the basis of 16S rDNA sequence analysis and physiological-biochemical characteristics. It produced catechol-species siderophore at a molecular weight of 488.59 Da and a maximum amount of 55.27 μg/ml with glucose as a carbon source and asparagine as a nitrogen source at a C/N ratio of 10:1, 30°C and pH 7. The siderophore exhibited prominent antagonistic activity against Fusarium oxysporum with a maximum inhibition rate of 95.24% and had also suppressive effects on other kinds of 11 phytopathogenic fungi in the absence of FeCl3·6H2O. Spore germination was completely inhibited by 50 μl of the siderophorecontaining solution, and the ultrastructures of mycelia and spores were also considerably suppressed by siderophore treatment as established by electron microscopy observation. These results indicate that the siderophore produced by Pseudomonas syringae BAF.1 could be potentially used for biocontrol of pathogenic Fusarium oxysporum.

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An in vitro study of the antifungal activity of Trichoderma virens 7b and a profile of its non-polar antifungal components released against Ganoderma boninense: Lee Pei Lee Angel , Mohd Termizi Yusof , Intan Safinar Ismail , Bonnie Tay Yen Ping , Intan Nur Ainni Mohamed Azni , Norman Hj Kamarudin , Shamala Sundram; J. Microbiol. 2016;54(11):732-744. Published online October 29, 2016; DOI: https://doi.org/10.1007/s12275-016-6304-4

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Abstract

Ganoderma boninense is the causal agent of a devastating disease affecting oil palm in Southeast Asian countries. Basal stem rot (BSR) disease slowly rots the base of palms, which radically reduces productive lifespan of this lucrative crop. Previous reports have indicated the successful use of Trichoderma as biological control agent (BCA) against G. boninense and isolate T. virens 7b was selected based on its initial screening. This study attempts to decipher the mechanisms responsible for the inhibition of G. boninense by identifying and characterizing the chemical compounds as well as the physical mechanisms by T. virens 7b. Hexane extract of the isolate gave 62.60% ± 6.41 inhibition against G. boninense and observation under scanning electron microscope (SEM) detected severe mycelial deformation of the pathogen at the region of inhibition. Similar mycelia deformation of G. boninense was observed with a fungicide treatment, Benlate® indicating comparable fungicidal effect by T. virens 7b. Fraction 4 and 5 of hexane active fractions through preparative thin layer chromatography (P-TLC) was identified giving the best inhibition of the pathogen. These fractions comprised of ketones, alcohols, aldehydes, lactones, sesquiterpenes, monoterpenes, sulphides, and free fatty acids profiled through gas chromatography mass spectrometry detector (GC/MSD). A novel antifungal compound discovery of phenylethyl alcohol (PEA) by T. virens 7b is reported through this study. T. virens 7b also proved to be an active siderophore producer through chrome azurol S (CAS) agar assay. The study demonstrated the possible mechanisms involved and responsible in the successful inhibition of G. boninense.

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The Pseudomonas aeruginosa extracellular secondary metabolite, Paerucumarin, chelates iron and is not localized to extracellular membrane vesicles: Uzma Qaisar , Cassandra J. Kruczek , Muhammed Azeem , Nasir Javaid , Jane A. Colmer-Hamood , Abdul N. Hamood; J. Microbiol. 2016;54(8):573-581. Published online August 2, 2016; DOI: https://doi.org/10.1007/s12275-016-5645-3

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Abstract

Proteins encoded by the Pseudomonas aeruginosa pvcA-D operon synthesize a novel isonitrile functionalized cumarin termed paerucumarin. The pvcA-D operon enhances the expression of the P. aeruginosa fimbrial chaperone/usher pathway (cup) genes and this effect is mediated through paerucumarin. Whether pvcA-D and/or paerucumarin affect the expression of other P. aeruginosa genes is not known. In this study, we examined the effect of a mutation in pvcA-D operon the global transcriptome of the P. aeruginosa strain PAO1- UW. The mutation reduced the expression of several ironcontrolled genes including pvdS, which is essential for the expression of the pyoverdine genes. Additional transcriptional studies showed that the pvcA-D operon is not regulated by iron. Exogenously added paerucumarin enhanced pyoverdine production and pvdS expression in PAO1-UW. Iron-chelation experiments revealed that purified paerucumarin chelates iron. However, exogenously added paerucumarin significantly reduced the growth of a P. aeruginosa mutant defective in pyoverdine and pyochelin production. In contrast to other secondary metabolite, Pseudomonas quinolone signal (PQS), paerucumarin is not localized to the P. aeruginosa membrane vesicles. These results suggest that paerucumarin enhances the expression of iron-controlled genes by chelating iron within the P. aeruginosa extracellular environment. Although paerucumarin chelates iron, it does not function as a siderophore. Unlike PQS, paerucumarin is not associated with the P. aeruginosa cell envelope.

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Role of Heavy Metal Resistant Ochrobactrum sp. and Bacillus spp. Strains in Bioremediation of a Rice Cultivar and Their PGPR Like Activities: Sanjeev Pandey , Pallab Kumar Ghosh , Sisir Ghosh , Tarun Kumar De , Tushar Kanti Maiti; J. Microbiol. 2013;51(1):11-17. Published online March 2, 2013; DOI: https://doi.org/10.1007/s12275-013-2330-7

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Abstract: The present study demonstrates the metal toxicity ameliorating and growth promoting abilities of three different bacterial isolates when applied to rice as host plant. The three bacterial strains included a cadmium resistant Ochrobactrum sp., a lead resistant Bacillus sp. and an arsenic resistant Bacillus sp. designated as CdSP9, PbSP6, and AsSP9, respectively. When these isolates were used as inocula applied to metaltreated rice plants of variety Satabdi, the germination percentage, relative root elongation (RRE), amylase and protease activities were increased. The toxic effect of metal was reduced in presence of these bacteria. The overall biomass and root/shoot ratio were also enhanced by bacterial inoculation. Hydroponic studies showed that the superoxide dismutase (SOD) activity and malondialdehyde (MDA) level, which had been increased in the presence of metal stress in rice roots, were lowered by the bacterial inoculation. In addition, all three strains were 1-aminocyclopropane-1-carboxylate (ACC) deaminase and catalase positive, whereas siderophore producing ability was lacking in PbSP6. However, both PbSP6 and AsSP9 were protease positive and could hydrolyse starch. The data indicate that these bacteria have promise for bioremediation as well as for plant growth promotion.

The Role of a Dark Septate Endophytic Fungus, Veronaeopsis simplex Y34, in Fusarium Disease Suppression in Chinese Cabbage: Rida O. Khastini , Hiroyuki Ohta , Kazuhiko Narisawa; J. Microbiol. 2012;50(4):618-624. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2105-6

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Abstract: The soil-inhabiting fungal pathogen Fusarium oxysporum has been an increasing threat to Chinese cabbage (Brassica campestris L.). A dark septate endophytic fungus, Veronaeopsis simplex Y34, isolated from Yaku Island, Japan, was evaluated in vitro for the ability to suppress Fusarium disease. Seedlings grown in the presence of the endophyte showed a 71% reduction in Fusarium wilt disease and still had good growth. The disease control was achieved through a synergetic effect involving a mechanical resistance created by a dense network of V. simplex Y34 hyphae, which colonized the host root, and siderophore production acting indirectly to induce a resistance mechanism in the plant. Changes in the relative abundance of the fungal communities in the soil as determined by fluorescently labelled T-RFs (terminal restriction fragments), appeared 3 weeks after application of the fungus. Results showed the dominance of V. simplex Y34, which became established in the rhizosphere and out-competed F. oxysporum.

Cyclic AMP-Receptor Protein Activates Aerobactin Receptor IutA Expression in Vibrio vulnificus: Choon-Mee Kim , Seong-Jung Kim , Sung-Heui Shin; J. Microbiol. 2012;50(2):320-325. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-2056-y

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Abstract: The ferrophilic bacterium Vibrio vulnificus can utilize the siderophore aerobactin of Escherichia coli for iron acquisition via its specific receptor IutA. This siderophore piracy by V. vulnificus may contribute to its survival and proliferation, especially in mixed bacterial environments. In this study, we examined the effects of glucose, cyclic AMP (cAMP), and cAMP-receptor protein (Crp) on iutA expression in V. vulnificus. Glucose dose-dependently repressed iutA expression. A mutation in cya encoding adenylate cyclase required for cAMP synthesis severely repressed iutA expression, and this change was recovered by in trans complementing cya or the addition of exogenous cAMP. Furthermore, a mutation in crp encoding Crp severely repressed iutA expression, and this change was recovered by complementing crp. Accordingly, glucose deprivation under iron-limited conditions is an environmental signal for iutA expression, and Crp functions as an activator that regulates iutA expression in response to glucose availability.

Iron Homeostasis in Brucella abortus: the Role of Bacterioferritin: Marta A. Almirón , Rodolfo A. Ugalde; J. Microbiol. 2010;48(5):668-673. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0145-3

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Abstract: Brucella abortus is the etiological agent of bovine brucellosis, an infectious disease of humans and cattle. Its pathogenesis is mainly based on its ability to survive and multiply inside macrophages. It has been demonstrated that if B. abortus ferrochelatase cannot incorporate iron into protoporphyrin IX to synthesize heme, the intracellular replication and virulence in mice is highly attenuated. Therefore, it can be hypothesized that the unavailability of iron could lead to the same attenuation in B. abortus pathogenicity. Thus, the purpose of this work was to obtain a B. abortus derivative unable to keep an internal iron pool and test its ability to replicate under iron limitation. To achieve this, we searched for iron-storage proteins in the genome of brucellae and found bacterioferritin (Bfr) as the sole ferritin encoded. Then, a B. abortus bfr mutant was built up and its capacity to store iron and replicate under iron limitation was investigated. Results indicated that B. abortus Bfr accounts for 70% of the intracellular iron content. Under iron limitation, the bfr mutant suffered from enhanced iron restriction with respect to wild type according to its growth retardation pattern, enhanced sensitivity to oxidative stress, accelerated production of siderophores, and altered expression of membrane proteins. Nonetheless, the bfr mutant was able to adapt and replicate even inside eukaryotic cells, indicating that B. abortus responds to internal iron starvation before sensing external iron availability. This suggests an active role of Bfr in controlling iron homeostasis through the availability of Bfr-bound iron.

Comparison of the Bacterial Community and Characterization of Plant Growth-Promoting Rhizobacteria from Different Genotypes of Chrysopogon zizanioides (L.) Roberty (Vetiver) Rhizospheres: Juliana Mendes Monteiro , Renata Estebanez Vollu , Marcia Reed Rodrigues Coelho , Celuta Sales Alviano , Arie Fitzgerald Blank , Lucy Seldin; J. Microbiol. 2009;47(4):363-370. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0048-3

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Abstract: Molecular approaches [PCR-denaturing gradient gel electrophoresis (DGGE)] were used to determine whether three different vetiver (Chrysopogon zizanioides) genotypes, commercially used in Brazil and considered economically important over the world, select specific bacterial populations to coexist in their rhizospheres. DGGE profiles revealed that the predominant rhizospheric bacterial community hardly varies regarding the vetiver genotype. Moreover, using traditional cultivation methods, bacterial strains were isolated from the different rhizospheres. Colonies presenting different morphologies (83) were selected for determining their potential for plant growth promotion. More than half of the strains tested (57.8%) were amplified by PCR using nifH-based primers, specific for the enzyme nitrogenase reductase. The production of siderophores was observed in 88% of the strains, while the production of antimicrobial substances was detected in only 14.5% of the isolates when Micrococcus sp. was used as the indicator strain. Production of indole-3-acetic acid and the solubilization of phosphate were observed in 55.4% and 59% of the isolates, respectively. In total, 44 strains (53%) presented at least three characteristics of plant growth promotion and were submitted to amplified ribosomal DNA restriction analysis. Twenty-four genetic groups were formed at 100% similarity and one representative of each group was selected for their identification by partial 16S rRNA gene sequencing. They were affiliated with the genera Acinetobacter, Comamonas, Chryseobacterium, Klebsiella, Enterobacter, Pantoea, Dyella, Burkholderia, or Pseudomonas. These strains can be considered of great importance as possible biofertilizers in vetiver.

Staphylococcus aureus Siderophore-Mediated Iron-Acquisition System Plays a Dominant and Essential Role in the Utilization of Transferrin-Bound Iron: Ra-Young Park , Hui-Yu Sun , Mi-Hwa Choi , Young-Hoon Bai , Sung-Heui Shin; J. Microbiol. 2005;43(2):183-190.; DOI: https://doi.org/2163 [pii]

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Abstract: Staphylococcus aureus is known to be capable of utilizing transferrin-bound iron, via both siderophore- and transferrin-binding protein (named IsdA)-mediated iron-acquisition systems. This study was designed in order to determine which iron-acquisition system plays the essential or dominant role with respect to the acquisition of iron from human transferrin, in the growth of S. aureus. Holotransferrin (HT) and partially iron-saturated transferrin (PT), but not apotransferrin (AT), were found to stimulate the growth of S. aureus. S. aureus consumed most of the transferrin-bound iron during the exponential growth phase. Extracellular proteases were not, however, involved in the liberation of iron from transferrin. Transferrin-binding to the washed whole cells via IsdA was not observed during the culture. The expression of IsdA was observed only in the deferrated media with AT, but not in the media supplemented with PT or HT. In contrast, siderophores were definitely produced in the deferrated media with PT and HT, as well as in the media supplemented with AT. The siderophores proved to have the ability to remove iron directly from transferrin, but the washed whole cells expressing IsdA did not. In the bioassay, the growth of S. aureus on transferrin-bound iron was stimulated by the siderophores alone. These results demonstrate that the siderophore-mediated iron-acquisition system plays a dominant and essential role in the uptake of iron from transferrin, whereas the IsdA-mediated iron-acquisition system may play only an ancillary role in the uptake of iron from transferrin.

Growth of Staphylococcus aureus with Defective Siderophore Production in Human Peritoneal Dialysate Solution: Ra-Young Park , Hui-Yu Sun , Mi-Hwa Choi , Young-Hoon Bae , Sung-Heui Shin; J. Microbiol. 2005;43(1):54-61.; DOI: https://doi.org/2137 [pii]

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Abstract: In this study, we attempted to determine the effects of iron-availability and the activity of the bacterial iron-uptake system (IUS) on the growth of Staphylococcus aureus in human peritoneal dialysate (HPD) solution. A streptonigrin-resistant S. aureus (SRSA) strain, isolated from S. aureus ATCC 6538, exhibited defective siderophore production, thereby resulting in ineffective uptake of iron from low iron-saturated transferrin. The growth of both strains was stimulated in HPD solution supplemented with FeCl_3 and holotransferrin, but growth was inhibited in HPD solution which had been supplemented with apotransferrin and dipyridyl. The SRSA strain grew less robustly than did its parental strain in both iron-supplemented HPD solution and regular HPD solution. These results indicate that iron-availability and siderophore-mediated IUS activity in particular, the ability to produce siderophores and thus capture iron from low iron-saturated transferrin play critical roles in the growth of S. aureus in HPD solution. Our results also indicated that the possibility of using iron chelators as therapeutic or preventive agents warrants further evaluation.

cloning of Gene Encoding for Siderophore biosynthesis in Fluorescent Pseudomonas sp.: Koh, Han Cheol , Ha, Sung Cheol , Na, Jung A , Kim, Ho Sang , Yeo, Myeong Gu , Lee, Jung Sup , Kim, Sung Jun , Park, yeal; J. Microbiol. 1995;33(1):28-33.

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Abstract: Pseudomonas sp. strain PY002, isolated from soil, was mutagenized with a transposon Tn5(21). To screening of siderophore biosunthesis defective mutant, 138 kanamycin resistant mutants were tested of growth on MKB medium supplemented with iron chelator(bipydidyl and EDDHA) and in vitro antibiosis. Among 138 mutants, 32 mutants do not excreted a siderophore and lose their antibiotic activity. So, these mutants were designated Flu^-Sid^-. A gene bank of DNA from Pseudomonas sp. strain PY002 was constructed using the broad-host range conjugative cosmid pLAFR3. The recombinant cosmids contained insert DNA averaging 21 kb in length and the frequence of transduction into E. coli HB101 per 1㎍ of insert DNA was 9 × 10³. Nonfluorescent mutants were complemented by mating the gene bank en masse and identifying the 108 fluorescent transconjugants. Restriction enzyme analysis of these complemented transconjugants revealed three different types and they were named pCOM61, pCOM91 and pCOM97. Sizes of their insert DNA were 30kb, 26kb and 28kb, respectively.

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