Attenuation Regulation as a Term Rewriting System

The classical attenuation regulation of gene expression in bacteria is considered. We propose to represent the secondary RNA structure in the leader region of a gene or an operon by a term, and we give a probabilistic term rewriting system modeling the whole process of such a regulation.Comment: to appea

Ribosome reinitiation at leader peptides increases translation of bacterial proteins

Part 1. RNA secondary structures of 5'-untranslated regions of proteins with the PF00270 and PF00271 domains in Corynebacterium diphtheria, C. glutamicum, and Bifidobacterium animalis. Figure S1.1. RNA duplex in the region from the stop codon of the leader gene to the start codon of the structural gene encoding helicase in C. diphtheria; Figure S1.2. RNA hairpin overlapping the Shine-Dalgarno sequence in the helicase in C. glutamicum; Figure S1.3. RNA hairpin overlapping two nucleotides of the helicase start codon in B. animalis and Streptomyces griseus. Part 2. Frequency of the leader-structural gene pairs as a function of the leader gene stop codon in Spirochaetales, Acidobacteria, Deinococcus-Thermus group, and Planctomycetes. Figure S2.1. Acidobacteria; Figure S2.2. Deinococcus–Thermus group; Figure S2.3. Planctomycetes; Figure S2.4. Spirochaetales; Figure S2.5. Actinobacteria. Part 3. Sequence logo of the 30-nt 5'-leader regions of all structural genes in Actinobacteria. (PDF 365 kb

Network Topologies That Can Achieve Dual Function of Adaptation and Noise Attenuation.

Many signaling systems execute adaptation under circumstances that require noise attenuation. Here, we identify an intrinsic trade-off existing between sensitivity and noise attenuation in the three-node networks. We demonstrate that although fine-tuning timescales in three-node adaptive networks can partially mediate this trade-off in this context, it prolongs adaptation time and imposes unrealistic parameter constraints. By contrast, four-node networks can effectively decouple adaptation and noise attenuation to achieve dual function without a trade-off, provided that these functions are executed sequentially. We illustrate ideas in seven biological examples, including Dictyostelium discoideum chemotaxis and the p53 signaling network and find that adaptive networks are often associated with a noise attenuation module. Our approach may be applicable to finding network design principles for other dual and multiple functions

방선균 Streptomyces coelicolor에서 전사인자 WblC를 통한 항생제 저항성의 유도

학위논문 (박사) -- 서울대학교 대학원 : 자연과학대학 생명과학부, 2020. 8. 석영재.Many antibiotics target bacterial translation, a fundamental metabolic process of protein synthesis. Translation-inhibitory antibiotics interfere with ribosome and other translational apparatus by diverse mode of action. Bacteria exhibit intrinsic resistance to antibiotics by utilizing indigenous genetic factors. Many types of intrinsic resistance mechanisms are known, but it has been observed that there are many undiscovered mechanisms as well. Actinomycetes of the gram-positive phylum Actinobacteria include environmental microbes, animal and plant symbionts, and pathogens. Actinomycetes include Streptomyces, which not only produce most of the commercial antibiotics but retains many antibiotic resistance mechanisms, and Mycobacterium, which includes major pathogens causing antibiotic resistance problems such as M. tuberculosis. WblC or WhiB7 of actinomycetes is a factor conferring intrinsic resistance to translation-inhibitory antibiotics. WblC (WhiB7), along with HrdB (SigA) is known to bind to target gene promoters and activate transcription. WblC (WhiB7) target gene products execute multiple antibiotic resistance mechanisms. However, the composition of WblC (WhiB7) regulon varies greatly among species as well as encompasses many genes of unknown functions. Moreover, there were several problems in the mode of defining WhiB7 regulon. On the other hand, while wblC (whiB7) is thought to be regulated by ribosome-mediated transcriptional attenuation, experimental evidences were insufficient and, most of all, no explanations were suggested regarding how the transcriptional attenuation is suppressed during antibiotic stress. The regulatory targets of WblC in Streptomyces coelicolor were examined in this study. 7.8% of all S. coelicolor genes exhibited WblC-dependent changes in transcript level during antibiotic stress, and 312 of these genes were confirmed as WblC regulon with observed direct binding of WblC to the promoters. As in mycobacteria, promoters of 288 WblC-upregulated regulon genes had 2 promoter sequence elements and a WblC-binding site in common, and showed WblC binding and transcriptional activation correlated to the degree of conservation of these common sequences. On the contrary, promoters of 24 WblC-downregulated regulon genes had no consensus sequence and exhibited no recruitment of HrdB by WblC. Meanwhile, WblC also regulated expression of many noncoding RNAs other than the regulon genes. Many of the WblC regulon products were identified to associate with ribosome and function as novel antibiotic resistance factors. S. coelicolor WblC regulon consists of multiple known antibiotic resistance genes and several overrepresented functional groups of genes, especially those involved in translation. WblC caused increase in global intracellular translation rate and a corresponding increase in growth rate at low-concentration antibiotic stress conditions, which may be due to diminished effective antibiotic concentration or stimulation of translation by WblC regulon products. Proteins showing both antibiotic stress- and WblC-dependent increase in ribosome association were identified, most of which were products of WblC regulon and many of which were reported to relieve translation stress. Respective mutation of 3 ribosome associate protein-coding WblC regulon genes resulted in increased susceptibility to erythromycin and/or tetracycline, which were recovered by complementation of the mutated genes. This study also deals with the mechanism how transcriptional attenuation is suppressed during antibiotic induction of wblC. First, the sequence elements of transcriptional attenuation were confirmed to be conserved among most of the wblC leader sequences of actinomycetes. Then, transcriptional termination caused by the Rho-independent terminator (RIT) of the leader sequence was verified to attenuate wblC expression. Putative antiterminator RNA structures were conserved in wblC leader sequences of sub-clades of actinomycetes, and the putative antiterminator of S. coelicolor actually functioned as an mechanism facilitating transcription readthrough during antibiotic stress. Lastly, it was found that amino acid starvation can also induce wblC expression by suppressing premature transcription termination, implying that ribosome-mediated attenuation of wblC responds to diverse translation deficiencies.세균의 기초 대사인 단백질 합성 과정, 즉 번역은 수많은 항생제의 작용 표적이다. 번역 저해 항생제들은 다양한 작용 방식으로 리보솜을 중심으로 한 번역 기구들의 기능을 저해한다. 세균은 유전적 변화를 통한 저항성 획득 외에도 고유의 유전인자를 활용해 항생제에 대한 내재적 저항성을 보이곤 한다. 여러 유형의 내재적 저항성 기작이 알려져 있으나 아직 밝혀지지 않은 기작도 많이 존재함이 세균들에서 관찰되고 있다. 그람 양성 방선균문 (Actinobacteria)의 방선균목 (actinomycetes) 은 환경 미생물, 동식물 공생체, 병원균들을 포함한다. 상용 항생제 대다수를 생합성하는 한편 항생제 저항성 기작을 다수 보유하는 Streptomyces 속과 결핵균 등 항생제 저항성 문제를 일으키는 주요 병원균이 속한 Mycobacterium 속이 방선균목에 포함된다. WblC 혹은 WhiB7은 번역 저해 항생제에 대한 방선균의 내재적 저항성 인자이다. WblC (WhiB7)는 시그마 인자 HrdB (SigA)와 함께 표적 유전자 프로모터에 결합해 전사를 활성화한다고 알려져 있다. WblC (WhiB7) 표적 유전자 산물들은 다수의 항생제 저항성 기작을 수행한다. 하지만 WblC (WhiB7) 조절 표적 유전자군 (조절군)의 구성은 종 간에 매우 상이할 뿐 아니라 다수의 기능 불명 유전자를 포함한다. 또한 기존의 WhiB7 조절군의 정의 방식에는 몇 가지 문제점들이 있었다. 한편 wblC (whiB7)는 리보솜 매개 전사 감쇠 기작에 의한 발현 조절을 받는다고 여겨지는데 이에 대한 실험적 증명이 아직 부족하며 무엇보다 항생제 스트레스 시 어떻게 전사 감쇠가 억제되는지는 제시된 바가 없다. 본 연구는 Streptomyces coelicolor에서 WblC의 조절 표적들을 규명하였다. S. coelicolor 유전자의 7.8%가 항생제 스트레스 상황에서 WblC에 의한 전사량 변화를 보였으며, 이들 중 312개 유전자가 항생제 스트레스 상황에서 WblC의 직접적 프로모터 결합이 관찰되는 WblC 조절군 (regulon) 으로 파악되었다. WblC에 의해 발현이 증가하는 288개 조절군 유전자들의 프로모터들은 mycobacteria에서와 같이 2개의 프로모터 서열 인자와 WblC 결합 부위를 공통적으로 지니고 있었고, 이들 공통 서열의 보존성에 상응하는 WblC 결합 및 전사 활성화 정도를 보였으며, WblC 의존적 HrdB 결합 증가가 관찰되었다. 반면 WblC에 의한 발현 감소를 보이는 24개 조절군 유전자들의 프로모터들에서는 공통 서열이 발견되지 않았으며 WblC에 의한 HrdB 결합 유도도 일어나지 않았다. 한편 WblC는 조절군 유전자들 외에도 다수의 noncoding RNA 발현을 조절하였다. WblC 조절군 산물 다수가 리보솜에 결합하며 새로운 항생제 저항성 인자들로 작용함도 확인하였다. S. coelicolor의 WblC 조절군은 기존에 알려진 항생제 저항성 유전자들을 다수 포함하는 한편 몇몇 기능 유형의 유전자들을 높은 비율로 포함하고 있었으며, 특히 다수의 단백질 합성 관여 유전자들을 포함하였다. WblC는 저농도 항생제 스트레스 상황에서 세포 내 전반적 번역 속도 증가와 이에 상응하는 생장 속도 증진을 유발했는데, 이는 WblC 조절군 산물들에 의한 유효 항생제 농도 저감 혹은 번역 촉진에 기인할 수 있다. 항생제 스트레스와 WblC에 의존적인 리보솜 결합량 증가가 일어나는 단백질들을 파악해 본 결과 대부분이 WblC 조절군의 산물이었으며 이들 중 상당수가 번역 스트레스의 해소에 관여한다는 보고들을 찾을 수 있었다. 리보솜 결합 단백질들을 암호화하는 3개 유전자의 변이가 erythromycin, tetracycline에 대한 저항성의 감소를 일으켰으며 변이된 유전자의 보완 시 항생제 저항성이 회복되었다. 본 연구는 또한 항생제에 의한 wblC 발현 유도 중 전사 감쇠가 억제되는 기작을 다루었다. 먼저 대다수의 방선균 wblC 선도 서열 (leader sequence)에 전사 감쇠의 서열 인자들이 보존되어 있음을 확인하였다. 그리고 선도 서열 내 Rho 비의존적 종결자가 일으키는 전사 종결이 wblC 발현 감쇠를 일으킴을 검증하였다. 방선균목 하위 분류군들의 선도 서열 내에 보존된 항종결인자 (antiterminator) 추정 RNA 구조가 발견되었고, S. coelicolor의 해당 항종결인자 추정 서열은 실제로 항생제 스트레스 상황에서 전사 감쇠를 억제하는 기작으로 작용하였다. 마지막으로, 아미노산 고갈 역시 전사 조기 종결을 억제해 wblC 발현을 유도함을 발견함으로써 wblC의 리보솜 매개 전사 감쇠는 다양한 번역 결함에 반응함을 제시하였다.Chapter 1. Introduction 1 1-1. Translation-inhibitory antibiotics 1 1-2. Intrinsic antibiotic resistance of bacteria 2 1-3. Intrinsic antibiotic resistance of actinomycetes 3 1-4. WblC/WhiB7, a transcriptional regulator of intrinsic antibiotic resistance 4 1-5. Antibiotic resistance genes of WblC/WhiB7 regulon 7 1-6. Criteria of defining WblC/WhiB7 regulon 8 1-7. Regulation of antibiotic resistance by ribosome-mediated attenuation 9 1-8. Ribosome-mediated transcriptional attenuation of wblC/whiB7 12 Chapter 2. Materials and Methods 13 2-1. Strains, plasmids, and reagents 13 2-2. Culture conditions 19 2-3. RNA sequencing (RNA-seq) 19 2-4. Chromatin immunoprecipitation-sequencing (ChIP-seq) 20 2-5. In silico sequence analyses 20 2-6. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) 21 2-7. In silico Analyses of gene-protein functions 22 2-8. In vivo 35S-methionine/cysteine incorporation assay 23 2-9. Ribosome isolation 23 2-10. Liquid chromatography-tandem mass spectrometry 24 2-11. Construction of mutant strains 25 2-12. Introduction of genes via integrative vectors 26 2-13. Minimum inhibitory concentration (MIC) test 27 2-14. S1 nuclease protection assay 27 2-15. Probe preparation for S1 nuclease protection assay 28 2-16. Quantitative reverse transcription-PCR (qRT-PCR) 29 Chapter 3. WblC-regulated targets in S. coelicolor 30 3-1. Direct target genes controlled by WblC 30 3-2. Promoter sequence motif of WblC-upregulated regulon 33 3-3. Different modes of WblC binding to activated and repressed regulon promoters 37 3-4. Noncoding RNA targets controlled by WblC 39 Chapter 4. Antibiotic resistance conferred by ribosome-associated proteins of WblC regulon 41 4-1. Functions of WblC-upregulated regulon gene products and relationship with antibiotic resistance 41 4-2. Maintenance of translation rate by WblC during antibiotic stress 48 4-3. Association of WblC-upregulated gene products with ribosome 51 4-4. Contribution of ribosome-associated WblC regulon gene products to intrinsic resistance 57 Chapter 5. The mechanism of wblC induction by translation stress 59 5-1. Conservation of ribosome-mediated transcriptional attenuation of wblC/whiB7 orthologs 59 5-2. Attenuation caused by leader RIT 61 5-3. Conservation of putative antiterminator structure in wblC leader sequences 64 5-4. Transcription readthrough caused by antiterminator formation during antibiotic stress 69 5-5. Induction of wblC by amino acid starvation 72 Chapter 6. Discussion 77 6-1. Transcriptional regulation by WblC 77 6-2. Antibiotic resistance and functions of WblC regulon 78 6-3. The mechanism of wblC induction by translation stress 79 Bibliography 81 Appendix 97 List of abbreviations 97 Abstract in Korean 99Docto

Adaptive Mechanisms of Niche Remodeling in Streptococcus pyogenes

The Gram-positive bacterium Streptococcus pyogenes is a remarkably successful pathogen, capable of infecting numerous tissue sites within its human host. The ability of S. pyogenes to invade these different niches is, in part, due to the species’ ability to monitor various physical and chemical signals in its environment and alter its transcriptional profile in response to these differential conditions. As a member of the lactic acid bacteria, S. pyogenes has a simple fermentative metabolism and relies exclusively on a combination of homo-lactic and mixed acid fermentation as a means of generating energy in the cell. As a consequence of its fermentative metabolism, S. pyogenes produces several organic acid end products that, over time, accumulate in the surrounding environment, causing a substantial reduction in pH. Thus, growth of the bacterium itself results in a significant remodeling of its local tissue environment. It also indicates that over the course of infection, it must both adapt to its self-inflicted acid stress as well as exploit alternative carbon sources for survival. Although pH has been identified as a signal utilized by S. pyogenes to induce global transcriptional changes, the specific regulatory mechanisms behind this transcriptional remodeling have largely remained unclear. To further characterize the process of S. pyogenes’ pH adaptive response we have identified several novel pH-sensitive transcriptional regulators and analyzed their contribution to gene expression and S. pyogenes pathogenesis. The malic enzyme pathway, which allows the cell to utilize malate as a carbon source for growth, consists of four genes in two adjacent operons, with the regulatory TCS MaeKR being required for the expression of the genes encoding a malate permease (maeP) and malic enzyme (maeE). Results show that expression of the maePE operon is influenced independently by external malate concentrations and pH in a MaeK-dependent mechanism. The ME genes are additionally regulated by a unique CcpA-independent form of catabolite repression which involves the PTS proteins PtsI and HPr. Furthermore, in vivo experiments demonstrate that loss of any individual ME gene has a significant effect on the outcome of a soft tissue infection. The secreted toxins SPN and SLO have been shown to contribute to S. pyogenes cytotoxicity and virulence in multiple models of pathogenesis, however little information is known about the specific regulatory mechanism that control expression of these toxins. Our work has determined that the growth-phase pattern of expression of the spn/slo operon is regulated by environmental pH. Additionally, this regulation requires both the CovRS two-component system as well as an additional protein, RocA. Additional data suggests that RocA does not function as a traditional histidine kinase, despite high structural and sequence homology to known histidine kinases such as CovS. However, all three regulatory proteins are required for the pH-mediated regulation of this virulence operon

Control Theory for Synthetic Biology: Recent Advances in System Characterization, Control Design, and Controller Implementation for Synthetic Biology

Living organisms are differentiated by their genetic material-millions to billions of DNA bases encoding thousands of genes. These genes are translated into a vast array of proteins, many of which have functions that are still unknown. Previously, it was believed that simply knowing the genetic sequence of an organism would be the key to unlocking all understanding. However, as DNA sequencing technology has become affordable, it has become clear that living cells are governed by complex, multilayered networks of gene regulation that cannot be deduced from sequence alone. Synthetic biology as a field might best be characterized as a learn-by-building approach, in which scientists attempt to engineer molecular pathways that do not exist in nature. In doing so, they test the limits of both natural and engineered organisms