Deborah A. Siegele

Global Analysis of Escherichia coli Gene Expression during the Acetate-Induced Acid Tolerance Response

The ability of Escherichia coli to survive at low pH is strongly affected by environmental factors, such as composition of the growth medium and growth phase. Exposure to short-chain fatty acids, such as acetate, proprionate, and butyrate, at neutral or nearly neutral pH has also been shown to increase acid survival of E. coli and Salmonella enterica serovar Typhimurium. To investigate the basis for acetate-induced acid tolerance in E. coli O157:H7, genes whose expression was altered by exposure to acetate were identified using gene arrays. The expression of 60 genes was reduced by at least twofold; of these, 48 encode components of the transcription-translation machinery. Expression of 26 genes increased twofold or greater following treatment with acetate. This included six genes whose products are known to be important for survival at low pH. Five of these genes, as well as six other acetate-induced genes, are members of the E. coli RpoS regulon. RpoS, the stress sigma factor, is known to be required for acid tolerance induced by growth at nonlethal low pH or by entry into stationary phase. Disruption of the rpoS gene by a transposon insertion mutation also prevented acetate-induced acid tolerance. However, induction of RpoS expression did not appear to be sufficient to activate the acid tolerance response. Treatment with either NaCl or sodium acetate (pH 7.0) increased expression of an rpoS::lacZ fusion protein, but only treatment with acetate increased acid survival.

High-throughput, quantitative analyses of genetic interactions in E. coli.

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 Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor–driven conjugation, which allows for high-throughput generation of double mutants in Escherichia 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 previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli.

The Gene Ontology's Reference Genome Project: A Unified Framework for Functional Annotation across Species

The Gene Ontology (GO) is a collaborative effort that provides structured vocabularies for annotating the molecular function, biological role, and cellular location of gene products in a highly systematic way and in a species-neutral manner with the aim of unifying the representation of gene function across different organisms. Each contributing member of the GO Consortium independently associates GO terms to gene products from the organism(s) they are annotating. Here we introduce the Reference Genome project, which brings together those independent efforts into a unified framework based on the evolutionary relationships between genes in these different organisms. The Reference Genome project has two primary goals: to increase the depth and breadth of annotations for genes in each of the organisms in the project, and to create data sets and tools that enable other genome annotation efforts to infer GO annotations for homologous genes in their organisms. In addition, the project has several important incidental benefits, such as increasing annotation consistency across genome databases, and providing important improvements to the GOs logical structure and biological content.

The terminase of bacteriophage λ: Functional domains for cosB binding and multimer assembly☆

Terminase is a protein complex involved in lambda DNA packaging. The subunits of terminase, gpNul and gpA, are the products of genes Nul and A. The actions of terminase include DNA binding, prohead binding and DNA nicking. Phage 21 is a lambdoid phage that also makes a terminase, encoded by genes 1 and 2. The terminases of 21 and lambda are not interchangeable. This specificity involves two actions of terminase; DNA binding and prohead binding. In addition, the subunits of lambda terminase will not form functional multimers with the subunits of 21 terminase. lambda-21 hybrid phages can be produced as a result of recombination. We describe here lambda-21 hybrid phages that have hybrid terminase genes. The packaging specificities of the hybrids and the structure of their genes were compared in order to identify functional domains of terminase. The packaging specificities were determined in vivo by complementation tests and helper packaging experiments. Restriction enzyme site mapping and sequencing located the sites at which recombination occurred to produce the hybrid phages. lambda-21 hybrid 51 carries the lambda A gene, and a hybrid 1/Nul gene. The crossover that produced this phage occurred near the middle of the 1 and Nul genes. The amino-terminal portion of the hybrid protein is homologous to gp1 and the carboxy-terminal portion is homologous to gpNul. It binds to 21 DNA and forms functional multimers with gpA, providing evidence that the amino-terminal portion of gpNul is involved in DNA binding and the carboxy-terminal portion of gpNul is involved in the interaction with gpA. lambda-21 hybrid 54 has a hybrid 2/A gene. The amino terminus of the hybrid protein of lambda-21 hybrid 54 is homologous with gp2. This protein forms functional multimers only with gp1, providing evidence that the amino terminus of gpA is involved in the interaction with gpNul. These studies identify three functional domains of terminase.

A functional domain of bacteriophage λ terminase for prohead binding

Terminase is a multifunctional protein complex involved in DNA packaging during bacteriophage λ assembly. Terminase is made of gpNu1 and gpA, the products of the phage λ Nu1 and A genes. Early during DNA packaging terminase binds to λ DNA to form a complex called complex I. Terminase is required for the binding of proheads by complex I to form a DNA : terminase : prohead complex known as complex II. Terminase remains associated with the DNA during encapsidation. The other known role for terminase in packaging is the production of staggered nicks in the DNA thereby generating the cohesive ends. Lambdoid phage 21 has cohesive ends identical to those of λ . The head genes of λ and 21 show partial sequence homology and are analogous in structure, function and position. The terminases of λ and 21 are not interchangeable. At least two actions of terminase are involved in this specificity: (1) DNA binding; (2) prohead binding. The 1 and 2 genes at the left end of the 21 chromosome were identified as coding for the 21 terminase. gp1 and gp2 are analogous to gpNu1 and gpA, respectively. We have isolated a phage, λ -21 hybrid 33, which is the product of a crossover between λ and 21 within the terminase genes. λ -21 hybrid 33 DNA and terminase have phage 21 packaging specificity, as determined by complementation and helper packaging studies. The terminase of λ -21 hybrid 33 requires λ proheads for packaging. We have determined the position at which the crossover between λ DNA and 21 DNA occurred to produce the hybrid phage. λ -21 hybrid 33 carries the phage 21 1 gene and a hybrid phage 2/A gene. Sequencing of λ -21 hybrid 33 DNA shows that it encodes a protein that is homologous at the carboxy terminus with the 38 amino acids of the carboxy terminus of λ gpA; the remainder of the protein is homologous to gp2. The results of these studies define a specificity domain for prohead binding at the carboxy terminus of gpA.

Packaging of the bacteriophage lambda chromosome: dependence of cos cleavage on chromosome length.

Packaging of the bacteriophage lambda chromosome is polarized, proceeding from an initial cohesive end site (cos) in an A to R direction to a terminal cos site. We have performed helper packaging studies which indicate that the frequency of cleavage of the terminal cos site is dependent on the length of the chromosome being packaged. The helper packaging experiments involved infection of repressed lysogens containing a packagable chromosome by a heteroimmune helper. The prophage at which packaging terminated contained a cos duplication segment, so that failure to terminate packaging by cleavage of the first cos site results in packaging of a cos duplication chromosome. The frequency of packaging of chromosomes with the duplication decreases with increasing chromosome length, leading to the conclusion that cos cleavage depends on chromosome length. This result is most compatible with DNA packaging models in which cos cleavage follows packaging. No dependence of cleavage on the length of the distal duplication segment was found, indicating that extensive one-dimensional diffusion of DNA does not occur during packaging.

Packaging of the bacteriophage lambda chromosome: A role for base sequences outside cos

Abstract The genetic basis for the divergent packaging specificities of phages λ and 21 ( B. Hohn, 1975 , J. Mol. Biol., 98, 93–106) has been examined. A hybrid derivative of lambda containing a substitution of the 21 head genes and left chromosome terminus has 21 packaging specificity, so the divergent packaging specificities of λ and 21 result from differences in this region. Helper packaging experiments show that lambda and 21 can carry out cohesive end site (cos) cleavage on chromosomes of the opposite packaging specificity, indicating that cos cleavage per se is not the reason for the divergent packaging specificities. Sequencing studies, compatible with the helper packaging studies, show that the cohesive end sites of lambda and the λ-21 hybrid are identical. It is concluded that base sequences outside of the 22 base pair cos segment are responsible for the packaging specifities of λ and 21. The possible molecular basis for the packaging specificities of λ and 21 is discussed. Complementation studies between lambda head ambers and phage 21 show that only D and FII mutants can be complemented by 21. Since DNA packaging involves specific interactions between DNA and recognition proteins and the prehead, it is not surprising that few components are interchangeable between these two systems.

Cosmid DNA packaging in vivo.

The packaging of cosmid DNA into phage particles during phage lambda growth is described. Evidence is presented supporting the work of others that cosmid transducing phages contain linear multimers of cosmid DNA in which the number of cosmid copies is that required to make a packagable DNA length (greater than 0.77 of the lambda DNA length). The yield of cosmid transducing phages declines sharply as the number of cosmid copies required to make a packagable DNA length increases. The cosmid DNA replication that produces the packaging substrate shares with lambda rolling-circle replication a dependence on the lambda gam gene product.

Approaches to the Study of Survival and Death in Stationary-Phase Escherichia coli

When pure cultures of the bacterium Escherichia coli are grown in standard laboratory media, the organisms grow exponentially until conditions no longer support growth and the cells enter stationary phase. During growth the change in the number of viable cells is predominantly the result of cell division. There is little or no detectable cell death during exponential growth as judged by the close correlation between viable counts and total cells observed microscopically. Once growth is arrested, some cells survive while others die. The large questions to be addressed are: Why do some cells die during starvation and what is the cause of their death? Why do some cells survive during starvation and what functions are required for their survival? While these questions remain largely unanswered, the approaches that have been made possible in recent years by the application of molecular genetics have come to complement the predominantly physiological work that had been carried out earlier (reviewed in Dawes, 1989; Matin et al., 1989; Matin, 1991; Siegele and Kolter, 1992). Here we present descriptions of the main approaches our laboratory uses to address these questions. We describe the methodologies we use to incubate E. coli in stationary phase and the methodologies used to define, distinguish, and separate viable and nonviable cells in our experiments. We also describe genetic approaches we have taken to study cell physiology during stationary phase. This presentation should serve as an indication that much remains to be explored; the prospects of gaining new insights in the understanding of microbial death and survival make the work particularly exciting.

Heat shock transcription factor σ32 co-opts the signal recognition particle to regulate protein homeostasis in E. coli.

The bacterial heat shock transcription factor, σ32, maintains proper protein homeostasis only after it is targeted to the inner membrane by the signal recognition particle (SRP), thereby enabling integration of protein folding information from both the cytoplasm and cell membrane.