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author:("Zhang, zhong")
1.  Transposon-Mediated Adaptive and Directed Mutations and Their Potential Evolutionary Benefits 
Transposons, mobile genetic elements that can hop from one chromosomal location to another, are known to be both beneficial and deleterious to the cell that bears them. Their value in accelerating evolutionary adaptation is well recognized. We herein summarize published research dealing with these elements and then move on to review our own research efforts which focus on a small transposon that can induce mutations under the control of host factors in a process that phenotypically and mechanistically conforms to the definition of ‘directed mutation’. Directed mutations occur at higher frequencies when they are beneficial, being induced by the stress condition that they relieve. Here, we review evidence for transposon-mediated directed mutation in Escherichia coli. Deletion mutants in the crp gene can not grow on glycerol (Glp−); however, these cells mutate specifically to efficient glycerol utilization (Glp+) at rates that are greatly enhanced by the presence of glycerol or the loss of the glycerol repressor (GlpR). These rates are greatly depressed by glucose or by glpR overexpression. Of the four tandem GlpR-binding sites (O1–O4) in the control region of the glpFK operon, O4 (downstream) specifically controls glpFK expression while O1 (upstream) controls mutation rate. Mutation is due to insertion of the small transposon IS5 into a specific site just upstream of the glpFK promoter. Mutational control by the glycerol regulon repressor GlpR is independent of the selection and assay procedures, and IS5 insertion into other gene activation sites is unaffected by the presence of glycerol or the loss of GlpR. The results establish the principle of transposon-mediated directed mutation, identify a protein responsible for its regulation, and define essential aspects of the mechanism.
doi:10.1159/000333108
PMCID: PMC3697268  PMID: 22248543
Transposon; Insertion sequence; Directed mutation; Adaptive evolution; Transposition regulation
2.  Need-based activation of ammonium uptake in Escherichia coli 
E. coli uses an energetically costly ammonium uptake process to maintain growth in ammonium-limited conditions. Detailed analysis of this process reveals that cells employ a novel strategy to activate ammonium transport only as necessary for cell growth.
Ammonium transport by AmtB is activated abruptly upon reduction in the ammonium concentration in the medium, after the ammonium assimilation capacity is maximized.Under different growth conditions that provide cells with different growth rates even when ammonium is replete, ammonium transport by AmtB is employed only as necessary to maintain cell growth at the maximum rate.The known molecular interactions reveal an integral feedback mechanism underlying the need-based control of AmtB activity, mediated by α-ketoglutarate (aKG).The two signaling molecules, glutamine and aKG, provide seamless coordination between ammonium assimilation and ammonium transport.
The efficient sequestration of nutrients is vital for the growth and survival of microorganisms. Some nutrients, such as CO2 and NH3, are readily diffusible across the cell membrane. The large membrane permeability of these nutrients obviates the need of transporters when the ambient level is high. When the ambient level is low, however, maintaining a high intracellular nutrient level against passive back diffusion is both challenging and costly. Here, we study the delicate management of ammonium (NH4+/NH3) sequestration by E. coli cells using microfluidic chemostats. We find that as the ambient ammonium concentration is reduced, E. coli cells first maximize their ability to assimilate the gaseous NH3 diffusing into the cytoplasm and then abruptly activate ammonium transport. The onset of transport varies under different growth conditions, but always occurring just as needed to maintain growth. Quantitative modeling of known interactions reveals an integral feedback mechanism by which this need-based uptake strategy is implemented. This novel strategy ensures that the expensive cost of upholding the internal ammonium concentration against back diffusion is kept at a minimum.
doi:10.1038/msb.2012.46
PMCID: PMC3472687  PMID: 23010999
active transport; futile cycle; integral feedback; metabolic coordination; microfluidics
3.  Growth-rate dependent global effects on gene expression in bacteria 
Cell  2009;139(7):1366.
Summary
Bacterial gene expression depends not only on specific regulations but also directly on bacterial growth, because important global parameters such as the abundance of RNA polymerases and ribosomes are all growth-rate dependent. Understanding these global effects is necessary for a quantitative understanding of gene regulation and for the robust design of synthetic genetic circuits. The observed growth-rate dependence of constitutive gene expression can be explained by a simple model using the measured growth-rate dependence of the relevant cellular parameters. More complex growth dependences for genetic circuits involving activators, repressors and feedback control were analyzed, and salient features were verified experimentally using synthetic circuits. The results suggest a novel feedback mechanism mediated by general growth-dependent effects and not requiring explicit gene regulation, if the expressed protein affects cell growth. This mechanism can lead to growth bistability and promote the acquisition of important physiological functions such as antibiotic resistance and tolerance (persistence).
doi:10.1016/j.cell.2009.12.001
PMCID: PMC2818994  PMID: 20064380
growth rate; constitutive gene expression; gene regulation; genetic circuits; bistability; growth feedback; antibiotics; persister cells
4.  A Mechanism of Transposon-mediated Directed Mutation 
Molecular microbiology  2009;74(1):29-43.
Directed mutation is a proposed process that allows mutations to occur at higher frequencies when they are beneficial. Until now, the existence of such a process has been controversial. Here we describe a novel mechanism of directed mutation mediated by the transposon, IS5 in Escherichia coli. crp deletion mutants mutate specifically to glycerol utilization (Glp+) at rates that are enhanced by glycerol or the loss of the glycerol repressor (GlpR), depressed by glucose or glpR overexpression, and RecA-independent. Of the four tandem GlpR-binding sites (O1–O4) upstream of the glpFK operon, O4 specifically controls glpFK expression while O1 primarily controls mutation rate in a process mediated by IS5 hopping to a specific site on the E. coli chromosome upstream of the glpFK promoter. IS5 insertion into other gene activation sites is unaffected by the presence of glycerol or the loss of GlpR. The results establish an example of transposon-mediated directed mutation, identify the protein responsible, and define the mechanism involved.
doi:10.1111/j.1365-2958.2009.06831.x
PMCID: PMC2973706  PMID: 19682247
Directed mutation; insertion sequence; transposition regulation; transposon; transposable genetic element
5.  Precise Excision of IS5 from the Intergenic Region between the fucPIK and the fucAO Operons and Mutational Control of fucPIK Operon Expression in Escherichia coli ▿  
Journal of Bacteriology  2010;192(7):2013-2019.
Excision of transposable genetic elements from host DNA occurs at low frequencies and is usually imprecise. A common insertion sequence element in Escherichia coli, IS5, has been shown to provide various benefits to its host by inserting into specific sites. Precise excision of this element had not previously been demonstrated. Using a unique system, the fucose (fuc) regulon, in which IS5 insertion and excision result in two distinct selectable phenotypes, we have demonstrated that IS5 can precisely excise from its insertion site, restoring the wild-type phenotype. In addition to precise excision, several “suppressor” insertion, deletion, and point mutations restore the wild-type Fuc+ phenotype to various degrees without IS5 excision. The possible bases for these observations are discussed.
doi:10.1128/JB.01085-09
PMCID: PMC2838058  PMID: 20097855
6.  A Novel Mechanism of Transposon-Mediated Gene Activation 
PLoS Genetics  2009;5(10):e1000689.
Transposable Insertion Sequences (IS elements) have been shown to provide various benefits to their hosts via gene activation or inactivation under stress conditions by appropriately inserting into specific chromosomal sites. Activation is usually due to derepression or introduction of a complete or partial promoter located within the element. Here we define a novel mechanism of gene activation by the transposon IS5 in Escherichia coli. The glycerol utilization operon, glpFK, that is silent in the absence of the cAMP-Crp complex, is activated by IS5 when inserted upstream of its promoter. High-level expression is nearly constitutive, only mildly dependent on glycerol, glucose, GlpR, and Crp, and allows growth at a rate similar to or more rapid than that of wild-type cells. Expression is from the glpFK promoter and dependent on (1) the DNA phase, (2) integration host factor (IHF), and (3) a short region at the 3′ end of IS5 harboring a permanent bend and an IHF binding site. The lacZYA operon is also subject to such activation in the absence of Crp. Thus, we have defined a novel mechanism of gene activation involving transposon insertion that may be generally applicable to many organisms.
Author Summary
Transposons are “jumping genes” that can move from one location within a genome to another. Insertion of a transponson changes the DNA sequence and therefore gives rise to mutations that can activate or inactivate gene expression. Here, we demonstrate for the first time that one such transposon, Insertion Sequence 5 (IS5), when positioned upstream of a metabolic operon (glpFK) of E. coli, can activate the otherwise cryptic expression of the operon. This effect is due solely to a short region at the 3′ end of IS5 that harbors a permanent bend and an overlapping nucleoid protein binding site, both of which are required for maximal gene expression. We demonstrate the importance of phasing and conclude that DNA looping probably plays a role. We also show that another operon, the E. coli lactose operon (lacZYA), can be similarly activated by IS5. Although this is the first study to show that unique sequences within a transposon are necessary and sufficient to activate a downstream silent promoter, similar mechanisms of gene activation may occur for other operons.
doi:10.1371/journal.pgen.1000689
PMCID: PMC2753651  PMID: 19834539
7.  Correction: Quantitative Characteristics of Gene Regulation by Small RNA 
PLoS Biology  2008;6(1):10.1371/journal.pbio.0060005.
doi:10.1371/journal.pbio.0060005
PMCID: PMC2214803
8.  Quantitative Characteristics of Gene Regulation by Small RNA 
PLoS Biology  2007;5(9):e229.
An increasing number of small RNAs (sRNAs) have been shown to regulate critical pathways in prokaryotes and eukaryotes. In bacteria, regulation by trans-encoded sRNAs is predominantly found in the coordination of intricate stress responses. The mechanisms by which sRNAs modulate expression of its targets are diverse. In common to most is the possibility that interference with the translation of mRNA targets may also alter the abundance of functional sRNAs. Aiming to understand the unique role played by sRNAs in gene regulation, we studied examples from two distinct classes of bacterial sRNAs in Escherichia coli using a quantitative approach combining experiment and theory. Our results demonstrate that sRNA provides a novel mode of gene regulation, with characteristics distinct from those of protein-mediated gene regulation. These include a threshold-linear response with a tunable threshold, a robust noise resistance characteristic, and a built-in capability for hierarchical cross-talk. Knowledge of these special features of sRNA-mediated regulation may be crucial toward understanding the subtle functions that sRNAs can play in coordinating various stress-relief pathways. Our results may also help guide the design of synthetic genetic circuits that have properties difficult to attain with protein regulators alone.
Author Summary
The activation of stress response programs, while crucial for the survival of a bacterial cell under stressful conditions, is costly in terms of energy and substrates and risky to the normal functions of the cell. Stress response is therefore tightly regulated. A recently discovered layer of regulation involves small RNA molecules, which bind the mRNA transcripts of their targets, inhibit their translation, and promote their cleavage. To understand the role that small RNA plays in regulation, we have studied the quantitative aspects of small RNA regulation by integrating mathematical modeling and quantitative experiments in Escherichia coli. We have demonstrated that small RNAs can tightly repress their target genes when their synthesis rate is smaller than some threshold, but have little or no effect when the synthesis rate is much larger than that threshold. Importantly, the threshold level is set by the synthesis rate of the small RNA itself and can be dynamically tuned. The effect of biochemical properties—such as the binding affinity of the two RNA molecules, which can only be altered on evolutionary time scales—is limited to setting a hierarchical order among different targets of a small RNA, facilitating in principle a global coordination of stress response.
In bacteria, small RNAs can regulate the expression of genes at the translational level. The many advantages of this type of control include a tuneable threshold response and resistance to biochemical noise.
doi:10.1371/journal.pbio.0050229
PMCID: PMC1994261  PMID: 17713988
9.  Functional Interactions between the Carbon and Iron Utilization Regulators, Crp and Fur, in Escherichia coli 
Journal of Bacteriology  2005;187(3):980-990.
In Escherichia coli, the ferric uptake regulator (Fur) controls expression of the iron regulon in response to iron availability while the cyclic AMP receptor protein (Crp) regulates expression of the carbon regulon in response to carbon availability. We here identify genes subject to significant changes in expression level in response to the loss of both Fur and Crp. Many iron transport genes and several carbon metabolic genes are subject to dual control, being repressed by the loss of Crp and activated by the loss of Fur. However, the sodB gene, encoding superoxide dismutase, and the aceBAK operon, encoding the glyoxalate shunt enzymes, show the opposite responses, being activated by the loss of Crp and repressed by the loss of Fur. Several other genes including the sdhA-D, sucA-D, and fumA genes, encoding key constituents of the Krebs cycle, proved to be repressed by the loss of both transcription factors. Finally, the loss of both Crp and Fur activated a heterogeneous group of genes under σS control encoding, for example, the cyclopropane fatty acid synthase, Cfa, the glycogen synthesis protein, GlgS, the 30S ribosomal protein, S22, and the mechanosensitive channel protein, YggB. Many genes appeared to be regulated by the two transcription factors in an apparently additive fashion, but apparent positive or negative cooperativity characterized several putative Crp/Fur interactions. Relevant published data were evaluated, putative Crp and Fur binding sites were identified, and representative results were confirmed by real-time PCR. Molecular explanations for some, but not all, of these effects are provided.
doi:10.1128/JB.187.3.980-990.2005
PMCID: PMC545712  PMID: 15659676
10.  Transcriptome Analysis of Crp-Dependent Catabolite Control of Gene Expression in Escherichia coli 
Journal of Bacteriology  2004;186(11):3516-3524.
We report here the transcriptome analyses of highly expressed genes that are subject to catabolite repression or activation mediated by the cyclic AMP receptor protein (Crp). The results reveal that many operons encoding enzymes of central carbon metabolic pathways (e.g., Krebs cycle enzymes), as well as transporters and enzymes that initiate carbon metabolism, are subject to direct Crp-mediated catabolite repression. By contrast, few enzyme-encoding genes (direct regulation) but many ribosomal protein- and tRNA-encoding genes (indirect regulation) are subject to Crp-dependent glucose activation. Additionally, Crp mediates strong indirect catabolite repression of many cytoplasmic stress response proteins, including the major chaperone proteins, five ATP-dependent protease complexes, and several cold and heat shock proteins. These results were confirmed by (i) phenotypic analyses, (ii) real-time PCR studies, (iii) reporter gene fusion assays, and (iv) previously published reports about representative genes. The results serve to define and extend our appreciation of the Crp regulon.
doi:10.1128/JB.186.11.3516-3524.2004
PMCID: PMC415760  PMID: 15150239
11.  The Ascorbate Transporter of Escherichia coli 
Journal of Bacteriology  2003;185(7):2243-2250.
The sgaTBA genes of Escherichia coli encode a putative 12-transmembrane α-helical segment (12 TMS) transporter, an enzyme IIB-like protein and an enzyme IIA-like protein of the phosphotransferase system (PTS), respectively. We show that all three proteins as well as the energy-coupling PTS proteins, enzyme I and HPr, are required for the anaerobic utilization and uptake of l-ascorbate in vivo and its phosphoenolpyruvate-dependent phosphorylation in vitro. The transporter exhibits an apparent Km for l-ascorbate of 9 μM and is highly specific. The sgaTBA genes are regulated at the transcriptional level by the yjfQ gene product, as well as by Crp and Fnr. The yjfR gene product is essential for l-ascorbate utilization and probably encodes a cytoplasmic l-ascorbate 6-phosphate lactonase. We conclude that SgaT represents a novel prototypical enzyme IIC that functions with SgaA and SgaB to allow phosphoryl transfer from HPr(his-P) to l-ascorbate via the phosphoryl transfer pathway: PEP → enzyme I-P → HPr-P → IIA-\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{ \,\substack{ ^{SgaA} \\ P \\ }\, }\end{equation*}\end{document} → IIB-\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{ \,\substack{ ^{SgaB} \\ P \\ }\, }\end{equation*}\end{document}\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{ \,\substack{ ^{IIC} \\ {\rightarrow} \\ }\, }\end{equation*}\end{document}SgaTl-ascorbate-6-P.
doi:10.1128/JB.185.7.2243-2250.2003
PMCID: PMC151508  PMID: 12644495
12.  A Second Quorum-Sensing System Regulates Cell Surface Properties but Not Phenazine Antibiotic Production in Pseudomonas aureofaciens 
The root-associated biological control bacterium Pseudomonas aureofaciens 30-84 produces a range of exoproducts, including protease and phenazines. Phenazine antibiotic biosynthesis by phzXYFABCD is regulated in part by the PhzR-PhzI quorum-sensing system. Mutants defective in phzR or phzI produce very low levels of phenazines but wild-type levels of exoprotease. In the present study, a second genomic region of strain 30-84 was identified that, when present in trans, increased β-galactosidase activity in a genomic phzB::lacZ reporter and partially restored phenazine production to a phzR mutant. Sequence analysis identified two adjacent genes, csaR and csaI, that encode members of the LuxR-LuxI family of regulatory proteins. No putative promoter region is present upstream of the csaI start codon and no lux box-like element was found in either the csaR promoter or the 30-bp intergenic region between csaR and csaI. Both the PhzR-PhzI and CsaR-CsaI systems are regulated by the GacS-GacA two-component regulatory system. In contrast to the multicopy effects of csaR and csaI in trans, a genomic csaR mutant (30-84R2) and a csaI mutant (30-84I2) did not exhibit altered phenazine production in vitro or in situ, indicating that the CsaR-CsaI system is not involved in phenazine regulation in strain 30-84. Both mutants also produced wild-type levels of protease. However, disruption of both csaI and phzI or both csaR and phzR eliminated both phenazine and protease production completely. Thus, the two quorum-sensing systems do not interact for phenazine regulation but do interact for protease regulation. Additionally, the CsaI N-acylhomoserine lactone (AHL) signal was not recognized by the phenazine AHL reporter 30-84I/Z but was recognized by the AHL reporters Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136(pCF240). Inactivation of csaR resulted in a smooth mucoid colony phenotype and formation of cell aggregates in broth, suggesting that CsaR is involved in regulating biosynthesis of cell surface components. Strain 30-84I/I2 exhibited mucoid colony and clumping phenotypes similar to those of 30-84R2. Both phenotypes were reversed by complementation with csaR-csaI or by the addition of the CsaI AHL signal. Both quorum-sensing systems play a role in colonization by strain 30-84. Whereas loss of PhzR resulted in a 6.6-fold decrease in colonization by strain 30-84 on wheat roots in natural soil, a phzR csaR double mutant resulted in a 47-fold decrease. These data suggest that gene(s) regulated by the CsaR-CsaI system also plays a role in the rhizosphere competence of P. aureofaciens 30-84.
doi:10.1128/AEM.67.9.4305-4315.2001
PMCID: PMC93161  PMID: 11526037

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