dicA promoter DNA에 붙는 H-NS 단백질에 의한 dicA 유전자의 발현 조절

윤상훈,이연호,임헌만,Yun, Sang Hoon,Lee, Yonho,Lim, Heon M. 한국미생물학회 2019 미생물학회지 Vol.55 No.3

H-NS는 대장균에서 DNA 결합 단백질로 수많은 유전자의 발현에 영향을 주는 것으로 잘 알려져 있다. DicA 단백질은 dicF, dicB의 발현을 억제하여 대장균의 분열을 조절한다. dicA의 발현에 Cnu, H-NS의 관여 여부는 CnuK9E 돌연변이가 $37^{\circ}C$에서 dicA의 발현을 억제하여 대장균이 길게 자라는 현상을 일으키며 처음 알려졌다. 하지만 Cnu와 H-NS 두 단백질이 어떻게 dicA의 발현을 조절하는지에 대한 분자적인 기작 연구는 잘 되어있지 않다. 본 연구에서 H-NS가 dicA와 dicC 유전자의 프로모터 부근에 염기서열 특이적으로 결합하며, $37^{\circ}C$ 보다 $25^{\circ}C$에서 DNA 더 잘 결합하는 것을 확인하였다. 그리고 EMSA를 통해 Cnu는 H-NS의 DNA 결합의 oligomeric state를 변화시키는 방식으로 작용하는 것을 보여주었다. In vivo transcription assay와 real time PCR을 통해 H-NS가 제거된 대장균에서 dicA 프로모터 활성이 높아지고, 분열 초기 dicA의 발현이 조절 받지 못하고 증가하는 것으로 보아, H-NS는 dicA의 발현에 억제자로서 기능한다. H-NS binds to promoter DNA and works as a general transcription silencer. DicA protein, by binding to the promoter DNA of dicA, activates dicA expression and at the same time inhibits expression of dicF and dicB, thus, exerting cell division control in Escherichia coli. H-NS complexed with a nucleoid protein Cnu was known to be involved in dicA expression. However, the exact nature of H-NS binding to dicA promoter DNA and the consequences of H-NS binding in expression of dicA is not clear. In this study, we explored the DNA binding activity of H-NS on the promoter DNA of dicA and found that H-NS binding occurs exclusively to the dicA promoter DNA. We never observed, however, H-NS binding at the vicinity of the dicA promoter. Temperature dependent oligomerization of H-NS was observed during DNA binding and the Cnu protein enhances the oligomerization process of H-NS binding. In vivo measurement of dicA expression in an hns deleted strain showed that dicA expression increased. These results demonstrated that H-NS binds specifically to dicA promoter DNA and functions as a transcription silencer.

The mechanism underlying Ler-mediated alleviation of gene repression by H-NS

Academic Press 2017 Biochemical and biophysical research communication Vol. No.

<P><B>Abstract</B></P> <P>Secretion of effector proteins in Enteropathogeneic <I>Escherichia coli</I> (EPEC) and Enterohemorrhagic <I>Escherichia coli</I> (EHEC) is mediated by a specialized type III secretion system, components of which are encoded in the LEE operons 1 to 5. H-NS, a global repressor in <I>E</I>. <I>coli</I>, silences the expression of LEE operons. Ler, a master regulator in LEE operons, shares 24% amnio acid identity and 44% amino acid similarity to H-NS. Interestingly, rather than a gene silencer, its main role has been characterized as an antagonizing protein that relieves H-NS-mediated transcriptional silencing. In the previous study we reported molecular mechanism for the repression of <I>LEE5</I> promoter in EPEC and EHEC by H-NS as a protein interaction between upstream DNA-bound H-NS and the αCTD of promoter-bound RNA polymerase. The mechanism underlying Ler-mediated alleviation of the genes repression by H-NS is largely unknown. We examined regulatory effect of these proteins on <I>LEE</I>5p activity using various in vitro tools. Our results revealed that binding affinity of Ler to the <I>LEE5</I>p DNA is about 40 folds greater than that of H-NS as determined by surface plasmon resonance. We verified that Ler binding removed H-NS bound to the same stretch of DNA on <I>LEE5</I> promoter resulting in a derepression.</P> <P><B>Highlights</B></P> <P> <UL> <LI> H-NS specifically represses <I>LEE5</I> promoter activity and Ler alleviates the repression by H-NS. </LI> <LI> Binding affinity of Ler to the <I>LEE5</I> promoter DNA is about 40 folds greater than that of H-NS. </LI> <LI> Ler binding can remove H-NS bound to the same sites on <I>LEE5</I> promoter region. </LI> </UL> </P>

대장균 4227BL21에서 대량 발현된 대장균 H-NS 단백질의 정제

박은규,김우연 中央大學校 遺傳工學硏究所 1997 遺傳工學硏究論集 Vol.10 No.1

H-NS is one of the most abundant uncleoid-associated DNA-binding protiens (H1a). H-NS consists of 136 amino acids with a single tryptophanyl residue at position 108. H-NS has a general effect on transcription by compacting DNA. It has been known to act directly as repressor or activator for a number of specific genes. In order to gain further insight into the complex function of H-NS, H-NS was overexpressed in E. coli 4227BL21, which contains T7 RNA polymerase gene under lac promoter. It was transformed with pPD3 containing H-NS gene under T7 RNA polymerase promoter. The transformant was grown with vigorous aeration and H-NS protein induced with IPTG. The overexpressed H-NS protein was purified by ammonium sulfate precipitation and DNA-cellulose column chromatography

H-NS Silences Gene Expression of LeuO, the Master Regulator of the Cyclic(Phe-Pro)-dependent Signal Pathway, in Vibrio vulnificus

( Na-young Park ),( Keun-woo Lee ),( Kun-soo Kim ) 한국미생물 · 생명공학회 2020 Journal of microbiology and biotechnology Vol.30 No.6

The histone-like nucleoid structuring protein (H-NS) is an abundant global regulator of environmentally controlled gene expression. Herein, we demonstrate that H-NS represses the expression of LeuO, the master regulator of the cyclic(Phe-Pro)-dependent signaling pathway, by directly binding to the upstream region of the gene. H-NS binds to a long stretched region (more than 160-bp long), which overlaps with binding sites for ToxR and LeuO. A high quantity of H-NS outcompetes ToxR for binding to the cis-acting element of leuO. However, our footprinting analyses suggests that the binding of H-NS is relatively weaker than LeuO or ToxR at the same molarity. Considering that the DNA nucleotide sequences of the upstream regions of leuO genes are highly conserved among various Vibrio, such patterns as those found in V. vulnificus would be a common feature in the regulation of leuO gene expression in Vibrionaceae. Taken together, these results suggest that, in species belonging to Vibrionaceae, H-NS regulates the expression of leuO as a basal stopper when cFP-ToxR mediated signaling is absent.

박테리아의 히스톤 유사 단백질에 의한 유전자 발현 조절

박신애,이정신,Park, Shinae,Lee, Jung-Shin 한국미생물학회 2018 미생물학회지 Vol.54 No.1

원핵 세포는 핵양체 결합 단백질(NAP)로 알려진 다양한 히스톤 유사 단백질을 가지고 있다. 이들은 DNA의 AT-rich 서열에 결합하여, DNA 자체를 감싸거나, 구부리거나, 떨어져 있는 DNA 가닥을 연결시키는 다리 역할을 하여, 결국에는 원핵 생물의 유전자 발현을 조절한다. NAP는 특히 전사의 억제 기능을 가지고 있기 때문에, 유전자 발현 억제에 있어서 이들의 역할과, 구체적인 메커니즘을 밝히는 것을 매우 중요한 일이다. 본 논문에서는 잘 알려져 있는 NAP인 H-NS와 HU에 대하여 정리하였고, 특히 E. coli와 Salmonella Typhimurium에서 이들의 유전자 발현에 대한 기능을 요약하였다. H-NS는 이들의 올리고머화와 필라멘트 구조 형성을 통하여 Salmonella와 같은 사람에 감염하는 병원성 세균의 독성유전자 발현을 억제할 수 있고, 이런 기능을 수행하였을 때 다른 NAP와 함께 작용할 수 있다. 최근에 H-NS는 사람에게 typhoid fever와 systemic disease를 발생시키는 독성물질인, typhoid toxin의 발현 또한 조절할 수 있음이 밝혀졌다. Salmonella에서 HU 또한 독성 유전자뿐만 아니라, 이들의 생리적 기능에 중요한 유전자들의 발현을 조절할 수 있다. 따라서, H-NS와 HU와 같은 NAP들이 원핵 생물의 독성 유전자 발현의 분자적인 메커니즘을 밝히는데 중요한 요소임을 제시한다. A prokaryotic cell has various histone-like proteins also known as nucleoid-associated proteins (NAPs). These proteins bind AT-rich sequence at DNA, which induce DNA wrapping, bending, and bridging, and subsequently regulate the gene expression in bacteria. Because NAPs function in transcriptional silencing of virulence genes, it is important to study their roles in gene silencing and specific mechanisms of these proteins. In this review, we discussed two well-known NAPs, H-NS, and HU, and summarized their roles for gene expression in Escherichia coli and Salmonella Typhimurium. Through the oligomerization and filamentation of H-NS, it represses the expression of virulence genes in human pathogenic bacteria, such as Salmonella Typhimurium, and it works with other NAPs positively or negatively. Recently, H-NS also regulates typhoid toxin expression, which causes typhoid fever and systemic disease in human. Additionally, HU regulates the expression of genes related to both virulence and physiology of Salmonella. Therefore, we suggest that NAPs like H-NS and HU are crucial factors to reveal the molecular mechanisms of virulence gene expression in bacteria.

Identification of High-Specificity H-NS Binding Site in LEE5 Promoter of Enteropathogenic Esherichia coli (EPEC)

Abhay Prasad Bhat,신민상,최현일 한국미생물학회 2014 The journal of microbiology Vol.52 No.7

Histone-like nucleoid structuring protein (H-NS) is a small but abundant protein present in enteric bacteria and is involved in compaction of the DNA and regulation of the transcription. Recent reports have suggested that H-NS binds to a specific AT rich DNA sequence than to intrinsically curved DNA in sequence independent manner. We detected two high-specificity H-NS binding sites in LEE5 promoter of EPEC centered at -110 and -138, which were close to the proposed consensus H-NS binding motif. To identify H-NS binding sequence in LEE5 promoter, we took a random mutagenesis approach and found the mutations at around -138 were specifically defective in the regulation byH-NS. It was concluded that H-NS exertsmaximumrepression via the specific sequence at around -138 and ubsequently contacts α subunit of RNAP through oligomerization.

A Mutational Study of Cnu Reveals Attractive Forces between Cnu and H-NS

윤상훈,임헌만,지상천,전흥진,Xun Wang,이영훈,최병석 한국분자세포생물학회 2012 Molecules and cells Vol.33 No.2

Cnu is a small 71-amino acid protein that complexes with H-NS and binds to a specific sequence in the replication origin of the E. coli chromosome. To understand the me-chanism of interaction between Cnu and H-NS, we used bacterial genetics to select and analyze Cnu variants that cannot complex with H-NS. Out of 2,000 colonies, 40 Cnu variants were identified. Most variants (82.5%) had a single mutation, but a few variants (17.5%) had double amino acid changes. An in vitro assay was used to identify Cnu variants that were truly defective in H-NS binding. The changes in these defective variants occurred exclusively at charged amino acids (Asp, Glu, or Lys) on the surface of the protein. We propose that the attractive force that governs the Cnu-H-NS interaction is an ionic bond, unlike the hydrophobic interaction that is the major attractive force in most proteins.

Inefficient Transmissibility of NS-Truncated H3N8 Equine Influenza Virus in Dogs

( Woon Sung Na ),( Man Ki Song ),( Min Joo Yeom ),( Nanuri Park ),( Bo Kyu Kang ),( Hyoung Joon Moon ),( Dae Gwin Jeong ),( Jeong Ki Kim ),( Dae Sub Song ) 한국미생물 · 생명공학회 2015 Journal of microbiology and biotechnology Vol.25 No.3

H3N8 equine influenza virus (EIV) causes respiratory diseases in the horse population, and it has been demonstrated that EIV can transmit into dogs owing to its availability on receptors of canine respiratory epithelial cells. Recently, we isolated H3N8 EIV from an EIV-vaccinated horse that showed symptoms of respiratory disease, and which has a partially truncated nonstructural gene (NS). However, it is not clear that the NS-truncated EIV has an ability to cross the host species barrier from horses to dogs as well. Here, we experimentally infected the NS-truncated H3N8 EIV into dogs, and monitored their clinical signs and viral load in respiratory organs to determine the virus``s transmissibility.

myEvalSVC: an Integrated Simulation Framework for Evaluation of H.264/SVC Transmission

( Chih-heng Ke ) 한국인터넷정보학회 2012 KSII Transactions on Internet and Information Syst Vol.6 No.1

The ever-increasing demand for H.264 scalable video coding (H.264/SVC) distribution motivates researchers to devise ways to enhance the quality of video delivered on the Internet. Furthermore, researchers and practitioners in general depend on computer simulators to analyze or evaluate their designed network architecture or proposed protocols. Therefore, a complete toolset, which is called myEvalSVC, for evaluating the delivered quality of H.264/SVC transmissions in a simulated environment is proposed to help the network and video coding research communities. The toolset is based on the H.264 Scalable Video coding streaming Evaluation Framework (SVEF) and extended to connect to the NS2 simulator. With this combination, people who work on video coding can simulate the effects of a more realistic network on video sequences resulting from their coding schemes, while people who work on network technology can evaluate the impact of real video streams on the proposed network architecture or protocols. To demonstrate the usefulness of the proposed new toolset, examples of H.264/SVC transmissions over 802.11 and 802.11e are provided.

Detection of the Avian Influenza Viruses Nonstructural Protein 1 for Distinction between Vaccinated and Infected Chickens Using Synthetic Peptide-Based ELISA

Lee, Hyung-Tae,Jung, Kyoung-Hwa,Park, Ji-Hyun,Ha, Gun-Woo,Oh, Jin-Sik,Oh, Youn-Kyoung,Chai, Young-Gyu 대한미생물학회 2010 Journal of Bacteriology and Virology Vol.40 No.4

Avian influenza (AI) virus infects both animal and human. Low pathogenic AI virus infections (some H7 and H9 subtypes) have been reported all over the world and pose a potential threat to the poultry industry. Vaccination is the most effective way to prevent virus infection. However, vaccination makes it difficult to differentiate between vaccinated chickens and infected chickens. In order to differentiate vaccinated chickens from naturally infected chickens, we adopted synthetic peptide-based enzyme-linked immunosorbent assay (ELISA) using the peptide sequences from nonstructural protein 1 (NS1) of H9N2. Five synthetic peptides were designed using Protein Variability Sever (http://imed.med.ucm.es/PVS/) and synthesized. NS1-1 ~ NS1-4 peptides failed to detect serum antibodies from both vaccinated and naturally infected chickens. NS1-5 peptide from the C-terminal NS1 protein detected serum antibody from naturally infected chickens but not vaccinated chickens. These results imply that NS1-5 peptide may be a useful tool to differentiate naturally infected chicken from vaccinated chicken as being used in the synthetic peptide-based ELISA.