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As antimicrobial resistance (AMR) continues to increase, the therapeutic use of phages has re-emerged as an attractive alternative. However, knowledge of phage resistance development and bacterium-phage interaction complexity are still not fully interpreted. In this study, two lytic T4-like and T7-like phage infecting model Escherichia coli strain C600 are selected, and host genetic determinants involved in phage susceptibility and resistance are also identified using TraDIS strategy. Isolation and identification of the lytic T7-like show that though it belongs to the phage T7 family, genes encoding replication and transcription protein exhibit high differences. The TraDIS results identify a huge number of previously unidentified genes involved in phage infection, and a subset (six in susceptibility and nine in resistance) are shared under pressure of the two kinds of lytic phage. Susceptible gene wbbL has the highest value and implies the important role in phage susceptibility. Importantly, two susceptible genes QseE (QseE/QseF) and RstB (RstB/RstA), encoding the similar two-component system sensor histidine kinase (HKs), also identified. Conversely and strangely, outer membrane protein gene ompW, unlike the gene ompC encoding receptor protein of T4 phage, was shown to provide phage resistance. Overall, this study exploited a genome-wide fitness assay to uncover susceptibility and resistant genes, even the shared genes, important for the E. coli strain of both most popular high lytic T4-like and T7-like phages. This knowledge of the genetic determinants can be further used to analysis the behind function signatures to screen the potential agents to aid phage killing of MDR pathogens, which will greatly be valuable in improving the phage therapy outcome in fighting with microbial resistance., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

We re-examined data from the classic Luria-Delbrück fluctuation experiment, which is often credited with establishing a Darwinian basis for evolution. We argue that, for the Lamarckian model of evolution to be ruled out by the experiment, the experiment must favor pure Darwinian evolution over both the Lamarckian model and a model that allows both Darwinian and Lamarckian mechanisms (as would happen for bacteria with CRISPR-Cas immunity). Analysis of the combined model was not performed in the original 1943 paper. The Luria-Delbrück paper also did not consider the possibility of neither model fitting the experiment. Using Bayesian model selection, we find that the Luria-Delbrück experiment, indeed, favors the Darwinian evolution over purely Lamarckian. However, our analysis does not rule out the combined model, and hence cannot rule out Lamarckian contributions to the evolutionary dynamics.

CRISPR-Cas systems provide prokaryotes with adaptive defense against bacteriophage infections. Given an enormous variety of strategies used by phages to overcome their hosts, one can expect that the efficiency of protective action of CRISPR-Cas systems against different viruses should vary. Here, we created a collection of Escherichia coli strains with type I-E CRISPR-Cas system targeting various positions in the genomes of bacteriophages λ, T5, T7, T4 and R1-37 and investigated the ability of these strains to resist the infection and acquire additional CRISPR spacers from the infecting phage. We find that the efficiency of CRISPR-Cas targeting by the host is determined by phage life style, the positions of the targeted protospacer within the genome, and the state of phage DNA. The results also suggest that during infection by lytic phages that are susceptible to CRISPR interference, CRISPR-Cas does not act as a true immunity system that saves the infected cell but rather enforces an abortive infection pathway leading to infected cell death with no phage progeny release., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The dynamics of host susceptibility to parasites are often influenced by trade-offs between the costs and benefits of resistance. We assayed changes in the resistance to three viruses in six lines of Escherichia coli that had been evolving for almost 45,000 generations in their absence. The common ancestor of these lines was completely resistant to T6, partially resistant to T6* (a mutant of T6 with altered host range), and sensitive to λ. None of the populations changed with respect to resistance to T6, whereas all six evolved increased susceptibility to T6*, probably ameliorating a cost of resistance. More surprisingly, however, the majority of lines evolved complete resistance to λ, despite not encountering that virus during this period. By coupling our results with previous work, we infer that resistance to λ evolved as a pleiotropic effect of a beneficial mutation that downregulated an unused metabolic pathway. The strong parallelism between the lines implies that selection had almost deterministic effects on the evolution of these patterns of host resistance. The opposite outcomes for resistance to T6* and λ demonstrate that the evolution of host resistance under relaxed selection cannot be fully predicted by simple trade-off models., (© 2010 The Author(s). Journal compilation © 2010 The Society for the Study of Evolution.)

Mosaic genome design, considered evidence of horizontal gene transfer, is prominent in T-even phage tail fiber genes involved in host recognition. The possibility of direct gene transfer was assessed through superinfection with two virulent phages T2 and PP01, which caused host recognition shift. Two recombinant phages designated as TPr03 and TPr04 were isolated. PCR-restriction fragment length polymorphism analysis and sequence analysis suggested that 18% of the TPr03 and 38% of the TPr04 genome derived from PP01. Both isolates showed host ranges identical to PP01. The results suggested the possibility of generating various recombinant phages by intentional dual infections and of the occasional occurrence in nature of generation of phage showing new characteristics through superinfection, followed by the genomic recombination.

Infection by tailed dsDNA phages is initiated by release of the viral DNA from the capsid and its polarized injection into the host. The driving force for the genome transport remains poorly defined. Among many hypothesis [1], it has been proposed that the internal pressure built up during packaging of the DNA in the capsid is responsible for its injection [2-4]. Whether the energy stored during packaging is sufficient to cause full DNA ejection or only to initiate the process was tested on phage T5 whose DNA (121,400 bp) can be released in vitro by mere interaction of the phage with its E. coli membrane receptor FhuA [5-7]. We present a fluorescence microscopy study investigating in real time the dynamics of DNA ejection from single T5 phages adsorbed onto a microfluidic cell. The ejected DNA was fluorescently stained, and its length was measured at different stages of the ejection after being stretched in a hydrodynamic flow. We conclude that DNA release is not an all-or-none process but occurs in a stepwise fashion and at a rate reaching 75,000 bp/sec. The relevance of this stepwise ejection to the in vivo DNA transfer is discussed.

For obligately lytic bacteriophage (phage) a trade-off exists between fecundity (burst size) and latent period (a component of generation time). This trade-off occurs because release of phage progeny from infected bacteria coincides with destruction of the machinery necessary to produce more phage progeny. Here we employ phage mutants to explore issues of phage latent-period evolution as a function of the density of phage-susceptible bacteria. Theory suggests that higher bacterial densities should select for shorter phage latent periods. Consistently, we have found that higher host densities (>/== approximately 10(7) bacteria/ml) can enrich stocks of phage RB69 for variants that display shorter latent periods than the wild type. One such variant, dubbed sta5, displays a latent period that is approximately 70 to 80% of that of the wild type-which is nearly as short as the RB69 eclipse period-and which has a corresponding burst size that is approximately 30% of that of the wild type. We show that at higher host densities (>/== approximately 10(7) bacteria/ml) the sta5 phage can outcompete the RB69 wild type, though only under conditions of direct (same-culture) competition. We interpret this advantage as corresponding to slightly faster sta5 population growth, resulting in multifold increases in mutant frequency during same-culture growth. The sta5 advantage is lost, however, given indirect (different-culture) competition between the wild type and mutant or given same-culture competition but at lower densities of phage-susceptible bacteria (

By following the kinetics of abortive and productive synthesis in single-round transcription assays, we confirm the existence of two general classes of initial transcribing complexes (ITCs), which we term "productive ITC" and "unproductive ITC". The productive ITCs are able to escape from the promoter rapidly to produce full-length transcripts, but only after carrying out an obligate series of abortive initiation steps. The unproductive ITCs were found to synthesize mostly abortive transcripts of 2-3 nucleotides and escape from the promoter extremely slowly, if at all. Formation of the unproductive ITC is not due to the inactive RNA polymerase. Instead, RNA polymerase molecules recovered from both the productive and unproductive ITC fractions were shown to carry out abortive and productive synthesis with both the partitioning tendency and transcription kinetics similar to those of the original enzyme. Our results suggest that early transcription complexes are partitioned into the productive and unproductive ITCs most likely during the formation of open promoter complexes. The extent of partitioning varies with individual promoter sequences and is dependent on the nature and concentration of the initiating nucleotide. Thus, multiple classes of ITCs can be formed during promoter binding and transcript initiation.

RNA chain initiation and promoter escape is the latter stage of transcription initiation. This stage is characterized by several well-defined biochemical events: synthesis and release of short RNA products ranging 2 to 15 nucleotides in length, release of the sigma subunit from the enzyme-promoter complex, and initial translocation of the polymerase away from the promoter. In this paper, we report the use of a steady-state transcription assay with [gamma-(32)P]ATP labeling to subject the RNA chain initiation-promoter escape reaction to quantitative analysis. The specific parameters we follow to describe the chain initiation-promoter escape process include the abortive and productive rates, the abortive probability, the abortive:productive ratio, and the maximal size of the abortive product. In this study, we measure these parameters for three bacteriophage promoters transcribed by Escherichia coli RNA polymerase: T7 A1, T5 N25, and T5 N25(antiDSR). Our studies show that all three promoters form substantial amounts of abortive products under all conditions we tested. However, each of the promoters shows distinct differences from the others when the various parameters are compared. At 100 microM NTP, in a 10 min reaction, the abortive and productive yields are 87 and 13%, respectively, for T7 A1; 97 and 3%, respectively, for T5 N25; and 99.4 and 0.6%, respectively, for T5 N25(antiDSR). These values correspond to approximately 7, 32, and 165 abortive transcripts per productive transcript for the three promoters, respectively. The yield of most of the abortive products is not affected by the elevated concentration of the NTP substrate corresponding to the next template-specified nucleotide; hence, abortive products are not normally formed through a simple process of "kinetic competition". Instead, formation of abortive products appears to be determined by intrinsic DNA signals embedded in the promoter recognition region and the initial transcribed sequence region of each promoter.

Abortive initiation and promoter escape are two principal biochemical reactions occurring in the latter stage of transcript initiation. We have analyzed the influences of individual DNA elements within the promoter recognition region (PRR) on these reactions by measuring the quantitative initiation parameters that describe abortive initiation and promoter escape; these parameters are the abortive rate, the productive rate, the abortive:productive ratio, the abortive probability, and the maximum size of abortive transcripts. Changes in the individual DNA elements within the PRR can have a substantial effect on each of these parameters. The discriminator region and the -10 element primarily influence the abortive probability at positions 2-5 and 6-10, respectively, while the -10 and -35 conserved hexamers and the spacer region affect the abortive probability at positions 11-15. Surprisingly, transcription of a consensus promoter invariably gives a higher abortive yield, a higher abortive probability, a longer abortive ladder, and a lower productive rate than promoter variants carrying even a single deviation in the consensus hexamers. These results suggest that strong RNA polymerase-PRR interactions stall the polymerase at the promoter, thereby reducing the rate of promoter escape and consequently enhancing the extent of abortive initiation.

Trade-offs among the abilities of organisms to respond to different environmental factors are often assumed to play a major role in the coexistence of species. There has been extensive theoretical study of the role of such trade-offs in ecological communities but it has proven difficult to study such trade-offs experimentally. Microorganisms are ideal model systems with which to experimentally study the causes and consequences of ecological trade-offs. In model communities of E. coli B and T-type bacteriophage, a trade-off in E. coli between resistance to bacteriophage and competitive ability is often observed. This trade-off can allow the coexistence of different ecological types of E. coli. The magnitude of this trade-off affects, in predictable ways, the structure, dynamics and response to environmental change of these communities. Genetic factors, environmental factors, and gene-by-environment interactions determine the magnitude of this trade-off. Environmental control of the magnitude of trade-offs represents one avenue by which environmental change can alter community properties such as invasability, stability and coexistence.

The fatal bacterial infection caused by inhalation of the Bacillus anthracis spores results from the synthesis of protein toxins-protective antigen (PA), lethal factor (LF), and edema factor (EF)--by the bacterium. PA is the target-cell binding protein and is common to the two effector molecules, LF and EF, which exert their toxic effects once they are translocated to the cytosol by PA. PA is the major component of vaccines against anthrax since it confers protective immunity. The large-scale production of recombinant protein-based anthrax vaccines requires overexpression of the PA protein. We have constitutively expressed the protective antigen protein in E. coli DH5alpha strain. We have found no increase in degradation of PA when the protein is constitutively expressed and no plasmid instability was observed inside the expressing cells. We have also scaled up the expression by bioprocess optimization using batch culture technique in a fermentor. The protein was purified using metal-chelate affinity chromatography. Approximately 125 mg of recombinant protective antigen (rPA) protein was obtained per liter of batch culture. It was found to be biologically and functionally fully active in comparison to PA protein from Bacillus anthracis. This is the first report of constitutive overexpression of protective antigen gene in E. coli.

Unfolded outer membrane protein A (OmpA) of Escherichia coli spontaneously inserts and refolds into lipid bilayers upon dilution of denaturing urea. In the accompanying paper, we have developed a new technique, time-resolved distance determination by fluorescence quenching (TDFQ), which is capable of monitoring the translocation across lipid bilayers of fluorescence reporter groups such as tryptophan in real time [Kleinschmidt, J. H., and Tamm, L. K. (1999) Biochemistry 38, 4996-5005]. Specifically, we have shown that wild-type OmpA, which contains five tryptophans, inserts into lipid bilayers via three structurally distinct membrane-bound folding intermediates. To take full advantage of the TDFQ technique and to further dissect the folding pathway, we have made five different mutants of OmpA, each containing a single tryptophan and four phenylalanines in the five tryptophan positions of the wild-type protein. All mutants refolded in vivo and in vitro and, as judged by SDS-PAGE, trypsin fragmentation, and Trp fluorescence, their refolded state was indistinguishable from the native state of OmpA. TDFQ analysis of the translocation across the lipid bilayer of the individual Trps of OmpA yielded the following results: Below 30 degrees C, all Trps started from a far distance from the bilayer center and then gradually approached a distance of approximately 10 A from the bilayer center. In a narrow temperature range between 30 and 35 degrees C, Trp-15, Trp-57, Trp-102, and Trp-143 were detected very close to the center of the lipid bilayer in the first few minutes and then moved to greater distances from the center. When monitored at 40 degrees C, which resolved the last steps of OmpA refolding, these four tryptophans crossed the center of the bilayer and approached distances of approximately 10 A from the center after refolding was complete. In contrast Trp-7 approached the 10 A distance from a far distance at all temperatures and was never detected to cross the center of the lipid bilayer. The translocation rates of Trp-15, Trp-57, Trp-102, and Trp-143 which are each located in different outer loop regions of the four beta-hairpins of the eight-stranded beta-barrel of OmpA were very similar to one another. This result and the common distances of these Trps from the membrane center observed in the third membrane-bound folding intermediate provide strong evidence for a synchronous translocation of all four beta-hairpins of OmpA across the lipid bilayer and suggest that OmpA inserts and folds into lipid bilayers by a concerted mechanism.

Infection of Escherichia coli cells with bacteriophage T1 induces synthesis of a bacteriophage-specific DNA methyltransferase (M.EcoT1, EC No: 2.1.1.72) with a specificity for adenine residues in the sequence 5'-GATC-3'. Purification of M.EcoT1 allowed the determination of the coding sequence of the gene (Schneider-Scherzer et al., 1990). The peptide of the entire coding sequence was over-expressed as a histidine-hexapeptide tagged protein in E. coli. Affinity purification using a Ni2+ chelating (Ni-NTA) resin yielded a recombinant enzyme with almost the same enzymatic properties as the protein purified from T1 infected E. coli cells. Interestingly, in both purification procedures, a protein with a molecular weight of 50000 was found to copurify with M.EcoT1. The N-terminal amino acid sequence identified these proteins in both cases as E. coli enolase (EC No: 4.2.1.11).

Primary structures of phage T5- and Escherichia coli-encoded tRNA(Phe) are distinct at four out of 11 positions known as identity elements for E. coli phenylalanyl-tRNA synthetase (FRS). In order to reveal structural requirements for FRS recognition, aminoacylation of wild-type phage T5 tRNA(Phe) gene transcript and mutants containing substitutions of the identity elements at positions 20, 34, 35 and 36 was compared with E. coli tRNA(Phe) gene transcript. The wild-type phage T5 transcript can be aminoacylated with the same catalytic efficiency as the E. coli counterpart. However, the maximal aminoacylation rate for T5 and E. coli transcripts was reached at different Mg2+ concentrations: 4 and 15 mM, respectively. Aminoacylation assays with tRNA(Phe) mutants revealed that (i) phage transcripts with the substituted anticodon bases at positions 35 and 36 were efficient substrates for aminoacylation at 15 mM Mg2+ but not at optimal 4 mM Mg2+; (ii) any change of G34 in phage transcripts dramatically decreased the aminoacylation efficiency at both 4 and 15 mM Mg2+ whereas G34A mutation in the E. coli transcript exhibits virtually no influence on aminoacylation rate at 15 mM Mg2+; (iii) substitution of A20 with U in the phage transcript caused no significant change in the aminoacylation rate at both Mg2+ concentrations; (iv) phage transcripts with double substitutions A20U+A35C and A20U+A36C were very poor substrates for FRS. Collectively, the results indicate the non-identical mode of tRNA(Phe) recognition by E. coli FRS at low and high Mg2+ concentrations. Probably, along with identity elements, the local tRNA conformation is essential for recognition by FRS.

Activities of RNA polymerases from wild type B/r strain of E. coli and its pleiotropic rpoB403 rif-r mutant were studied using five various DNA matrices and various groups of early region promotors of T4 and T7 phage DNA. The mutation differentially affects RNA polymerase-catalyzed RNA synthesis depending on the promotor used. Relative efficiency of A3 promotor of T7 phage DNA is enhanced in case of the mutant enzyme. rpoB403 mutation can be used for the classification analysis of promotors differing in their interaction with the enzyme.

The process of RNA chain initiation by RNA polymerases plays a central role in the regulation of transcription. In this complex phase of transcription, short oligomers are synthesized and released from the enzyme-promoter complex in a reaction termed abortive initiation. The polymerase undergoes many cycles of abortive initiation prior to completion of the initiation process, which is signaled by the translocation of the enzyme away from the promoter, release of sigma factor, and formation of an elongation complex in which the RNA is stably bound. We have studied the parameters that affect escape from the promoter by Escherichia coli RNA polymerase for the phage T7 A1 promoter, the phage T5 N25 promoter, and the chimeric promoter T5 N25antiDSR. The latter site contains a synthetic initial transcribed region that reduces its ability to synthesize RNA both in vivo and in vitro. Clearance from T5 N25antiDSR can be stimulated up to 10-fold in vitro by addition of the E. coli transcript cleavage factor GreA or GreB, but these factors have little effect on transcription from the normal T7 A1 or T5 N25 promoters. Using an E. coli strain lacking GreA and GreB, we were also able to show stimulation of transcription by the Gre factors from the T5 N25antiDSR promotor in vivo. The stimulation of RNA chain initiation by Gre factors, together with their known biochemical properties in the transcription elongation reaction, suggests some specific models for steps in the transcription initiation reaction.

We examined the role of plasmid-based phage defence in maintaining plasmids, using colicin plasmids in Escherichia coli as a model system. Experimental data indicated that the possession of a colicin plasmid can confer limited protection against bacteriophages. A continuous culture model, using these experimental values, indicated that the observed limited protection alone could selectively maintain colicin plasmids, without requiring a competitive advantage due to colicinogeny. Phage defence might explain the current maintenance of colicin plasmids, given the naturally occurring high levels of resistance to colicins. This model also suggests that many plasmids might be maintained in natural populations, in part, by phage resistance, including 'cryptic' plasmids for which no phenotype is known.

Phage T5 requires 0.1 mM calcium to produce phage progeny in Escherichia coli cells. Decreasing calcium below 0.1 mM at time phage DNA was transferred depleted the bacteria of K+, caused membrane depolarization, perturbation of phage DNA transfer and resulted in a low internal ATP level. Our data suggest that calcium controls the conformation of the channel involved in the transfer of phage DNA through the host envelope and that below 0.1 mM calcium the channel remains open. This creates an energetic state of the host unfavorable to the synthesis of phage components and leads to abortion of the infectious process.

This article presents the development of a set of new expression vectors for overproduction of proteins in Escherichia coli. The vectors, pETUBI-ES1, 2 and 3, allow in-frame cloning of any sequence with the ubiquitin gene driven by the strong T7f10 promoter. Combination of the T7 expression system with ubiquitin fusion appears to have a synergistic effect on protein overproduction. Large amounts of stable RNA are produced by T7 RNA polymerase, and fusion of ubiquitin to the N-terminus of target proteins seems to confer more efficient translation, better folding or protection against proteolytic degradation. The ubiquitin part can be utilized for purification of the fusion protein, after which it can be easily removed from the fusion product by ubiquitin-specific proteases. The advantage of combining both systems is demonstrated by the synthesis of large quantities (up to 40-50% of the total protein) of the human ERCC1 protein that hitherto was refractory to overproduction in various other E. coli and yeast expression systems.

Transposon mutagenesis, using IS50L::phoA(Tn-phoA), was performed in a K54/O4/H5 blood isolate of Escherichia coli (CP9), to generate a library of random mutants. Five hundred and twenty-six independent CP9 TnphoA mutants were isolated with active gene fusions to alkaline phosphatase. From this mutant library, eight capsule-deficient strains were detected and were found to have a single copy of TnphoA. Sixteen additional capsule deficient mutants with TnphoA inserts were subsequently obtained that did not possess active PhoA fusions. In conjunction with the initial eight capsule-deficient isolates we have defined genes on three different XbaI fragments as being involved in capsule production. Generalized transduction with the bacteriophage T4 established that these insertions were responsible for the loss of capsule and that they are linked. These capsule-deficient strains can be used to assess the pathogenic role of the K54 capsular polysaccharide.

DNA supercoiling is known to influence the pattern of gene expression in prokaryotes. Thus the mechanism of transcription initiation and the topological state of the template are intimately related. Using in vitro reconstituted transcription assays, composed of purified RNA polymerase and promoters in their natural topological state, we have conducted a detailed study of transcription initiation from T7 early promoters including the following steps: the formation of ternary complexes, acquisition of rifampicin resistance, release of sigma factor and the capacity for RNA chain elongation in complexes. We determined the order of these events and the length of the transcripts when each step occurred during initiation of transcription on supercoiled templates. The length of the transcripts varied in a promoter-specific manner. Analysis of abortive products formed during the initiation showed that stronger promoters go to the elongation mode at transcript lengths shorter than that required for weaker promoters.

Using a rifampicin-resistant RNA polymerase with altered specificity to different promoters, the D promoter of T7 phage DNA with increased affinity to the mutant enzyme was chosen. This promoter and the T7 A1 promoter with unchanged affinity as well as some nonpromoter DNA fragments were used to compare temperature-induced conformational transitions of RNA polymerase in the course of complex formation. Conformational alterations of RNA polymerase were monitored by the fluorescent label method. It was shown that RNA polymerase undergoes a set of conformational transitions during complex formation with each promoter, some of which were similar by the character of change to spectral parameters of the label (reflecting RPi and, probably, RPo formation). The local structure of complexes formed above 33 degrees C differs for A1 and D. The conformational analysis reveals at least one temperature-dependent stage upon nonspecific interaction of the enzyme with nonpromoter DNA at 13-16 degrees C. Models of functional organization of the enzyme recognizing center and some features of the structure of the promoters which may be essential for their recognition are discussed.

Molecular matchmakers are a class of proteins that use the energy released from the hydrolysis of adenosine triphosphate to cause a conformational change in one or both components of a DNA binding protein pair to promote formation of a metastable DNA-protein complex. After matchmaking the matchmaker dissociates from the complex, permitting the matched protein to engage in other protein-protein interactions to bring about the effector function. Matchmaking is most commonly used under circumstances that require targeted, high-avidity DNA binding without relying solely on sequence specificity. Molecular matchmaking is an extensively used mechanism in repair, replication, and transcription and most likely in recombination and transposition reactions, too.

Gene 5.5 of coliphage T7 is one of the most highly expressed genes during T7 infection. Gene 5.5 protein, purified from cells overexpressing the cloned gene, purifies with the nucleoid protein H-NS of Escherichia coli during three chromatographic steps. A fusion protein of gene 5.5 protein and maltose binding protein also purifies with H-NS. The fusion protein binds to the DNA-H-NS complex and abolishes H-NS-mediated inhibition of transcription by Escherichia coli and T7 RNA polymerases in vitro. Expression of gene 5.5 also relieves the repression of the Escherichia coli proU promoter by H-NS in vivo. The change of leucine to proline at residue 30 of gene 5.5 protein abolishes the interaction between gene 5.5 protein and H-NS.

Strains of Escherichia coli have been produced which express very high levels of the tRNA(1Leu) isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA(1Leu) gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA(1Leu). As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA(1Leu). We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA(1Leu) was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m1G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA(1Leu) operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA(1Leu) levels. This level of tRNA(1Leu) titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.

An experimental approach is presented for the creation of an artificial and functional repressor/operator interaction that does not involve polypeptides. This in vitro approach confers oligonucleotide regulation upon a bacteriophage T7 RNA polymerase promoter by introducing an overlapping homopurine operator that can be recognized by oligonucleotide-directed DNA triple-helix formation. Recognition of optimized operator sequences in either of two triple-helix motifs is shown to efficiently inhibit T7 RNA polymerase transcription initiation in both a promoter- and oligonucleotide-specific manner. Inhibition due to triple helices of the pyrimidine motif is pH-dependent, as expected. Inhibition by purine motif triple helices is not pH-dependent and occurs efficiently under optimum T7 RNA polymerase transcription conditions. Repression by triple-helix formation can be observed rapidly after addition of purine motif repressor oligonucleotides, even when polymerase has been given prior access to the promoter. The mechanism of repression is shown to be occlusion of polymerase from the promoter rather than trapping of the polymerase in unproductive preinitiation or initiation complexes. In contrast to their inhibition of T7 RNA polymerase initiation, the triple-helical complexes studied here do not detectably inhibit transcription elongation.

Spinach glycolate oxidase (GAO) was expressed in Escherichia coli using the T7 RNA polymerase promotor. The enzyme accounts for approx. 1% of the soluble protein fraction and is expressed as a soluble and active enzyme. Comparison with GAO expressed in Saccharomyces cerevisiae (Macheroux, P., Massey, V., Thiele, D.J. and Volokita, M. (1991) Biochemistry 30, 4612-4619) showed that the GAO expressed in E. coli has identical physico-chemical features to the wild-type enzyme, but is expressed at a level approx. 15-fold higher than in the yeast system.

The poliovirus nonstructural proteins 2B, 2C, 2C3A, 2C3AB, 3A, and 3AB have been cloned and efficiently expressed in Escherichia coli cells. Each individual protein, or combinations of some of them, were cloned using polymerase chain reaction techniques and correspond to the genuine poliovirus protein plus an additional methionine. The system used to express them uses pET vectors containing the promoter of gene 10 of phage T7. Expression of protein 2C in BL21 (DE3) pLysS cells, which express the T7 lysozyme, is not toxic, and the bacteria synthesize this protein for several hours after induction. In contrast, the expression of proteins 2B, 3A, or 3AB is not tolerated by BL21 (DE3) pLysS cells which could make them only for a limited period of time. Protein 3AB was particularly toxic and induced a rapid lysis of the recombinant clone after its induction with isopropyl-1-thio-beta-D-galactopyranoside alone or with both isopropyl-1-thio-beta-D-galactopyranoside and rifampicin. Further analyses showed that 3AB induced profound modifications in membrane permeability to o-nitrophenyl-beta-D-galactopyranoside, labeled uridine, and nonpermeant translation inhibitors. Cloning and expression of proteins 2B, 3A, and 3AB in BL21 (DE3) cells that do not contain the T7 lysozyme lead to a more sustained expression of these proteins without detectable cell lysis. Changes in permeability to low molecular weight compounds such as radioactive uridine, o-nitrophenyl-beta-D-galactopyranoside, and hygromycin B readily appeared upon induction of 2B, 3A, and 3AB. Our results indicate that the poliovirus nonstructural polypeptides 2B and 3A (or 3AB) are lytic for the bacteria. In fact, both proteins 2B and 3A contain hydrophobic domains in a potential amphipathic helix; this is one characteristic shared with a number of membrane-active peptides.

Phage T4-induced anticodon nuclease triggers cleavage-ligation of the host tRNA(Lys). The enzyme is encoded in latent form by the optional Escherichia coli locus prr and is activated by the product of the phage stp gene. Anticodon nuclease latency is attributed to the masking of the core function prrC by flanking elements homologous with type I restriction-modification genes (prrA-hsdM and prrD-hsdR). Activation of anticodon nuclease in extracts of uninfected prr+ cells required synthetic Stp, ATP and GTP and appeared to depend on endogenous DNA. Stp could be substituted by a small, heat-stable E. coli factor, hinting that anticodon nuclease may be mobilized in cellular situations other than T4 infection. Hsd antibodies recognized the anticodon nuclease holoenzyme but not the prrC-encoded core. Taken together, these data indicate that Hsd proteins partake in the latent ACNase complex where they mask the core factor PrrC. Presumably, this masking interaction is disrupted by Stp in conjunction with Hsd ligands. The Hsd-PrrC interaction may signify coupling and mutual enhancement of two prokaryotic restriction systems operating at the DNA and tRNA levels.

The action of rifampicin on the RNA chain initiation catalysed by E. coli RNA polymerase over different templates has been studied. The steady-state formation of dinucleoside tetraphosphate under the condition of abortive initiation reaction was assayed. It was observed that rifampicin shows a spectrum of inhibitory effects on transcription initiation at different promoters. At two different promoters with a pyrimidine nucleotide at the 5'-initiation site, e.g. rrnB P2 having CTP and lacP2 having UTP, the effect of rifampicin on the abortive synthesis of the first phosphodiester bond was found to be total, even at low concentrations of the antibiotic. On the other hand, in most cases the effect of rifampicin on the abortive synthesis with a purine nucleotide at the 5'-initiation site was found to be only partial, with the exception of the T7A2 promoter, where rifampicin stimulates the abortive synthesis of pppGpC. It was also noticed that if there was a purine nucleotide at the second position of a dinucleotide which had already been synthesised by the enzyme, then further addition of the third nucleotide was not blocked in the presence of rifampicin. It appeared that a purine nucleotide at the initiation site or at the product terminus site of a translocated dinucleotide behaved similarly towards rifampicin. In the same way, if this position was occupied by a pyrimidine, rifampicin would inhibit further phosphodiester synthesis, even at a very low concentration. The stimulatory effect of rifampicin at the T7A2 promoter was presumably because here a ternary complex containing the promoter, enzyme and the abortive transcript pppGpC was initially stable, but dissociated upon addition of rifampicin, resulting in the rapid turn-over of the product.

The development of bacteriophage T7 was examined in an Escherichia coli double mutant defective for the two major apurinic, apyrimidinic endonucleases (exonuclease III and endonuclease IV, xth nfo). In cells infected with phages containing apurinic sites, the defect in repair enzymes led to a decrease of phage survival and a total absence of bacterial DNA degradation and of phage DNA synthesis. These results directly demonstrate the toxic action of apurinic sites on bacteriophage T7 at the intracellular level and its alleviation by DNA repair. In addition, untreated T7 phage unexpectedly displayed reduced plating efficiency and decreased DNA synthesis in the xth nfo double mutant.

Intron mobility requires cleavage of an intronless allele by an intron-encoded endonuclease, followed by transfer of the intron into the cleaved recipient. The mobile phage introns provide an opportunity to identify accessory functions involved in the intron inheritance process. To test for trans and cis requirements of mobility in Escherichia coli, we have exploited the td intron of phage T4 in both phage T4 and lambda genetic backgrounds. Mobility depends on host or phage recombinase functions, RecA or UvsX, respectively. The process also requires a phage-encoded 5'----3' exonuclease activity and associated annealing function that can be provided by phage lambda. Finally, host-encoded 3'----5' exonuclease activities are also implicated in intron inheritance. We demonstrated further that restriction enzymes could substitute for the intron-encoded endonuclease, indicating that the endonuclease does not have an essential role in recombination. Neither the precise position nor the nature of the double-strand break was critical to intron transfer. These features provide insight into the recombination pathway and are factors impacting on the spread of introns throughout natural populations.

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, the product of the Escherichia coli aroA gene, has been overproduced in E. coli BL21(lambda DE3) under the control of the T7 gene 10 promoter and ribosome binding site, to a level of approximately 50% of total cell protein. EPSP synthase is the primary target of the post-emergence herbicide, glyphosate, commonly known as Roundup. A simple two step purification is described, which results in 99% pure homogeneous protein (as determined by PAGE). The integrity of the protein has been compared with previously characterized protein from E. coli AB2829(pKD501) by determination of its kinetic parameters, N-terminal protein and DNA sequences, amino acid analysis and 13C-NMR spectroscopy. This new overproducing strain readily provides the gram quantities of highly pure protein required for NMR studies of the active site and the development of novel time-resolved solid-state NMR techniques currently underway in this laboratory.

The bacteriophage T4-encoded MotA protein is critical for transcription from T4 middle-mode promoters. However, a direct interaction of this protein with a middle-mode promoter has not previously been demonstrated. We have cloned the motA gene and overexpressed the gene product using the T7 expression system. A simple procedure was then developed to purify the MotA protein to homogeneity. Using the purified protein we have demonstrated that MotA protein binds to the -30 region of the middle-mode promoter PuvsY. This promoter has previously been shown to be a necessary component of a T4 replication origin, and thus MotA is also a T4 origin-binding protein. Modified RNA polymerase purified from T4-infected cells was used to establish middle-mode transcription in vitro. Transcription from PuvsY was markedly enhanced by the addition of MotA protein, whether or not the template contained the cytosine modifications characteristic of T4 DNA. However, transcription from PuvsY was apparently independent of the MotA protein when unmodified RNA polymerase from uninfected cells was used.

Transcriptional regulation of the bacteriophage T4 late genes requires the participation of three DNA polymerase accessory proteins that are encoded by T4 genes 44, 62, and 45, and that act at an enhancer-like site. Transcriptional activation by these DNA replication proteins also requires the function of an RNA polymerase-bound coactivator protein that is encoded by T4 gene 33 and a promoter recognition protein that is encoded by T4 gene 55. Transcriptional activation in DNA constructs, in which the enhancer and a T4 late promoter can be segregated on two rings of a DNA catenane, has now been analyzed. The ability of an interposed DNA-binding protein to affect communication between the enhancer and the promoter was also examined. Together, these experiments demonstrate that this transcription-activating signal is conveyed between its enhancer and a T4 late promoter by a DNA-tracking mechanism. Alternative activation mechanisms relying entirely on through-space interactions of enhancer-bound and promoter-bound proteins are excluded.

The lytic coliphage T7 encodes a serine/threonine-specific protein kinase which supports viral reproduction under suboptimal growth conditions. Expression of the protein kinase in T7-infected Escherichia coli cells results in the phosphorylation of 30S ribosomal subunit protein S1, and initiation factors IF1, IF2, and IF3, as determined by high-resolution two-dimensional gel electrophoresis and specific immunoprecipitation analysis. Phosphorylation occurs either exclusively on threonine (IF1, IF3, S1) or on serine and threonine (IF2). There is no phosphorylation of these proteins in uninfected cells or in cells infected with T7 lacking the protein kinase function. Phosphorylation of the initiation factors coincides with the onset of T7 late protein synthesis, occurring 9-12-min postinfection. T7 late protein synthesis, otherwise inhibited in ColIb plasmid-containing cells, is specifically supported by expression of the protein kinase. These results provide the first evidence for the functional involvement of protein phosphorylation in the control of bacterial translation.

Two new plasmid expression vectors, pTacT7 and pTacT7L, have been constructed, which incorporate between the tac promoter and a downstream NcoI-HindIII polylinker sequence a synthetic sequence derived from the region upstream from gene 10 of bacteriophage T7 (g10-L). This sequence was recently shown to act as a translational enhancer (Olins et al., 1988) and was termed "Epsilon" (Enhancer of Protein Synthesis Initiation) element (Olins and Rangwala, 1989). In this communication we describe in detail the construction of ptacT7 and ptacT7L. Furthermore, we present evidence that the "Epsilon" element is able to enhance 3 to 20-fold the expression levels of two poorly expressed test genes encoding the human placental proteins PP9 and PP15. On the other hand, the expression levels of two highly expressed test genes encoding the human placental proteins PP4 and FXIIIa could not be further enhanced by the presence of the "Epsilon" element. These experiments show that the T7 gene 10 leader sequence can be utilized to improve the expression yields of otherwise poorly expressed heterologous genes in Escherichia coli.

Recombinant plasmid DNAs containing long DNA inserts that can be propagated in Escherichia coli would be useful in the analysis of complex genomes. We tested a bacteriophage T4 in vitro DNA packaging system that has the capacity to package about 170 kb of DNA into its capsid for cloning long DNA fragments. We first asked whether the T4 in vitro system can package foreign DNA such as concatemerized lambda imm434 DNA and phage P1-pBR322 hybrid DNA. The data suggest that the T4 system can package foreign DNA as efficiently as the mature phage T4 DNA. We then tested the system for its ability to clone foreign DNA fragments using the P1-pBR322 hybrid vectors constructed by Sternberg [Proc. Natl. Acad. Sci. USA 87 (1990) 103-107]. E. coli genomic DNA fragments were ligated with the P1 vectors containing two directly oriented loxP sites, and the ligated DNA was packaged by the T4 in vitro system. The packaged DNA was then transduced into E. coli expressing the phage P1 cyclization recombination protein recombinase to circularize the DNA by recombination between the loxP sites situated at the ends of the transduced DNA molecule. Clones with long DNA inserts were obtained by using this approach, and these were maintained as single-copy plasmids under the control of the P1 plasmid replicon. Clones with up to about 122-kb size inserts were recovered using this approach.

Overexpression of glycoprotein-encoding genes in Escherichia coli sometimes results in toxicity to the host and low protein yields. One possible explanation for this phenomenon is the presence of hydrophobic amino acid (aa) domains approx. 15-20 aa in length in the overproduced protein. As an initial test of this hypothesis, regions of hydrophobicity located within the envelope glycoproteins of HIV-1 and HTLV-1 were identified by computer analysis, and subsequently deleted by site-directed mutagenesis. The parent and modified envelope genes were expressed in bacteria using both lambda pL and T7 inducible expression systems. Removal of the hydrophobic domains reduced the apparent toxicity and significantly increased the accumulation of recombinant protein from undetectable levels to approx. 10-15% of total cellular protein.

This report provides a purification method for the two proteins, 51 kDa and 33 kDa, both encoded by the same mcrB gene of the McrBC restriction system in Escherichia coli K-12. The two proteins were produced in large quantity using a T7 expression system and copurified to near homogeneity by DEAE-Sepharose and Affi-Gel blue column chromatography. The N-terminal amino acid sequences of these purified McrB proteins were the same as those predicted from the mcrB DNA sequence by Ross et al. [J. Bacteriol. 171 (1989b) 1974-1981]. The 33-kDa protein totally overlaps the C-terminal part of the 51-kDa protein.

The gene (cytA) coding for the 27 kDa polypeptide of the Bacillus thuringiensis var. israelensis mosquito larvicidal delta-endotoxin, was cloned into a plasmid containing the T7 bacteriophage promoter. The plasmid was used to transform an Escherichia coli strain containing the T7 RNA polymerase gene 1, under the control of lacP. Loss of colony-forming ability without substantial lysis, associated with immediate inhibition of DNA synthesis, was observed after induction of transformed cells. The cytA gene product may kill E. coli cells by disrupting their chromosome replicating apparatus.

Synthesis of the gamma-subunit of DNA polymerase III holoenzyme depends on precise and efficient translational frameshifting to the -1 frame at a specific site in the dnaX gene of Escherichia coli. In vitro mutagenesis of this frameshift site demonstrated the importance of an A AAA AAG heptanucleotide sequence, which allows two adjacent tRNAs to retain a stable interaction with mRNA after they slip to the -1 position. The AAG lysine codon present in the 3' half of this heptanucleotide was a key element for highly efficient frameshifting. A tRNA(Lys) with a CUU anticodon, which has a strong affinity for AAG lysine codons, is present in eukaryotic cells but absent in E. coli. Expression in E. coli of a mutant tRNA(Lys) with a CUU anticodon specifically inhibited the frameshifting at the AAG codon, suggesting that the absence of this tRNA in E. coli contributes to the efficiency of the dnaX frameshift.

Phage T5 injects its DNA into Escherichia coli cells in two steps; 8% of the chromosome is first injected, and then there is a pause during which proteins encoded by this DNA fragment are synthesized allowing the remaining DNA to be injected. Using a potassium-selective electrode, we show that the injection of the two DNA fragments is associated with an efflux in two steps, of cytoplasmic potassium. The rate of efflux is linearly related to the number of added phages suggesting that each phage induces the formation of at least one channel in the inner membrane. The first efflux occurs even in depolarized cells suggesting that the insertion and the opening of the channel can take place in the absence of the electrochemical gradient of protons (delta mu H+). The channel is in a closed configuration during the time required for the synthesis of the phage-encoded proteins; this closing and the second efflux are prevented by the depolarization of the cell. The insertion of the channel in the inner membrane requires a fluid membrane. The results obtained suggest that the function of this channel is to translocate phage T5 DNA.

Endoribonuclease RNase E has an important role in the processing and degradation of bacteriophage T4 and Escherichia coli mRNAs. We have undertaken a mutational analysis of the -71 RNase E processing site of T4 gene 32. A series of mutations were introduced into a synthetic T4 sequence cloned on a plasmid, and their effects on processing were analyzed in vivo. The same mutations were transferred into T4 by homologous recombination. In both the plasmid and the phage contexts the processing of the transcripts was similarly affected by the mutations. Partially purified RNase E has also been used to ascertain the effect of these mutations on RNase E processing in vitro. The hierarchy of the efficiency of processing of the various mutant transcripts was the same in vivo and in vitro. These results and an analysis of all of the known putative RNase E sites suggest a consensus sequence RAUUW (R = A or G; W = A or U) at the cleavage site. Modifications of the stem-loop structure downstream of the -71 site indicate that a secondary structure is required for RNase E processing. Processing by RNase E was apparently inhibited by sequences that sequester the site in secondary structure.