Journal of Microbiology

Volume 48(1); February 2010

Research Support, Non-U.S. Gov'ts

Endophytic Fungi Diversity of Aquatic/Riparian Plants and Their Antifungal Activity In Vitro: Hai-Yan Li , Chun-An Zhao , Chen-Jian Liu , Xiao-Fei Xu; J. Microbiol. 2010;48(1):1-6. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-009-0163-1

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Two hundred and fourteen endophytic fungi were isolated from 500 segments of aquatic/riparian plants Ottelia acuminata, Myriophyllum verticillatum, Equisetum arvense, Cardamine multijuga, and Impatiens chinensis. They were identified to 31 taxa in which Cladosporium, Fusarium, and Geotrichum were the dominant genera. Among all isolates, 169 (79%) were anamorphic fungi, 1 (0.5%) was an teleomorphic ascomycete and 44 (21%) were sterile mycelia. There were significant differences in the colonization frequency of endophytes between the five plant species (X~2=51.128, P<0.001, Chi-square test). The riparian plants harboured more endophytes than the submerged plants. The antifungal activity of these isolates against Fusarium solani and Phytophthora nicotianae in vitro were tested and 28 (13.1%) isolates showed antifungal activities with more than 30% growth inhibition rate against the two pathogens.

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Microbial Community Response to a Simulated Hydrocarbon Spill in Mangrove Sediments: Rodrigo Gouvêa Taketani , Natália Oliveira Franco , Alexandre Soares Rosado , Jan Dirk van Elsas; J. Microbiol. 2010;48(1):7-15. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-009-0147-1

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Abstract: In this study, we examined the hypothesis that the microbial communities in mangrove sediments with different chemical and historical characteristics respond differently to the disturbance of a hydrocarbon spill. Two different mangrove sediments were sampled, one close to an oil refinery that had suffered a recent oil spill and another that had not been in contact with oil. Based on the sampled sediment, two sets of mesocosms were built, and oil was added to one of them. They were subjected to mimicked mangrove conditions and monitored for 75 days. Archaeal and bacterial communities were evaluated through PCRDGGE. Both communities showed the emergence of small numbers of novel bands in response to oil pollution. 16S rRNA gene clone libraries were constructed from both mesocosms before the addition of oil and at day 75 after oil addition. LIBSHUFF analysis showed that both mangrove-based mesocosms contained similar communities at the start of the experiment and that they were different from the initial one, as well as from each other, after 75 days. These results hint at a role of environmental history that is not obvious from community diversity indicators, but is apparent from the response to the applied stress.

Isolation and Characterization of Marine Pigmented Bacteria from Norwegian Coastal Waters and Screening for Carotenoids with UVA-Blue Light Absorbing Properties: Marit H. Stafsnes , Kjell D Josefsen , Geir Kildahl-Andersen , Svein Valla , Trond E. Ellingsen , Per Bruheim; J. Microbiol. 2010;48(1):16-23. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-009-0118-6

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Abstract: Microbial culture collections are important resources for isolation of natural compounds with novel properties. In this study, a culture collection of around 1,500 pigmented heterotrophic bacteria was established. The bacteria were isolated from the sea surface microlayer at different sampling sites along the mid-part of the Norwegian coast. The bacterial isolates produced pigments of various coloration (e.g. golden, yellow, red, pink and orange). Methanol extracts of sixteen isolates were characterized with LC-Diodearray-TOF mass spectrometry analysis. The number of pigments per isolate varied considerably, and a tentative identification of the pigments was performed based on UV-absorbance profile and molecular formula assignation based on the accurate mass determination. The LC-MS analyses evealed that most of the pigments probably were carotenoids. Furthermore, we developed a high throughput LC-MS method for characterization and screening of a larger sub-fraction (300 isolates) of the culture collection. The aim was to screen and identify bacterial isolates producing carotenoids that absorb light in the UVA-Blue light. Six of the bacterial strains were selected for detailed investigation, including 16s rRNA sequencing, preparative HPLC for purification of major carotenoids and subsequent structural elucidation with NMR. Among the identified carotenoids were zeaxanthin, nostoxanthin and sarcinaxanthin, some with novel glycosylation patterns.

Expression of Recombinant Hybrid Peptide Hinnavin II/α-Melanocyte-Stimulating Hormone in Escherichia coli: Purification and Characterization: Son Kwon Bang , Chang Soo Kang , Man-Deuk Han , In Seok Bang; J. Microbiol. 2010;48(1):24-29. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-009-0317-1

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Abstract

The increasing problem of antibiotic resistance among pathogenic bacteria requires novel strategies for the construction of multiple, joined genes of antimicrobial agents. The strategy used in this study involved synthesis of a cDNA-encoding hinnavin II/α-melanocyte-stimulating hormone (hin/MSH) hybrid peptide, which was cloned into the pET32a (+) vector to allow expression of the hybrid peptide as a fusion protein in Escherichia coli BL21 (DE3). The resulting expression of fusion protein Trx-hin/MSH could reach up to 20% of the total cell proteins. More than 50% of the target protein was in a soluble form. The target fusion protein from the soluble fraction, Trx-hin/MSH, was easily purified by Ni2+-chelating chromatography. Then, enterokinase cleavage effectively cleaved the Trx-hin/MSH to release the combinant hin/MSH (rhin/MSH) hybrid peptide. After removing the contaminants, we purified the recombinant hybrid peptide to homogeneity by reversed-phase FPLC and obtained 210 mg of pure, active rhin/MSH from 800 ml of culture medium. Antimicrobial activity assay demonstrated that rhin/MSH had a broader spectrum of activity than did the parental hinnavin II or MSH against fungi and Gram-positive and Gram-negative bacteria. These results suggest an efficient method for producing high-level expression of various kinds of antimicrobial peptides that are toxic to the host, a reliable and simple method for producing different hybrid peptides for biological studies.

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Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds
Yunqi Ma, Chang-Joo Lee, Jang-Su Park
Antibiotics.2020; 9(9): 541. CrossRef
Design and high-level expression of a hybrid antimicrobial peptide LF15-CA8 in Escherichia coli
Xing-Jun Feng, Li-Wei Xing, Di Liu, Xue-Ying Song, Chun-Long Liu, Jing Li, Wen-Shan Xu, Zhong-Qiu Li
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Stenotrophomonas panacihumi sp. nov., Isolated from Soil of a Ginseng Field: Hoonbok Yi , Sathiyaraj Srinivasan , Myung Kyum Kim; J. Microbiol. 2010;48(1):30-35. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-010-0006-0

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Abstract

The study isolated a Gram-negative, rod-shaped, non-motile bacterium from the soil of a ginseng field in Daejeon, South Korea and characterized it to determine its taxonomic position. Phylogenetic analysis, based on the 16S rRNA gene sequence, revealed that strain MK06T belongs to the family Xanthomonadacea, and showed the highest degree of sequence similarity to Stenotrophomonas rhizophila e-p10T (98.6%), Xanthomonas campestris LMG 568T (98.0%), Stenotrophomonas maltophilia ATCC 1d3637T (97.3%), and Stenotrophomonas humi R-32729T (96.9%). Chemotaxonomic data revealed that strain MK06T possesses ubiquinone Q-8 as the predominant respiratory lipoquinone, which is common in the genus tenotrophomonas, and that the predominant fatty acids were 15:0 iso (41.1%), 15:0 anteiso (12.6%), and 17:1 iso ω9c (8.6%). The results of physiological and biochemical tests clearly demonstrated that strain MK06T represents a distinct species and supported its affiliation with the genus Stenotrophomonas. Based on these data, MK06T (KCTC, 22893T; JCM, 16536T; KEMB, 9004-002T) should be classified as the type strain for a novel species, for which we propose the name Stenotrophomonas panacihumi sp. nov.

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Revised taxonomic classification of the Stenotrophomonas genomes, providing new insights into the genus Stenotrophomonas
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Luz E. Ochoa-Sánchez, Pablo Vinuesa
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Antonie van Leeuwenhoek.2016; 109(6): 755. CrossRef
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Yong Li, YiXin Ying, WanLong Ding, Shilin Chen
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Camille Granada, Pedro Beschoren da Costa, Bruno Brito Lisboa, Luciano Kayser Vargas, Luciane M. P. Passaglia
Plant and Soil.2013; 373(1-2): 339. CrossRef
Conversion of Ginsenoside Rb1 and Taxonomical Characterization of Stenotrophomonas sp. 4KR4 from Ginseng Rhizosphere Soil
In-Hwa Jeon, Geon-Yeong Cho, Song-Ih Han, Sun Kyun Yoo, Kyung-Sook Whang
The Korean Journal of Microbiology.2013; 49(4): 369. CrossRef
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Acinetobacter brisouii sp. nov., Isolated from a Wetland in Korea: Rangasamy Anandham , Hang-Yeon Weon , Soo-Jin Kim , Yi-Seul Kim , Byung-Yong Kim , Soon-Wo Kwon; J. Microbiol. 2010;48(1):36-39. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-009-0132-8

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Abstract: A bacterial strain 5YN5-8T was isolated from peat layer on Yongneup in Korea. Cells of strain 5YN5-8T were strictly aerobic, Gram-negative, coccobacilli, non-spore forming, and non-motile. The isolate exhibited optimal growth at 28°C, pH 7.0, and 0-1% NaCl. Results of 16S rRNA gene sequence analyses indicated a close relationship of this isolate to Acinetobacter calcoaceticus (97.8% similarity for strain DSM 30006T). It also exhibited 94.4-97.8% 16S rRNA gene sequence similarities to the validly published Acinetobacter species. The value for DNA-DNA hybridization between strain 5YN5-8T and other members of the genus Acinetobacter ranged from 16 to 28%. Predominant cellular fatty acids were C18:1 ω9c, summed feature 4 containing C15:0 iso 2-OH and/or C16:1 ω7c, and C16:0. The DNA G+C content was 43.9 mol%. Phylogenetic, phenotypic, and chemotaxonomic data accumulated in this study revealed that the isolate could be classified in a novel species of the genus Acinetobacter. The name Acinetobacter brisouii sp. nov. is proposed for the novel species, with 5YN5-8T (=KACC 11602T =DSM 18516T) as the type strain.

Potentiation of Bacterial Killing Activity of Zinc Chloride by Pyrrolidine Dithiocarbamate: Eun-Kyoung Choi , Hye-Hyang Lee , Mi-Sun Kang , Byung-Gook Kim , Hoi-Soon Lim , Seon-Mi Kim , In-Chol Kang; J. Microbiol. 2010;48(1):40-43. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-009-0049-2

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Abstract

Zinc has antimicrobial activity and zinc salts including zinc chloride (ZnCl2) have been used for the control of oral malodor. In this study, we hypothesized that pyrrolidine dithiocarbamate (PDTC), a zinc ionophore, may enhance antimicrobial efficacy of ZnCl2. The bactericidal effectiveness of ZnCl2 alone (0.5-8 mM) or in combination with PDTC (1 or 10 μM) was evaluated by in vitro short (1 h) time-killing assays against Fusobacterium nucleatum and Porphyromonas gingivalis. Only a slight viability decrease was observed with ZnCl2 or PDTC alone after 1-h incubation. By contrast, combination of ZnCl2 and PDTC could achieve a more than 100-fold viability reduction compared with ZnCl2 or PDTC alone in F. nucleatum and P. gingivalis. Therefore, PDTC greatly enhanced the bactericidal activity of ZnCl2 against the oral malodor-producing bacteria. These results suggest that use of PDTC may be useful for enhancing bactericidal activity of antimalodor regimens of zinc salts.

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Synthesis and characterization of mixed ligand complexes of lomefloxacin drug and glycine with transition metals. Antibacterial, antifungal and cytotoxicity studies
Gehad G. Mohamed, Hanan F. Abd El-Halim, Maher M.I. El-Dessouky, Walaa H. Mahmoud
Journal of Molecular Structure.2011; 999(1-3): 29. CrossRef

Enterococcus faecium Isolated from Honey Synthesized Bacteriocin-Like Substances Active against Different Listeria monocytogenes Strains: Carolina Ibarguren , Raúl R. Raya , María C. Apella , M. Carina Audisio; J. Microbiol. 2010;48(1):44-52. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-009-0177-8

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Abstract: Four Enterococcus faecium strains, isolated from honeycombs (C1 and M2d strains) and feral combs (Mori1 and M1b strains) secreted antimicrobial substances active against fourteen different Listeria spp. strains. The antimicrobial compound(s) present in the cell free supernatant were highly thermostable (121°C for 15 min) and inactivated by proteolytic enzymes, but not by α-amylase and lipase, thus suggesting a peptidic nature. Since the structural bacteriocin gene determinants of enterocins A and B were PCR amplified from the four E. faecium isolates, only the bacteriocin produced by strain C1 was further characterized: it showed a broad band of approximately 4.0-7.0 kDa in SDS-PAGE and was bactericidal (4 log decrease) against L. monocytogenes 99/287. L. monocytogenes 99/287R, a clone spontaneously resistant to the enterocin produced by E. avium DSMZ17511 (ex PA1), was not inhibited by the enterocin-like compounds produced by strain C1. However, it was inhibited in mixed culture fermentations by E. faecium C1 and a bacteriostatic effect was observed. The bacteriocin-producer Enterococcus strains were not haemolytic; gelatinase negative and sensitive to vancomycin and other clinically relevant antibiotics.

Purification and Biochemical Properties of a Glucose-Stimulated β-D-Glucosidase Produced by Humicola grisea var. thermoidea Grown on Sugarcane Bagasse: Cesar Vanderlei Nascimento , Flávio Henrique Moreira Souza , Douglas Chodi Masui , Francisco Assis Leone , Rosane Marina Peralta , João Atílio Jorge , Rosa Prazeres Melo Furriel; J. Microbiol. 2010;48(1):53-62. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-009-0159-x

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Abstract: The effect of several carbon sources on the production of mycelial-bound β-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated β-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The β-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50°C, respectively. The purified enzyme was thermostable up to 60 min in water at 55°C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60°C. The enzyme hydrolyzed p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-galactopyranoside, p-nitrophenyl-β-D-fucopyranoside, p-nitrophenyl-β-D-xylopyranoside, o-nitrophenyl-β-Dgalactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-β-Dfucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude β-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea β-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.

Role of a Burkholderia pseudomallei Polyphosphate Kinase in an Oxidative Stress Response, Motilities, and Biofilm Formation: Suda Tunpiboonsak , Rungrawee Mongkolrob , Kaniskul Kitudomsub , Phawatwaristh Thanwatanaying , Witcha Kiettipirodom , Yanin Tungboontina , Sumalee Tungpradabkul; J. Microbiol. 2010;48(1):63-70. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-010-9138-5

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Abstract

Burkholderia pseudomallei, a motile and rod Gram-negative bacterium, is the causative agent of melioidosis. The bacterium is an intracellular pathogen and that motility is generally crucial for their survival in a natural environment and for systemic infection inside a host. We report here a role of B. pseudomallei polyphosphate kinase in virulence, such as an oxidative stress response, motilities and biofilm formation. The polyphosphate kinase (ppk) mutant is susceptible to hydrogen peroxide in an oxidative stress condition, unable to perform swimming, swarming motilities, and has lower density biofilm forming capacity than the wild-type strain. We also demonstrated that both polyphosphate kinase and motile flagella are essential and independently involved in biofilm formation. The B. pseudomallei flagellin (fliC) mutant and B. mallei, a nonmotile species, are shown to produce higher density biofilm formation than the ppk mutant, but less than wild type B. pseudomallei.

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Rapid Propagational Interactions of Slow Binding Inhibitor with RecA Protein Occur on the Longer Nucleoprotein Filaments: Jong-Il Kim; J. Microbiol. 2010;48(1):71-76. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-009-0306-4

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Abstract: RecA protein is a DNA-dependent ATPase. RecA protein-mediated ATP hydrolysis occurs throughout the filamentous nucleoprotein complexes of RecA and DNA. Nucleotide analog ATP[γS] may not act simply as a competitive inhibitor, leading to inhibition kinetic patterns that are informative. When a mixture of ATP and ATP[γS] is present at the beginning of reaction, a transient phase lasting several minutes is observed in which the system approaches the state characteristic of the new ATP/ATP[γS] ratio. This phase consists of a burst or lag in ATP hydrolysis, depending on whether ATP or ATP[γS] respectively, is added first. The transition phase reflects a slow conformational change in a RecA monomer or a general adjustment in the structure of RecA filaments. The RecA filaments formed on longer DNA cofactor were more sensitive, and respond more rapidly to ATP[γS] than on shorter DNA cofactors.

The Involvement of the nif-Associated Ferredoxin-Like Genes fdxA and fdxN of Herbaspirillum seropedicae in Nitrogen Fixation: André L.F. Souza , Adriana L. Invitti , Fabiane G.M. Rego , Rose A. Monteiro , Giseli Klassen , Emanuel M. Souza , Leda S. Chubatsu , Fábio O. Pedrosa , Liu U. Rigo; J. Microbiol. 2010;48(1):77-83. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-009-0077-y

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Abstract

The pathway of electron transport to nitrogenase in the endophytic β-Proteobacterium Herbaspirillum seropedicae has not been characterized. We have generated mutants in two nif-associated genes encoding putative ferredoxins, fdxA and fdxN. The fdxA gene is part of the operon nifHDKENXorf1orf2fdxAnifQmodABC and is transcribed from the nifH promoter, as revealed by lacZ gene fusion. The fdxN gene is probably cotranscribed with the nifB gene. Mutational analysis suggests that the FdxA protein is essential for maximum nitrogenase activity, since the nitrogenase activity of the fdxA mutant strain was reduced to about 30% of that of the wild-type strain. In addition, the fdxA mutation had no effect on the nitrogenase switch-off in response to ammonium. Nitrogenase activity of a mutant strain lacking the fdxN gene was completely abolished. This phenotype was reverted by complementation with fdxN expressed under lacZ promoter control. The results suggest that the products of both the fdxA and fdxN genes are probably involved in electron transfer during nitrogen fixation.

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Serotype Distribution and β-Lactam Resistance in Haemophilus influenzae Isolated from Patients with Respiratory Infections in Korea: Songmee Bae , Jaehoon Lee , Eunah Kim , Jaehwa Lee , Jaeyon Yu , Yeonho Kang; J. Microbiol. 2010;48(1):84-88. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-009-0212-9

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Abstract

Haemophilus influenzae is a frequent causative bacterial pathogen of respiratory tract infections. Resistance to β-lactam antibiotics has been a significant clinical problem in treatment for H. influenzae respiratory infections. This study describes the serotype, antibiotic resistance and distribution of TEM-1 or ROB-1 β-lactamase in H. influenzae isolates from local private hospitals from 2002 to 2004. Among the 100 H. influenzae respiratory isolates, only 7% were identified as serotypes a, b, e, and f, with the remaining 93% being nontypeable. Resistance to ampicillin, cefaclor, and tetracycline was 57%, 46%, and 16%, respectively. All strains were susceptible to azithromycin and ciprofloxacin, whereas amoxicillin/clavulanate, cefotaxime, and imipenem exhibited reduced susceptibilities of 99%, 99%, and 91%, respectively. All 57 ampicillinresistant strains (minimum inhibitory concentration, MIC≥4 µg/ml) were β-lactamase-positive and possessed the TEM-1 type β-lactamase. One β-lactamase-positive amoxicillin/clavulanate-resistant isolate that was resistant to ampicillin (MIC>128 µg/ml) had the TEM-1 type β-lactamase and not susceptible to cefaclor and cefotaxime. Analysis of penicillin binding protein 3 revealed six residues (Asp-350, Met-377, Ala-502, Asn-526, Val-547, and Asn-569)that were substituted by Asn, Ile, Val, Lys, Ile, and Ser, respectively.

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Salmonella enterica is highly diverse in terms of genome structure, which is at least partly due to the horizontal transfer of genetic elements from various sources. In this study, we examined the expression profiles of such genes in Salmonella Pathogenicity Islands (SPIs) and the cob/pdu locus, horizontally acquired large DNA segments, during growth under standard growth conditions. Transcripts from exponentially growing and early stationary phase Salmonellae were compared using various methods including cDNA microarray analysis. Nearly all genes encoded by SPIs and the cob/pdu locus were induced at the onset of the stationary phase in a stringent molecule ppGpp-dependent but stationary phase σ, σ38-independent manner. Although, it has been suggested that ppGpp acts in concert with DksA, we found the stationary phase induction of those SPI genes was not DksA dependent. It is suggested that ppGpp stimulates the expression of these stress-inducible genes encoded by horizontally acquired DNA, by itself or in concert with DksA.

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Transcriptional Regulation of hemO Encoding Heme Oxygenase in Clostridium perfringens: Sufi Hassan , Kaori Ohtani , Ruoyu Wang , Yonghui Yuan , Yun Wang , Yumi Yamaguchi , Tohru Shimizu; J. Microbiol. 2010;48(1):96-101. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-009-0384-3

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Abstract: A Gram-positive anaerobic pathogen, Clostridium perfringens, causes clostridial myonecrosis or gas gangrene in humans by producing numerous extracellular toxins and enzymes that act in concert to degrade host tissues. The ability of infectious bacteria to acquire sufficient iron during infection is essential for the pathogen to cause disease. In the C. perfringens strain 13 genome, a heme oxygenase gene homologue (CPE0214, hemO) was found and its role was examined. The purified recombinant HemO protein showed heme oxygenase activity that can convert heme to biliverdin. hemO transcription was induced in response to extracellular hemin in a dose-dependent manner. The global two-component VirR/VirS regulatory system and its secondary regulator VR-RNA had positive regulatory effects on the transcription of hemO. These data indicate that heme oxygenase may play important roles in iron acquisition and cellular metabolism, and that the VirR/VirS-VR-RNA system is also involved in the regulation of cellular iron homeostasis, which might be important for the survival of C. perfringens in a human host.

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