Search tips
Search criteria

Results 1-25 (25)

Clipboard (0)

Select a Filter Below

more »
Year of Publication
more »
1.  Unprecedented High-Resolution View of Bacterial Operon Architecture Revealed by RNA Sequencing 
mBio  2014;5(4):e01442-14.
We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3′ transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5′ ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread.
We precisely mapped the 5′ and 3′ ends of RNA transcripts across the E. coli K-12 genome by using a single-nucleotide analytical approach. Our resulting high-resolution transcriptome maps show that ca. one-third of E. coli operons are complex, with internal promoters and terminators generating multiple transcription units and allowing differential gene expression within these operons. We discovered extensive antisense transcription that results from more than 500 operons, which fully overlap or extensively overlap adjacent divergent or convergent operons. The genomic regions corresponding to these antisense transcripts are highly conserved in E. coli (including Shigella species), although it remains to be proven whether or not they are functional. Our observations of features unearthed by single-nucleotide transcriptome mapping suggest that deeper layers of transcriptional regulation in bacteria are likely to be revealed in the future.
PMCID: PMC4161252  PMID: 25006232
3.  Escherichia coli K-12: a cooperatively developed annotation snapshot—2005 
Nucleic Acids Research  2006;34(1):1-9.
The goal of this group project has been to coordinate and bring up-to-date information on all genes of Escherichia coli K-12. Annotation of the genome of an organism entails identification of genes, the boundaries of genes in terms of precise start and end sites, and description of the gene products. Known and predicted functions were assigned to each gene product on the basis of experimental evidence or sequence analysis. Since both kinds of evidence are constantly expanding, no annotation is complete at any moment in time. This is a snapshot analysis based on the most recent genome sequences of two E.coli K-12 bacteria. An accurate and up-to-date description of E.coli K-12 genes is of particular importance to the scientific community because experimentally determined properties of its gene products provide fundamental information for annotation of innumerable genes of other organisms. Availability of the complete genome sequence of two K-12 strains allows comparison of their genotypes and mutant status of alleles.
PMCID: PMC1325200  PMID: 16397293
4.  Colony-live —a high-throughput method for measuring microbial colony growth kinetics— reveals diverse growth effects of gene knockouts in Escherichia coli 
BMC Microbiology  2014;14:171.
Precise quantitative growth measurements and detection of small growth changes in high-throughput manner is essential for fundamental studies of bacterial cell. However, an inherent tradeoff for measurement quality in high-throughput methods sacrifices some measurement quality. A key challenge has been how to enhance measurement quality without sacrificing throughput.
We developed a new high-throughput measurement system, termed Colony-live. Here we show that Colony-live provides accurate measurement of three growth values (lag time of growth (LTG), maximum growth rate (MGR), and saturation point growth (SPG)) by visualizing colony growth over time. By using a new normalization method for colony growth, Colony-live gives more precise and accurate growth values than the conventional method. We demonstrated the utility of Colony-live by measuring growth values for the entire Keio collection of Escherichia coli single-gene knockout mutants. By using Colony-live, we were able to identify subtle growth defects of single-gene knockout mutants that were undetectable by the conventional method quantified by fixed time-point camera imaging. Further, Colony-live can reveal genes that influence the length of the lag-phase and the saturation point of growth.
Measurement quality is critical to achieving the resolution required to identify unique phenotypes among a diverse range of phenotypes. Sharing high-quality genome-wide datasets should benefit many researchers who are interested in specific gene functions or the architecture of cellular systems. Our Colony-live system provides a new powerful tool to accelerate accumulation of knowledge of microbial growth phenotypes.
PMCID: PMC4096534  PMID: 24964927
Growth kinetics; Phenotype screening; High-throughput; Single-gene knockout; Keio collection; Lag time of growth (LTG); Maximum growth rate (MGR); Saturation point growth (SPG)
6.  The tRNA Thiolation Pathway Modulates the Intracellular Redox State in Escherichia coli 
Journal of Bacteriology  2013;195(9):2039-2049.
We have performed a screening of hydroxyurea (HU)-sensitive mutants using a single-gene-deletion mutant collection in Escherichia coli K-12. HU inhibits ribonucleotide reductase (RNR), an enzyme that catalyzes the formation of deoxyribonucleotides. Unexpectedly, seven of the mutants lacked genes that are required for the incorporation of sulfur into a specific tRNA modification base, 5-methylaminomethyl-2-thiouridine (mnm5s2U), via persulfide relay. We found that the expression of RNR in the mutants was reduced to about one-third both in the absence and presence of HU, while sufficient deoxynucleoside triphosphate (dNTP) was maintained in the mutants in the absence of HU but a shortage occurred in the presence of HU. Trans-supply of an RNR R2 subunit rescued the HU sensitivity of these mutants. The mutants showed high intracellular ATP/ADP ratios, and overexpression of Hda, which catalyzes the conversion of DnaA-ATP to DnaA-ADP, rescued the HU sensitivity of the mutants, suggesting that DnaA-ATP represses RNR expression. The high intracellular ATP/ADP ratios were due to high respiration activity in the mutants. Our data suggested that intracellular redox was inclined toward the reduced state in these mutants, which may explain a change in RNR activity by reduction of the catalytically formed disulfide bond and high respiration activity by the NADH reducing potential. The relation between persulfide relay and intracellular redox is discussed.
PMCID: PMC3624572  PMID: 23457245
7.  Characterization of Peptide Chain Length and Constituency Requirements for YejABEF-Mediated Uptake of Microcin C Analogues ▿† 
Journal of Bacteriology  2011;193(14):3618-3623.
Microcin C (McC), a natural antibacterial compound consisting of a heptapeptide attached to a modified adenosine, is actively taken up by the YejABEF transporter, after which it is processed by cellular aminopeptidases, releasing the nonhydrolyzable aminoacyl adenylate, an inhibitor of aspartyl-tRNA synthetase. McC analogues with variable length of the peptide moiety were synthesized and evaluated in order to characterize the substrate preferences of the YejABEF transporter. It was shown that a minimal peptide chain length of 6 amino acids and the presence of an N-terminal formyl-methionyl-arginyl sequence are required for transport.
PMCID: PMC3133342  PMID: 21602342
8.  Transcription, Processing, and Function of CRISPR Cassettes in Escherichia coli 
Molecular microbiology  2010;77(6):1367-1379.
CRISPR/Cas, bacterial and archaeal systems of interference with foreign genetic elements such as viruses or plasmids, consist of DNA loci called CRISPR cassettes (a set of variable spacers regularly separated by palindromic repeats) and associated cas genes. When a CRISPR spacer sequence exactly matches a sequence in a viral genome, the cell can become resistant to the virus. The CRISPR/Cas systems function through small RNAs originating from longer CRISPR cassette transcripts. While laboratory strains of Escherichia coli contain a functional CRISPR/Cas system (as judged by appearance of phage resistance at conditions of artificial co-overexpression of Cas genes and a CRISPR cassette engineered to target a λ phage), no natural phage resistance due to CRISPR system function was observed in this best-studied organism and no E. coli CRISPR spacer matches sequences of well-studied E. coli phages. To better understand the apparently “silent” E. coli CRISPR/Cas system, we systematically characterized processed transcripts from CRISPR cassettes. Using an engineered strain with genomically located spacer matching phage λ we show that endogenous levels of CRISPR cassette and cas genes expression allow only weak protection against infection with the phage. However, derepression of the CRISPR/Cas system by disruption of the hns gene leads to high level of protection.
PMCID: PMC2939963  PMID: 20624226
9.  Genome-Wide Screens: Novel Mechanisms in Colicin Import and Cytotoxicity 
Molecular microbiology  2009;73(4):571-585.
Only two new genes (fkpA and lepB) have been identified to be required for colicin cytotoxicity in the last twenty-five years. Genome-wide screening using the “Keio collection” to test sensitivity to colicins A, B, D, E1, E2, E3, E7 and N from groups A and B, allowed identification of novel genes affecting cytotoxicity and provided new information on mechanisms of action. The requirement of lipopolysaccharide for colN cytotoxicity resides specifically in the LPS inner-core and first glucose. ColA cytotoxicity is dependent on gmhB and rffT genes, which function in the biosynthesis of LPS and ECA. Of the tol genes that function in the cytoplasmic membrane translocon, colE1 requires tolA and tolR but not tolQ for activity. Pal, which interacts with the Tol network, is not required for cytotoxicity of group A colicins. Except for TolQRA, no cytoplasmic membrane protein is essential for cytotoxicity of group A colicins, implying that TolQRA provides the sole pathway for their insertion into/through the cytoplasmic membrane. The periplasmic protease that cleaves between the receptor and catalytic domains of colE7 was not identified, implying either that the responsible gene is essential for cell viability, or that more than one gene-product has the necessary proteolysis function.
PMCID: PMC3100173  PMID: 19650773
ASKA; BtuB; Keio; OmpF; translocon
10.  Global Regulation by the Seven-component Pi Signaling System 
Current opinion in microbiology  2010;13(2):198-203.
This review concerns how Escherichia coli detects environmental inorganic orthophosphate (Pi) to regulate genes of the phosphate (Pho) regulon by the PhoR/PhoB two-component system (TCS). Pi control by the PhoR/PhoB TCS is a paradigm of a bacterial signal transduction pathway in which occupancy of a cell surface receptor(s) controls gene expression in the cytoplasm. The Pi signaling pathway requires seven proteins, all of which probably interact in a membrane-associated signaling complex. Our latest studies show that Pi signaling involves three distinct processes, which appear to correspond to different states of the sensory histidine kinase PhoR: an inhibition state, an activation state, and a deactivation state. We describe a revised model for Pi signal transduction of the E. coli Pho regulon.
PMCID: PMC2847643  PMID: 20171928
11.  The Escherichia coli K-12 ORFeome: a resource for comparative molecular microbiology 
BMC Genomics  2010;11:470.
Systems biology and functional genomics require genome-wide datasets and resources. Complete sets of cloned open reading frames (ORFs) have been made for about a dozen bacterial species and allow researchers to express and study complete proteomes in a high-throughput fashion.
We have constructed an open reading frame (ORFeome) collection of 3974 or 94% of the known Escherichia coli K-12 ORFs in Gateway® entry vector pENTR/Zeo. The collection has been used for protein expression and protein interaction studies. For example, we have compared interactions among YgjD, YjeE and YeaZ proteins in E. coli, Streptococcus pneumoniae, and Staphylococcus aureus. We also compare this ORFeome with other Gateway-compatible bacterial ORFeomes and show its utility for comparative functional genomics.
The E. coli ORFeome provides a useful resource for functional genomics and other areas of protein research in a highly flexible format. Our comparison with other ORFeomes makes comparative analyses straighforward and facilitates direct comparisons of many proteins across many genomes.
PMCID: PMC3091666  PMID: 20701780
13.  A tool-kit for high-throughput, quantitative analyses of genetic interactions in E. coli 
Nature methods  2008;5(9):781-787.
Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in S. cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here, we describe a method based on F-driven conjugation, which allows for high-throughput generation of double mutants in E. coli. This method, termed Genetic Interaction ANalysis Technology for E. coli (GIANT-coli), permits us to systematically generate and array double mutant cells on solid media, in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify new negative (synthetic sickness/lethality) and positive (suppressive/epistatic) relationships. Finally, we describe a complementary strategy for suppressor mutant identification on a genome-wide level. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli.
PMCID: PMC2700713  PMID: 19160513
14.  Escherichia coli Peptidase A, B, or N Can Process Translation Inhibitor Microcin C▿  
Journal of Bacteriology  2008;190(7):2607-2610.
The heptapeptide-nucleotide microcin C (McC) targets aspartyl-tRNA synthetase. Upon its entry into a susceptible cell, McC is processed to release a nonhydrolyzable aspartyl-adenylate that inhibits aspartyl-tRNA synthetase, leading to the cessation of translation and cell growth. Here, we surveyed Escherichia coli cells with singly, doubly, and triply disrupted broad-specificity peptidase genes to show that any of three nonspecific oligopeptidases (PepA, PepB, or PepN) can effectively process McC. We also show that the rate-limiting step of McC processing in vitro is deformylation of the first methionine residue of McC.
PMCID: PMC2293190  PMID: 18223070
15.  Analysis of Promoter Targets for Escherichia coli Transcription Elongation Factor GreA In Vivo and In Vitro▿ †  
Journal of Bacteriology  2007;189(24):8772-8785.
Transcription elongation factor GreA induces nucleolytic activity of bacterial RNA polymerase (RNAP). In vitro, transcript cleavage by GreA contributes to transcription efficiency by (i) suppressing pauses and arrests, (ii) stimulating RNAP promoter escape, and (iii) enhancing transcription fidelity. However, it is unclear which of these functions is (are) most relevant in vivo. By comparing global gene expression profiles of Escherichia coli strains lacking Gre factors and strains expressing either the wild type (wt) or a functionally inactive GreA mutant, we identified genes that are potential targets of GreA action. Data analysis revealed that in the presence of chromosomally expressed GreA, 19 genes are upregulated; an additional 105 genes are activated upon overexpression of the wt but not the mutant GreA. Primer extension reactions with selected transcription units confirmed the gene array data. The most prominent stimulatory effect (threefold to about sixfold) of GreA was observed for genes of ribosomal protein operons and the tna operon, suggesting that transcript cleavage by GreA contributes to optimal expression levels of these genes in vivo. In vitro transcription assays indicated that the stimulatory effect of GreA upon the transcription of these genes is mostly due to increased RNAP recycling due to facilitated promoter escape. We propose that transcript cleavage during early stages of initiation is thus the main in vivo function of GreA. Surprisingly, the presence of the wt GreA also led to the decreased transcription of many genes. The mechanism of this effect is unknown and may be indirect.
PMCID: PMC2168603  PMID: 17766423
16.  The X-ray crystal structures of two constitutively active mutants of the E. coli PhoB receiver domain give insights into activation 
Journal of molecular biology  2006;366(2):626-641.
The PhoR/PhoB two-component system is a key regulatory protein network enabling Escherichia coli to respond to inorganic phosphate (Pi) starvation conditions by turning on Pho regulon genes for more efficient Pi uptake and use of alternative phosphorus sources. Under environmental Pi depletion, the response regulator (RR) component, PhoB, is phosphorylated at the receiver domain (RD), a process that requires Mg2+ bound at the active site. Phosphorylation of the RD relieves the inhibition of the PhoB effector domain (ED), a DNA-binding region that binds to Pho regulon promoters to activate transcription. The molecular details of the activation are proposed to involve dimerisation of the RD and a conformational change in the RD detected by the ED. The structure of the PhoB RD shows a symmetrical interaction involving α1, loop β5α5 and N-terminus of α5 elements, also seen in the complex of PhoB RD with Mg2+, in which helix α4 highly increases its flexibility. PhoB RD in complex with Mg2+ and BeF3− (an emulator of the phosphate moiety) undergoes a dramatic conformational change on helix α4 and shows another interaction involving α4, β5 and α5 segments. We have selected a series of constitutively active PhoB mutants (PhoBCA) that are able to turn on the Pho regulon promoters in the absence phosphorylation and, as they cannot be inactivated, should therefore mimic the active RD state of PhoB and its functional oligomerisation. We have analysed the PhoBCA RD crystal structures of two such mutants, Asp53Ala/Tyr102Cys and Asp10Ala/Asp53Glu. Interestingly, both mutants reproduce the homodimeric arrangement through the symmetric interface encountered in the unbound and magnesium-bound wild-type PhoB RD structures. Besides, the mutant RD structures show a modified active site organization as well as changes at helix α4 that correlate with repositioning of surrounding residues, like the active-site events indicator Trp54, putatively redifining the interaction with the ED in the full-length protein.
PMCID: PMC1855202  PMID: 17182055
PhoB; two-component signal transduction; receiver domain; X-ray crystal structure; asp-phosphorylation
17.  Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110 
Molecular Systems Biology  2006;2:2006.0007.
With the goal of solving the whole-cell problem with Escherichia coli K-12 as a model cell, highly accurate genomes were determined for two closely related K-12 strains, MG1655 and W3110. Completion of the W3110 genome and comparison with the MG1655 genome revealed differences at 267 sites, including 251 sites with short, mostly single-nucleotide, insertions or deletions (indels) or base substitutions (totaling 358 nucleotides), in addition to 13 sites with an insertion sequence element or defective prophage in only one strain and two sites for the W3110 inversion. Direct DNA sequencing of PCR products for the 251 regions with short indel and base disparities revealed that only eight sites are true differences. The other 243 discrepancies were due to errors in the original MG1655 sequence, including 79 frameshifts, one amino-acid residue deletion, five amino-acid residue insertions, 73 missense, and 17 silent changes within coding regions. Errors in the original MG1655 sequence (<1 per 13 000 bases) were mostly within portions sequenced with out-dated technology based on radioactive chemistry.
PMCID: PMC1681481  PMID: 16738553
crp mutation; E. coli K-12 genome; E. coli K-12 pedigree; genome corrections; rpoS mutations
18.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection 
Molecular Systems Biology  2006;2:2006.0008.
We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants—the ‘Keio collection'—provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (
PMCID: PMC1681482  PMID: 16738554
bacterial functional genomics; E. coli/gene; essential gene; knockout mutants; resources; systems biology
19.  Phenotype MicroArray Analysis of Escherichia coli K-12 Mutants with Deletions of All Two-Component Systems 
Journal of Bacteriology  2003;185(16):4956-4972.
Two-component systems are the most common mechanism of transmembrane signal transduction in bacteria. A typical system consists of a histidine kinase and a partner response regulator. The histidine kinase senses an environmental signal, which it transmits to its partner response regulator via a series of autophosphorylation, phosphotransfer, and dephosphorylation reactions. Much work has been done on particular systems, including several systems with regulatory roles in cellular physiology, communication, development, and, in the case of bacterial pathogens, the expression of genes important for virulence. We used two methods to investigate two-component regulatory systems in Escherichia coli K-12. First, we systematically constructed mutants with deletions of all two-component systems by using a now-standard technique of gene disruption (K. A. Datsenko and B. L. Wanner, Proc. Natl. Acad. Sci. USA 97:6640-6645, 2000). We then analyzed these deletion mutants with a new technology called Phenotype MicroArrays, which permits assays of nearly 2,000 growth phenotypes simultaneously. In this study we tested 100 mutants, including mutants with individual deletions of all two-component systems and several related genes, including creBC-regulated genes (cbrA and cbrBC), phoBR-regulated genes (phoA, phoH, phnCDEFGHIJKLMNOP, psiE, and ugpBAECQ), csgD, luxS, and rpoS. The results of this battery of nearly 200,000 tests provided a wealth of new information concerning many of these systems. Of 37 different two-component mutants, 22 showed altered phenotypes. Many phenotypes were expected, and several new phenotypes were also revealed. The results are discussed in terms of the biological roles and other information concerning these systems, including DNA microarray data for a large number of the same mutants. Other mutational effects are also discussed.
PMCID: PMC166450  PMID: 12897016
20.  Escherichia coli phnN, Encoding Ribose 1,5-Bisphosphokinase Activity (Phosphoribosyl Diphosphate Forming): Dual Role in Phosphonate Degradation and NAD Biosynthesis Pathways 
Journal of Bacteriology  2003;185(9):2793-2801.
An enzymatic pathway for synthesis of 5-phospho-d-ribosyl α-1-diphosphate (PRPP) without the participation of PRPP synthase was analyzed in Escherichia coli. This pathway was revealed by selection for suppression of the NAD requirement of strains with a deletion of the prs gene, the gene encoding PRPP synthase (B. Hove-Jensen, J. Bacteriol. 178:714-722, 1996). The new pathway requires three enzymes: phosphopentomutase, ribose 1-phosphokinase, and ribose 1,5-bisphosphokinase. The latter activity is encoded by phnN; the product of this gene is required for phosphonate degradation, but its enzymatic activity has not been determined previously. The reaction sequence is ribose 5-phosphate → ribose 1-phosphate → ribose 1,5-bisphosphate → PRPP. Alternatively, the synthesis of ribose 1-phosphate in the first step, catalyzed by phosphopentomutase, can proceed via phosphorolysis of a nucleoside, as follows: guanosine + Pi → guanine + ribose 1-phosphate. The ribose 1,5-bisphosphokinase-catalyzed phosphorylation of ribose 1,5-bisphosphate is a novel reaction and represents the first assignment of a specific chemical reaction to a polypeptide required for cleavage of a carbon-phosphorus (C—P) bond by a C-P lyase. The phnN gene was manipulated in vitro to encode a variant of ribose 1,5-bisphosphokinase with a tail consisting of six histidine residues at the carboxy-terminal end. PhnN was purified almost to homogeneity and characterized. The enzyme accepted ATP but not GTP as a phosphoryl donor, and it used ribose 1,5-bisphosphate but not ribose, ribose 1-phosphate, or ribose 5-phosphate as a phosphoryl acceptor. The identity of the reaction product as PRPP was confirmed by coupling the ribose 1,5-bisphosphokinase activity to the activity of xanthine phosphoribosyltransferase in the presence of xanthine, which resulted in the formation of 5′-XMP, and by cochromatography of the reaction product with authentic PRPP.
PMCID: PMC154390  PMID: 12700258
21.  Conditional-Replication, Integration, Excision, and Retrieval Plasmid-Host Systems for Gene Structure-Function Studies of Bacteria 
Journal of Bacteriology  2001;183(21):6384-6393.
We have developed a series of powerful and versatile conditional-replication, integration, and modular (CRIM) plasmids. CRIM plasmids can be replicated at medium or high copy numbers in different hosts for making gene (or mutant) libraries. They can be integrated in single copies into the chromosomes of Escherichia coli and related bacteria to study gene function under normal physiological conditions. They can be excised from the chromosome, e.g., to verify that phenotypes are caused by their presence. Furthermore, they can be retrieved singly or en masse for subsequent molecular analyses. CRIM plasmids are integrated into the chromosome by site-specific recombination at one of five different phage attachment sites. Integrants are selected as antibiotic-resistant transformations. Since CRIM plasmids encode different forms of resistance, several can be used together in the same cell for stable expression of complex metabolic or regulatory pathways from diverse sources. Following integration, integrants are stably maintained in the absence of antibiotic selection. Each CRIM plasmid has a polylinker or one of several promoters for ectopic expression of the inserted DNA. Their modular design allows easy construction of new variants with different combinations of features. We also report a series of easily curable, low-copy-number helper plasmids encoding all the requisite Int proteins alone or with the respective Xis protein. These helper plasmids facilitate integration, excision (“curing”), or retrieval of the CRIM plasmids.
PMCID: PMC100134  PMID: 11591683
22.  Dual Transcriptional Regulation of the Escherichia coli Phosphate-Starvation-Inducible psiE Gene of the Phosphate Regulon by PhoB and the Cyclic AMP (cAMP)-cAMP Receptor Protein Complex 
Journal of Bacteriology  2000;182(19):5596-5599.
We have shown that the Escherichia coli phosphate-starvation-inducible psiE gene is regulated by both phosphate and the carbon source by using both lacZ and chloramphenicol acetyltransferase gene (cat) fusions. Yet, under all conditions tested, a single transcriptional start site lying 7 bp downstream of a predicted −10 region was revealed by primer extension analysis. DNase I footprinting showed that the PhoB transcriptional-activator protein protects two predicted pho boxes lying upstream of and near the −35 promoter region. Similar analysis showed that the cyclic AMP (cAMP)-cAMP receptor protein (cAMP-CRP) complex binds a region that overlaps with the downstream pho box. These results, together with measurements of the in vivo psiE promoter activity under various conditions, show that expression of the psiE gene is under direct positive and negative control by PhoB and cAMP-CRP, respectively.
PMCID: PMC111007  PMID: 10986267
23.  Multiple Factors Independently Regulate hilA and Invasion Gene Expression in Salmonella enterica Serovar Typhimurium 
Journal of Bacteriology  2000;182(7):1872-1882.
HilA activates the expression of Salmonella enterica serovar Typhimurium invasion genes. To learn more about regulation of hilA, we isolated Tn5 mutants exhibiting reduced hilA and/or invasion gene expression. In addition to expected mutations, we identified Tn5 insertions in pstS, fadD, flhD, flhC, and fliA. Analysis of the pstS mutant indicates that hilA and invasion genes are repressed by the response regulator PhoB in the absence of the Pst high-affinity inorganic phosphate uptake system. This system is required for negative control of the PhoR-PhoB two-component regulatory system, suggesting that hilA expression may be repressed by PhoR-PhoB under low extracellular inorganic phosphate conditions. FadD is required for uptake and degradation of long-chain fatty acids, and our analysis of the fadD mutant indicates that hilA is regulated by a FadD-dependent, FadR-independent mechanism. Thus, fatty acid derivatives may act as intracellular signals to regulate hilA expression. flhDC and fliA encode transcription factors required for flagellum production, motility, and chemotaxis. Complementation studies with flhC and fliA mutants indicate that FliZ, which is encoded in an operon with fliA, activates expression of hilA, linking regulation of hilA with motility. Finally, epistasis tests showed that PhoB, FadD, FliZ, SirA, and EnvZ act independently to regulate hilA expression and invasion. In summary, our screen has identified several distinct pathways that can modulate S. enterica serovar Typhimurium's ability to express hilA and invade host cells. Integration of signals from these different pathways may help restrict invasion gene expression during infection.
PMCID: PMC101869  PMID: 10714991
24.  Use of New Methods for Construction of Tightly Regulated Arabinose and Rhamnose Promoter Fusions in Studies of the Escherichia coli Phosphate Regulon 
Journal of Bacteriology  1998;180(5):1277-1286.
Escherichia coli genes regulated by environmental inorganic phosphate (Pi) levels form the phosphate (Pho) regulon. This regulation requires seven proteins, whose synthesis is under autogenous control, including response regulator PhoB, its partner, histidine sensor kinase PhoR, all four components of the Pi-specific transport (Pst) system (PstA, PstB, PstC, and PstS), and a protein of unknown function called PhoU. Here we examined the effects of uncoupling PhoB synthesis and PhoR synthesis from their normal controls by placing each under the tight control of the arabinose-regulated ParaB promoter or the rhamnose-regulated PrhaB promoter. To do this, we made allele replacement plasmids that may be generally useful for construction of ParaB or PrhaB fusions and for recombination of them onto the E. coli chromosome at the araCBAD or rhaRSBAD locus, respectively. Using strains carrying such single-copy fusions, we showed that a PrhaB fusion is more tightly regulated than a ParaB fusion in that a PrhaB-phoR+ fusion but not a ParaB-phoR+ fusion shows a null phenotype in the absence of its specific inducer. Yet in the absence of induction, both ParaB-phoB+ and PrhaB-phoB+ fusions exhibit a null phenotype. These data indicate that less PhoR than PhoB is required for transcriptional activation of the Pho regulon, which is consistent with their respective modes of action. We also used these fusions to study PhoU. Previously, we had constructed strains with precise ΔphoU mutations. However, we unexpectedly found that such ΔphoU mutants have a severe growth defect (P. M. Steed and B. L. Wanner, J. Bacteriol. 175:6797–6809, 1993). They also readily give rise to compensatory mutants with lesions in phoB, phoR, or a pst gene, making their study particularly difficult. Here we found that, by using ParaB-phoB+, PrhaB-phoB+, or PrhaB-phoR+ fusions, we were able to overcome the extremely deleterious growth defect of a Pst+ ΔphoU mutant. The growth defect is apparently a consequence of high-level Pst synthesis resulting from autogenous control of PhoB and PhoR synthesis in the absence of PhoU.
PMCID: PMC107018  PMID: 9495769
25.  Regulation of lac Operon Expression: Reappraisal of the Theory of Catabolite Repression 
Journal of Bacteriology  1978;136(3):947-954.
The physiological state of Escherichia coli with respect to (permanent) catabolite repression was assessed by measuring the steady-state level of β-galactosidase in induced or in constitutive cells under a variety of growth conditions. Four results were obtained. (i) Catabolite repression had a major effect on fully induced or constitutive expression of the lac gene, and the magnitude of this effect was found to be dependent on the promoter structure; cells with a wild-type lac promoter showed an 18-fold variation in lac expression, and cells with the lacP37 (formerly lac-L37) promoter exhibited several hundred-fold variation. (ii) Exogenous adenosine cyclic 3′,5′-monophosphoric acid (cAMP) could not abolish catabolite repression, even though several controls demonstrated that cAMP was entering the cells in significant amounts. (Rapid intracellular degradation of cAMP could not be ruled out.) (iii) Neither the growth rate nor the presence of biosynthetic products altered the degree of catabolite repression; all variation could be related to the catabolites present in the growth medium. (iv) Slowing by imposing an amino acid restriction decreased the differential rate of β-galactosidase synthesis from the wild-type lac promoter when bacteria were cultured in either the absence or presence of cAMP; this decreased lac expression also occurred when the bacteria harbored the catabolite-insensitive lacP5 (formerly lacUV5) promoter mutation. These findings support the idea that (permanent) catabolite repression is set by the catabolites in the growth medium and may not be related to an imbalance between catabolism and anabolism.
PMCID: PMC218529  PMID: 214424

Results 1-25 (25)