- [Protocol] Use of Cas9 Targeting and Red Recombination for Designer Phage Engineering
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Shin-Yae Choi , Danitza Xiomara Romero-Calle , Han-Gyu Cho , Hee-Won Bae , You-Hee Cho
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J. Microbiol. 2024;62(1):1-10. Published online February 1, 2024
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DOI: https://doi.org/10.1007/s12275-024-00107-2
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 Abstract
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Bacteriophages (phages) are natural antibiotics and biological nanoparticles, whose application is significantly boosted by
recent advances of synthetic biology tools. Designer phages are synthetic phages created by genome engineering in a way
to increase the benefits or decrease the drawbacks of natural phages. Here we report the development of a straightforward
genome engineering method to efficiently obtain engineered phages in a model bacterial pathogen, Pseudomonas aeruginosa.
This was achieved by eliminating the wild type phages based on the Streptococcus pyogenes Cas9 (SpCas9) and facilitating
the recombinant generation based on the Red recombination system of the coliphage λ (λRed). The producer (PD) cells of
P. aeruginosa strain PAO1 was created by miniTn7-based chromosomal integration of the genes for SpCas9 and λRed under
an inducible promoter. To validate the efficiency of the recombinant generation, we created the fluorescent phages from a
temperate phage MP29. A plasmid bearing the single guide RNA (sgRNA) gene for selectively targeting the wild type gp35
gene and the editing template for tagging the Gp35 with superfolder green fluorescent protein (sfGFP) was introduced into
the PD cells by electroporation. We found that the targeting efficiency was affected by the position and number of sgRNA.
The fluorescent phage particles were efficiently recovered from the culture of the PD cells expressing dual sgRNA molecules.
This protocol can be used to create designer phages in P. aeruginosa for both application and research purposes.
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Citations
Citations to this article as recorded by  -
Pilin regions that select for the small RNA phages in
Pseudomonas aeruginosa
type IV pilus
Hee-Won Bae, Hyeong-Jun Ki, Shin-Yae Choi, You-Hee Cho, Kristin N. Parent
Journal of Virology.2025;[Epub] CrossRef - Characteristics of bioaerosols under high-ozone periods, haze episodes, dust storms, and normal days in Xi’an, China
Yiming Yang, Liu Yang, Xiaoyan Hu, Zhenxing Shen
Particuology.2024; 90: 140. CrossRef - Airborne desert dust and aeromicrobiology over the Turkish Mediterranean coastline
Dale W. Griffin, Nilgün Kubilay, Mustafa Koçak, Mike A. Gray, Timothy C. Borden, Eugene A. Shinn
Atmospheric Environment.2007; 41(19): 4050. CrossRef
- Chemokine CCL6 Plays Key Role in the Inhibitory Effect of Vitamin A on Norovirus Infection
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Heetae Lee , Giljae Lee , You-Hee Cho , Youngcheon Song , GwangPyo Ko
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J. Microbiol. 2023;61(5):579-587. Published online May 26, 2023
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DOI: https://doi.org/10.1007/s12275-023-00047-3
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Abstract
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Norovirus (NoV) is the most common viral cause of acute gastroenteritis worldwide. Vitamin A has demonstrated the
potential to protect against gastrointestinal infections. However, the effects of vitamin A on human norovirus (HuNoV)
infections remain poorly understood. This study aimed to investigate how vitamin A administration affects NoV replication.
We demonstrated that treatment with retinol or retinoic acid (RA) inhibited NoV replication in vitro based on their effects
on HuNoV replicon-bearing cells and murine norovirus-1 (MNV-1) replication in murine cells. MNV replication in vitro
showed significant transcriptomic changes, which were partially reversed by retinol treatment. RNAi knockdown of CCL6,
a chemokine gene that was downregulated by MNV infection but upregulated by retinol administration, resulted in increased
MNV replication in vitro. This suggested a role of CCL6 in the host response to MNV infections. Similar gene expression
patterns were observed in the murine intestine after oral administration of RA and/or MNV-1.CW1. CCL6 directly decreased
HuNoV replication in HG23 cells, and might indirectly regulate the immune response against NoV infection. Finally, relative
replication levels of MNV-1.CW1 and MNV-1.CR6 were significantly increased in CCL6 knockout RAW 264.7 cells. This
study is the first to comprehensively profile transcriptomes in response to NoV infection and vitamin A treatment in vitro,
and thus may provide new insights into dietary prophylaxis and NoV infections.
- Repositioning of a mucolytic drug to a selective antibacterial against Vibrio cholerae
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In-Young Chung† , Bi-o Kim† , Hye-Jeong Jang† , You-Hee Cho
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J. Microbiol. 2020;58(1):61-66. Published online January 2, 2020
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DOI: https://doi.org/10.1007/s12275-020-9590-9
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64
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Abstract
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Drug repositioning, the approach to explore existing drugs
for use in new therapeutic indications, has emerged as an alternative
drug development strategy. In this study, we found
that a mucolytic drug, N-acetylcysteine (NAC) showed antibacterial
activity against Vibrio cholerae. NAC can provide
acid stress that selectively inhibited the growth of V. cholerae
among other bacterial pathogens. To address the antibacterial
mechanism of NAC against V. cholerae, six acr (acetylcysteine-
resistant) mutants were isolated from 3,118 random
transposon insertion clones. The transposon insertion sites
of the six mutants were mapped at the five genes. All these
mutants did not display NAC resistance under acidic conditions,
despite their resistance to NAC under alkaline conditions,
indicating that the NAC resistance directed by the
acr mutations was independent of the unusual pH-sensitivity
of V. cholerae. Furthermore, all these mutants displayed
attenuated virulence and reduced biofilm formation, suggesting
that the acr genes are required for pathogenesis of
V. cholerae. This study validates the relevance of drug repositioning
for antibacterials with new modes of action and will
provide an insight into a novel antibacterial therapy for V.
cholerae infections to minimize side effects and resistance
emergence.
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Citations
Citations to this article as recorded by - Identification of brevinin-1EMa-derived stapled peptides as broad-spectrum virus entry blockers
Mi Il Kim, Thanh K. Pham, Dahee Kim, Minkyung Park, Bi-o Kim, You-Hee Cho, Young-Woo Kim, Choongho Lee
Virology.2021; 561: 6. CrossRef
- Differential expression of the major catalase, KatA in the two wild type Pseudomonas aeruginosa strains, PAO1 and PA14
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Bi-o Kim , In-Young Chung , You-Hee Cho
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J. Microbiol. 2019;57(8):704-710. Published online June 11, 2019
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DOI: https://doi.org/10.1007/s12275-019-9225-1
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77
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10
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Abstract
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KatA is the major catalase required for hydrogen peroxide
(H2O2) resistance and acute virulence in Pseudomonas aeruginosa
PA14, whose transcription is governed by its dual
promoters (katAp1 and katAp2). Here, we observed that KatA
was not required for acute virulence in another wild type P.
aeruginosa strain, PAO1, but that PAO1 exhibited higher
KatA expression than PA14 did. This was in a good agreement
with the observation that PAO1 was more resistant
than PA14 to H2O2 as well as to the antibiotic peptide, polymyxin
B (PMB), supposed to involve reactive oxygen species
(ROS) for its antibacterial activity. The higher KatA expression
in PAO1 than in PA14 was attributed to both katAp1
and katAp2 transcripts, as assessed by S1 nuclease mapping.
In addition, it was confirmed that the PMB resistance is attributed
to both katAp1 and katAp2 in a complementary manner
in PA14 and PAO1, by exploiting the promoter mutants
for each -10 box (p1m, p2m, and p1p2m). These results provide
an evidence that the two widely used P. aeruginosa strains
display different virulence mechanisms associated with OxyR
and Anr, which need to be further characterized for better
understanding of the critical virulence pathways that may
differ in various P. aeruginosa strains.
-
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International Journal of Biological Macromolecules.2025; 306: 141430. CrossRef - Enhancing the compost maturation of deer manure and corn straw by supplementation via black liquor
Shijun Pan, Gang Wang, Yide Fan, Xiqing Wang, Juan Liu, Mingzhu Guo, Huan Chen, Sitong Zhang, Guang Chen
Heliyon.2023; 9(2): e13246. CrossRef - The small RNA PrrH of Pseudomonas aeruginosa regulates hemolysis and oxidative resistance in bloodstream infection
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Microbial Pathogenesis.2023; 180: 106124. CrossRef - Eco-evolutionary dynamics of experimental Pseudomonas aeruginosa populations under oxidative stress
Taoran Fu, Danna R. Gifford, Christopher G. Knight, Michael A. Brockhurst
Microbiology
.2023;[Epub] CrossRef - The Pseudomonas aeruginosa DksA1 protein is involved in H2O2 tolerance and within-macrophages survival and can be replaced by DksA2
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Journal of Fish Diseases.2022; 45(3): 421. CrossRef - Nitrite Promotes ROS Production to Potentiate Cefoperazone-Sulbactam-Mediated Elimination to Lab-Evolved and Clinical-Evolved Pseudomonas aeruginosa
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Microbiology Spectrum.2022;[Epub] CrossRef - Nitrate Respiration Promotes Polymyxin B Resistance in Pseudomonas aeruginosa
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Journal of Medical Microbiology
.2021;[Epub] CrossRef
- REVIEW] Antibacterial strategies inspired by the oxidative stress and response networks
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So Youn Kim , Chanseop Park , Hye-Jeong Jang , Bi-o Kim , Hee-Won Bae , In-Young Chung , Eun Sook Kim , You-Hee Cho
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J. Microbiol. 2019;57(3):203-212. Published online February 26, 2019
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DOI: https://doi.org/10.1007/s12275-019-8711-9
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149
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124
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114
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Abstract
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Oxidative stress arises from an imbalance between the excessive
accumulation of reactive oxygen species (ROS) and
a cell’s capability to readily detoxify them. Although ROS are
spontaneously generated during the normal oxygen respiration
and metabolism, the ROS generation is usually augmented
by redox-cycling agents, membrane disrupters, and
bactericidal antibiotics, which contributes their antimicrobial
bioactivity. It is noted that all the bacteria deploy an arsenal
of inducible antioxidant defense systems to cope with the
devastating effect exerted by the oxidative stress: these systems
include the antioxidant effectors such as catalases and
the master regulators such as OxyR. The oxidative stress response
is not essential for normal growth, but critical to survive
the oxidative stress conditions that the bacterial pathogens
may encounter due to the host immune response and/or
the antibiotic treatment. Based on these, we here define the
ROS-inspired antibacterial strategies to enhance the oxidative
stress of ROS generation and/or to compromise the bacterial
response of ROS detoxification, by delineating the ROSgenerating
antimicrobials and the core concept of the bacterial
response against the oxidative stress.
-
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- [PROTOCOL] Drosophila melanogaster as a polymicrobial infection model for Pseudomonas aeruginosa and Staphylococcus aureus
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Young-Joon Lee , Hye-Jeong Jang , In-Young Chung , You-Hee Cho
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J. Microbiol. 2018;56(8):534-541. Published online July 25, 2018
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DOI: https://doi.org/10.1007/s12275-018-8331-9
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Abstract
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Non-mammalian infection models have been developed over
the last two decades, which is a historic milestone to understand
the molecular basis of bacterial pathogenesis. They also
provide small-scale research platforms for identification of
virulence factors, screening for antibacterial hits, and evaluation
of antibacterial efficacy. The fruit fly, Drosophila melanogaster
is one of the model hosts for a variety of bacterial
pathogens, in that the innate immunity pathways and tissue
physiology are highly similar to those in mammals. We here
present a relatively simple protocol to assess the key aspects
of the polymicrobial interaction in vivo between the human
opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus
aureus, which is based on the systemic infection
by needle pricking at the dorsal thorax of the flies. After infection,
fly survival and bacteremia over time for both P.
aeruginosa and S. aureus within the infected flies can be monitored
as a measure of polymicrobial virulence potential.
The infection takes ~24 h including bacterial cultivation. Fly
survival and bacteremia are assessed using the infected flies
that are monitored up to ~60 h post-infection. These methods
can be used to identify presumable as well as unexpected phenotypes
during polymicrobial interaction between P. aeruginosa
and S. aureus mutants, regarding bacterial pathogenesis
and host immunity.
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- Superinfection Exclusion Reveals Heteroimmunity between Pseudomonas aeruginosa Temperate Phages
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In-Young Chung , Hee-Won Bae , Hye-Jung Jang , Bi-o Kim , You-Hee Cho
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J. Microbiol. 2014;52(6):515-520. Published online May 29, 2014
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DOI: https://doi.org/10.1007/s12275-014-4012-5
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71
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4
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Abstract
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Temperate siphophages (MP29, MP42, and MP48) were isolated from the culture supernatant of clinical Pseudomonas aeruginosa isolates. The complete nucleotide sequences and annotation of the phage genomes revealed the overall synteny
to the known temperate P. aeruginosa phages such as MP22, D3112, and DMS3. Genome-level sequence analysis showed the conservation of both ends of the linear genome and the divergence at the previously identified dissimilarity
regions (R1 to R9). Protein sequence alignment of the c repressor (ORF1) of each phage enabled us to divide the six phages into two groups: D3112 group (D3112, MP29, MP42, and MP48) and MP22 group (MP22 and DMS3). Superinfection
exclusion was observed between the phages belonging to the same group, which was mediated by the specific interaction between the c repressor and the cognate operator. Based on these, we suggest that the temperate siphophages prevalent in the clinical strains of P. aeruginosa represent at least two distinct heteroimmunity groups.
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- EDITORIAL] Molecular Microbiology in Antibacterial Research
-
You-Hee Cho
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J. Microbiol. 2014;52(3):185-187.
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DOI: https://doi.org/10.1007/s12275-014-4088-y
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73
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Abstract
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The special issue of Journal of Microbiology contains six reviews dealing with cutting edge research achievements in the fields of molecular microbiology focusing on antibacterial research. In a more specific sense, this special issue helps outline the progress of 21st-century basic molecular microbiology that can encompass related disciplines regarding a variety of interactions involving bacteria during bacterial pathogenesis and their control: sociomicrobiology (interaction between bacteria), immunology (interaction between bacteria and their hosts), and bacteriophage (phage) virology (interaction between bacteria and their parasites).
Recent advancements have rapidly been made in our understanding of the real situation regarding polymicrobial interactions during bacterial infection and in non-mammalian host infection models to uncover the molecular mechanisms of host-bacteria interactions, which will complement our growing knowledge about immune responses toward bacterial and environmental elicitors. Moreover, much attention
has recently been paid to phages and phage products as potential antibacterial therapeutics in the era of antibiotic resistance. Below, I summarize the individual contributions in these distinct categories.
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Citations
Citations to this article as recorded by - Defense and anti-defense mechanisms of bacteria and bacteriophages
Xiaoqing Wang, Sebastian Leptihn
Journal of Zhejiang University-SCIENCE B.2024; 25(3): 181. CrossRef
- Simultaneous Detection of Major Enteric Viruses Using a Combimatrix Microarray
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Ju-Mi Kim , Sung Yeon Kim , Young Bin Park , Hye Jin Kim , Byung Sup Min , Jae-Chang Cho , Jai Myung Yang , You-Hee Cho , GwangPyo Ko
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J. Microbiol. 2012;50(6):970-977. Published online October 27, 2012
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DOI: https://doi.org/10.1007/s12275-012-2228-9
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Abstract
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Various enteric viruses including norovirus, rotavirus, adenovirus, and astrovirus are the major etiological agents of food-borne and water-borne disease outbreaks and frequently cause non-bacterial gastroenteritis worldwide. Sensitive and
high-throughput detection methods for these viral pathogens are compulsory for diagnosing viral pathogens and subsequently improving public health. Hence, we developed a sensitive, specific, and high-throughput analytical assay to detect most major enteric viral pathogens using “Combimatrix” platform oligonucleotide probes. In order to detect four different enteric viral pathogens in a sensitive and simultaneous
manner, we first developed a multiplex RT-PCR assay targeting partial gene sequences of these viruses with fluorescent labeling for the subsequent microarray. Then, five olignonucleotides specific to each of the four major enteric viruses were selected for the microarray from the oligonulceotide pools targeting the specific genes obtained by multiplex PCR of these viruses. The oligonucleotide microarray
was evaluated against stool specimens containing single or mixed viral species. As a result, we demonstrated that the multiplex RT-PCR assay specifically amplified partial sequences of four enteric viruses and the subsequent microarray
assay was capable of sensitive and simultaneous detection of those viruses. The developed method could be useful for diagnosing enteric viruses in both clinical and
environmental specimens.
- Conserved Virulence Factors of Pseudomonas aeruginosa are Required for Killing Bacillus subtilis
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Shin-Young Park , Yun-Jeong Heo , Young-Seok Choi , Eric Deziel , You-Hee Cho
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J. Microbiol. 2005;43(5):443-450.
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DOI: https://doi.org/2277 [pii]
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Abstract
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The multi-host pathogen, Pseudomonas aeruginosa, possesses an extraordinary versatility which makes it capable of surviving the adverse conditions provided by environmental, host, and, presumably, competing microbial factors in its natural habitats. Here, we investigated the P. aeruginosa-Bacillus subtilis interaction in laboratory conditions and found that some P. aeruginosa strains can outcompete B. subtilis in mixed planktonic cultures. This is accompanied by the loss of B. subtilis viability. The bactericidal activity of P. aeruginosa is measured on B. subtilis plate cultures. The bactericidal activity is attenuated in pqsA, mvfR, lasR, pilB, gacA, dsbA, rpoS, and phnAB mutants. These results suggest that P. aeruginosa utilizes a subset of conserved virulence pathways in order to survive the conditions provided by its bacterial neighbors.
- Identification of [sigma]^B-Dependent Promoters Using Consensus-Directed Search of Streptomyces coelicolor Genome
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Eun-Jin Lee , You-Hee Cho , Hyo-Sub Kim , Jung-Hye Roe
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J. Microbiol. 2004;42(2):147-151.
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DOI: https://doi.org/2030 [pii]
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Abstract
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[sigma]^B plays an important role in both osmoprotection and proper differentiation in Streptomyces coelicolor A3(2). We searched for candidate members of the [sigma]^B regulon from the genome database, using the consensus promoter sequence (GNNTN_14-16GGGTAC/T). The list consists of 115 genes, and includes all the known [sigma]^B target genes and many other genes whose functions are related to stress protection and differentiation.
- Analysis of the Dual Promoters and the H 2 O 2 -responsive Element of the catA Gene Encoding Catalase A in Streptomyces coelicolor
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You-Hee Cho , Ji-Sook Hahn , Jung-Hye Roe
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J. Microbiol. 2000;38(4):239-244.
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Abstract
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The catA gene encodes the major catalase in Streptomyces coelicolor, whose production increases upon H_2O_2 treatment. Besides the previously identified primary promoter (catAp1), a minor promoter (catAp2) was newly assigned by S1 nuclease mapping. The catAp2 transcript was observed transiently upon entry into the stationary phase in liquid culture and upon differentiation on solid plates, whereas the level of catAp1 transcription did not change significantly during this growth transition. The catAp1 promoter was transcribed by the major vegetative RNA polymerase holoenzyme containing [sigma]^HrdB , whereas the catAp2 was transcribed in vitro by the holoenzyme containing [sigma]^R that is activated under oxidative conditions. The cis-element regulating the H_2 O_2 -inducibility of catAp1 was identified within the 23 bp inverted repeat sequence located between -65 and -43 of the catAp1 promoter. We named this sequence HRE (H_2O_2 -responsive element). The distal half of the inverted repeat was more crucial for H_2 O_2 ?pendent induction of the catAp1 transcript than the proximal half. HRE most likely serves as a binding site for the H_2 O_2 -responsive repressor CatR.
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