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Assembly of 30S ribosomal subunits from Escherichia coli has been dissected in detail using an in vitro system. Such studies have allowed characterization of the role for ribosomal protein S15 in the hierarchical assembly of 30S subunits; S15 is a primary binding protein that orchestrates the assembly of ribosomal proteins S6, S11, S18, and S21 with the central domain of 16S ribosomal RNA to form the platform of the 30S subunit. In vitro S15 is the sole primary binding protein in this cascade, performing a critical role during assembly of these four proteins. To investigate the role of S15 in vivo, the essential nature of rpsO, the gene encoding S15, was examined. Surprisingly, E. coli with an in-frame deletion of rpsO are viable, although at 37°C this ΔrpsO strain has an exaggerated doubling time compared to its parental strain. In the absence of S15, the remaining four platform proteins are assembled into ribosomes in vivo, and the overall architecture of the 30S subunits formed in the ΔrpsO strain at 37°C is not altered. Nonetheless, 30S subunits lacking S15 appear to be somewhat defective in subunit association in vivo and in vitro. In addition, this strain is cold sensitive, displaying a marked ribosome biogenesis defect at low temperature, suggesting that under nonideal conditions S15 is critical for assembly. The viability of this strain indicates that in vivo functional populations of 70S ribosomes must form in the absence of S15 and that 30S subunit assembly has a plasicity that has not previously been revealed or characterized.

We have constructed a set of plasmids that can be used to express recombineering functions in some gram-negative bacteria, thereby facilitating in vivo genetic manipulations. These plasmids include an origin of replication and a segment of the bacteriophage lambda genome comprising the red genes (exo, bet and gam) under their native control. These constructs do not require the anti-termination event normally required for Red expression, making their application more likely in divergent species. Some of the plasmids have temperature-sensitive replicons to simplify curing. In creating these vectors we developed two useful recombineering applications. Any gene linked to a drug marker can be retrieved by gap-repair using only a plasmid origin and target homologies. A plasmid origin of replication can be changed to a different origin by targeted replacement, to potentially alter its copy number and host range. Both these techniques will prove useful for manipulation of plasmids in vivo. Most of the Red plasmid constructs catalyzed efficient recombination in E. coli with a low level of uninduced background recombination. These Red plasmids have been successfully tested in Salmonella, and we anticipate that that they will provide efficient recombination in other related gram-negative bacteria.

The lysis-lysogeny decision of bacteriophage lambda (λ) is a paradigm for developmental genetic networks. There are three key features, which characterize the network. First, after infection of the host bacterium, a decision between lytic or lysogenic development is made that is dependent upon environmental signals and the number of infecting phages per cell. Second, the lysogenic prophage state is very stable. Third, the prophage enters lytic development in response to DNA-damaging agents. The CI and Cro regulators define the lysogenic and lytic states, respectively, as a bistable genetic switch. Whereas CI maintains a stable lysogenic state, recent studies indicate that Cro sets the lytic course not by directly blocking CI expression but indirectly by lowering levels of CII which activates cI transcription. We discuss how a relatively simple phage like λ employs a complex genetic network in decision-making processes, providing a challenge for theoretical modeling.

The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. It then presents support protocols that describe several two-step selection/counter-selection methods of making genetic alterations without leaving any unwanted changes in the targeted DNA, and a method for retrieving onto a plasmid a genetic marker (cloning by retrieval) from the Escherichia coli chromosome or a co-electroporated DNA fragment. Additional protocols describe methods to screen for unselected mutations, removal of the defective prophage from recombineering strains, and other useful techniques.

Bacteriophage lambda N and bacterial Nus proteins together with a unique site NUT in the leader of the early viral N gene transcript bind RNA polymerase (RNAP) and form a highly processive antitermination complex; N bound at NUT also represses N translation. In this study, we investigate whether N and NUT cause N translation repression as part of the antitermination complex by testing conditions that inhibit the formation of the N-modified transcription complex for their effect on N-mediated translation repression. We show that nus and nut mutations that in combination destabilize multiple interactions in the antitermination complex prevent N-mediated translation repression. Likewise, transcription of the nut-N region by T7 RNAP, which does not lead to the assembly of an effective antitermination complex when N is supplied, eliminates translation repression. We also demonstrate that a unique mutant beta subunit of RNAP reduces N-mediated translation repression, and that overexpression of transcription factor NusA suppresses this defect. We conclude that the N-modified RNAP transcription complex is necessary to repress N translation.

Recombinogenic engineering methodology, also known as recombineering, utilizes homologous recombination to create targeted changes in cellular DNA with great specificity and flexibility. In Escherichia coli, the Red recombination system from bacteriophage lambda has been used successfully to modify both plasmid and chromosomal DNA in a highly efficient manner, using either a linear double-stranded DNA fragment or a synthetic single-stranded oligonucleotide (SSO). The current model for Red/SSO-mediated recombination involves the SSO first annealing to a transient, single-stranded region of DNA before being incorporated into the chromosome or plasmid target. It has been observed previously, in both eukaryotes and prokaryotes, that mutations in the two strands of the DNA double helix are 'corrected' by complementary SSOs with differing efficiencies. Here we investigate further the factors that influence the strand bias as well as the overall efficiency of Red/ SSO-mediated recombination in E.coli. We show that the direction of DNA replication and the nature of the SSO-encoded mismatch are the main factors dictating the recombinational strand bias. However, the influence that the SSO-encoded mismatch exerts upon the recombinational strand bias is abolished in E.coli strains that are defective in mismatch repair (MMR). This reflects the fact that different base-base mispairs are corrected by the mutS/H/L-dependent MMR pathway with differing efficiencies. Furthermore, our data indicate that transcription has negligible influence on the strand bias. These results demonstrate for the first time that the interplay between DNA replication and MMR has a major effect on the efficiency and strand bias of Red/SSO-mediated recombination in E.coli., published_or_final_version

We demonstrate that the bacteriophage lambda Red functions efficiently recombine linear DNA or single-strand oligonucleotides (ss-oligos) into bacteriophage lambda to create specific changes in the viral genome. Point mutations, deletions, and gene replacements have been created. While recombineering with oligonucleotides, we encountered other mutations accompanying the desired point mutational change. DNA sequence analysis suggests that these unwanted mutations are mainly frameshift deletions introduced during oligonucleotide synthesis.

Transcription factor NusG is present in all prokaryotes, and orthologous proteins have also been identified in yeast and humans. NusG contains a 27-residue KOW motif, found in ribosomal protein L24 where it interacts with rRNA. NusG in Escherichia coli (EcNusG) is an essential protein and functions as a regulator of Rho-dependent transcription termination, phage lambda N and rRNA transcription antitermination, and phage HK022 Nun termination. Relative to EcNusG, Aquifex aeolicus NusG (AaNusG) and several other bacterial NusG proteins contain a variable insertion sequence of approximately 70 residues in the central region of the molecule. Recently, crystal structures of AaNusG in space groups P2(1) and I222 have been reported; the authors conclude that there are no conserved dimers among the contacting molecules in the crystals [Steiner, T., Kaiser, J. T., Marinkovic, S., Huber, R., and Wahl, M. C. (2002) EMBO J. 21, 4641-4653]. We have independently determined the structures of AaNusG also in two crystal forms, P2(1) and C222(1), and surprisingly found that AaNusG molecules form domain-swapped dimers in both crystals. Additionally, polymerization is also observed in the P2(1) crystal. A unique "ball-and-socket" junction dominates the intermolecular interactions within both oligomers. We believe that this interaction is a clue to the function of the molecule and propose a spring-loaded state in the functional cycle of NusG. The importance of the ball-and-socket junction for the function of NusG is supported by the functional analysis of site-directed mutants.

▪ Abstract In the past few years, in vivo technologies have emerged that, due to their efficiency and simplicity, may one day replace standard genetic engineering techniques. Constructs can be made on plasmids or directly on the Escherichia coli chromosome from PCR products or synthetic oligonucleotides by homologous recombination. This is possible because bacteriophage-encoded recombination functions efficiently recombine sequences with homologies as short as 35 to 50 base pairs. This technology, termed recombineering, is providing new ways to modify genes and segments of the chromosome. This review describes not only recombineering and its applications, but also summarizes homologous recombination in E. coli and early uses of homologous recombination to modify the bacterial chromosome. Finally, based on the premise that phage-mediated recombination functions act at replication forks, specific molecular models are proposed.

Efficient expression of most bacteriophage lambda early genes depends upon the formation of an antiterminating transcription complex to overcome transcription terminators in the early operons, p(L) and p(R). Formation of this complex requires the phage-encoded protein N, the first gene product expressed from the p(L) operon. The N leader RNA contains, in this order: the NUTL site, an RNase III-sensitive hairpin and the N ribosome-binding site. N bound to NUTL RNA is part of both the antitermination complex and an autoregulatory complex that represses the translation of the N gene. In this study, we show that cleavage of the N leader by RNase III does not inhibit antitermination but prevents N-mediated translation repression of N gene expression. In fact, by preventing N autoregulation, RNase III activates N gene translation at least 200-fold. N-mediated translation repression is extremely sensitive to growth rate, reflecting the growth rate regulation of RNase III expression itself. Given N protein's critical role in lambda development, the level of RNase III activity therefore serves as an important sensor of physiological conditions for the bacteriophage.

Transcription antitermination by the bacteriophage λ N protein is stimulated in vitro by the Escherichia coli NusG protein. Earlier work suggested that NusG was not required for N activity in vivo. Here we present evidence that NusG also stimulates N-mediated transcription antitermination in intact cells.

Induction of a lambda prophage causes the death of the host cell even in the absence of phage replication and lytic functions due to expression of functions from the lambda p(L) operon. We genetically modified the lambda prophage to determine which lambda p(L) operon functions were involved in cell killing. Viability assays and flow cytometry were used to monitor cell death and filamentation. The kil gene was shown to cause cell death and filamentation as described previously. Another killing activity was mapped within the p(L) operon to the gam gene. Inspection of the DNA sequence showed that there are two possible translation start points for both kil and gam. In both cases, the shorter of the two possible products could cause cell killing. The shorter products were also sufficient for the known filamentation and recombination activities of the respective Kil and Gam functions. The expression level of the p(L) operon is down-regulated by Cro repressor. In the absence of Cro, higher p(L) expression levels allow either Kil or Gam to be lethal or growth inhibitory, whereas at lowered expression in Cro-repressed conditions, only Kil is lethal. The filamentation function of Kil and recombination activity of Gam are unaffected at Cro-repressed levels of expression.

Recently, a highly efficient recombination system for chromosome engineering in Escherichia coli was described that uses a defective lambda prophage to supply functions that protect and recombine a linear DNA targeting cassette with its substrate sequence (Yu et al., 2000, Proc. Natl. Acad. Sci. USA 97, 5978-5983). Importantly, the recombination is proficient with DNA homologies as short as 30-50 bp, making it possible to use PCR-amplified fragments as the targeting cassette. Here, we adapt this prophage system for use in bacterial artificial chromosome (BAC) engineering by transferring it to DH10B cells, a BAC host strain. In addition, arabinose inducible cre and flpe genes are introduced into these cells to facilitate BAC modification using loxP and FRT sites. Next, we demonstrate the utility of this recombination system by using it to target cre to the 3' end of the mouse neuron-specific enolase (Eno2) gene carried on a 250-kb BAC, which made it possible to generate BAC transgenic mice that specifically express Cre in all mature neurons. In addition, we show that fragments as large as 80 kb can be subcloned from BACs by gap repair using this recombination system, obviating the need for restriction enzymes or DNA ligases. Finally, we show that BACs can be modified with this recombination system in the absence of drug selection. The ability to modify or subclone large fragments of genomic DNA with precision should facilitate many kinds of genomic experiments that were difficult or impossible to perform previously and aid in studies of gene function in the postgenomic era.

A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective λ prophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo . The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42°C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective λ prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.

The Escherichia coli nusG gene product is required for transcription termination by phage HK022 Nun protein at the lambda nutR site in vivo. We show that it is also essential for Nun termination at lambda nutL. Three recessive mis-sense nusG mutations have been isolated that inhibit termination by Nun at lambda nutR. The mutations are ineffective in a lambda pL nutL fusion, even when lambda nutR replaces lambda nutL. The mutant strains support lambda growth, indicating that lambda N antitermination activity is not impaired. Transcription arrest by Nun in vitro is stimulated by NusG protein at both lambda nutR and lambda nutL. Mutant NusG protein fails to enhance transcriptional arrest by Nun at either site. The mutant protein, like the wild-type protein, suppresses transcriptional pausing by RNA polymerase and stimulates Rho-dependent termination. These results imply that the role of NusG in Nun termination may be distinct from its roles in other transcription reactions.

Summary RNase III, a double-stranded RNA-specific endonuclease, is proposed to be one of Escherichia coli ’s global regulators because of its ability to affect the expression of a large number of unrelated genes by influencing post-transcriptional control of mRNA stability or mRNA translational efficiency. Here, we describe the phenotypes of bacteria carrying point mutations in rnc, the gene encoding RNase III. The substrate recognition and RNA-processing properties of mutant proteins were analysed in vivo by measuring expression from known RNase III-modulated genes and in vitro from the proteins’ binding and cleavage activities on known double-stranded RNA substrates. Our results show that although the point mutation rnc70 exhibited all the usual rnc null-like phenotypes, unlike other mutations, it was dominant over the wild-type allele. Multicopy expression of rnc70 could suppress a lethal phenotype of the wild-type rnc allele in a certain genetic background; it could also inhibit the RNase III-mediated activation of lN gene translation by competing for the RNA-binding site of the wild-type endonuclease. The mutant protein failed to cleave the standard RNase III substrates in vitro but exhibited an affinity for double-stranded RNA when passed through poly(rI):poly(rC) columns. Filter binding and gel-shift assays with purified Rnc70 showed that the mutant protein binds to known RNase III mRNA substrates in a site-specific manner. In vitro processing reactions with purified enzyme and labelled RNA showed that the in vivo dominant effect of the mutant enzyme over the wild-type was not necessarily caused by formation of mixed dimers. Thus, the rnc70 mutation generates a mutant RNase III with impaired endonucleolytic activity but without blocking its ability to recognize and bind double-stranded RNA substrates.

Inactivation of transcription factor ς 54 , encoded by rpoN ( glnF ), restores high-temperature growth in Luria-Bertani (LB) medium to strains containing the heat-sensitive cell division mutation ftsZ84 . Mutational defects in three other genes involved in general nitrogen control ( glnD , glnG , and glnL ) also suppress lethal filamentation. Since addition of glutamine to LB medium fully blocks suppression by each mutation, the underlying cause of suppression likely derives from a stringent response to the limitation of glutamine. This model is supported by several observations. The glnL mutation requires RelA-directed synthesis of the nutrient alarmone ppGpp to suppress filamentation. Artificially elevated levels of ppGpp suppress ftsZ84 , as do RNA polymerase mutations that reproduce global effects of the ppGpp-induced state. Both the glnF null mutation and an elevated copy number of the relA gene similarly affect transcription from the upstream (pQ) promoters of the ftsQAZ operon, and both of these genetic conditions increase the steady-state level of the FtsZ84 protein. Physiological suppression of ftsZ84 by a high salt concentration was also shown to involve RelA. Additionally, we found that the growth of a glnF or glnD strain on LB medium depends on RelA or supplemental glutamine in the absence of RelA function. These data expand the roles for ppGpp in the regulation of glutamine metabolism and the expression of FtsZ during cell division.

The two-step process of selection and counter-selection is a standard way to enable genetic modification and engineering of bacterial genomes using homologous recombination methods. The tetA and sacB genes are contained in a DNA cassette and confer a novel dual counter-selection system. Expression of tetA confers bacterial resistance to tetracycline (Tc(R)) and also causes sensitivity to the lipophillic chelator fusaric acid; sacB causes sensitivity to sucrose. These two genes are introduced as a joint DNA cassette into Escherichia coli by selection for Tc(R). A medium containing both fusaric acid and sucrose has been developed, in which, coexpression of tetA-sacB is orders of magnitude more sensitive as a counter-selection agent than either gene alone. In conjunction with the homologous recombination methods of recombineering and P1 transduction, this powerful system has been used to select changes in the bacterial genome that cannot be directly detected by other counter-selection systems.

The terminator tI is located approx. 280 nucleotides beyond the int gene of bacteriophage λ. Besides its role as a transcription terminator, tI may confer stability to the int message by protecting it from 3′ exonucleolytic degradation. In order to study the role of the tI sequence in transcription termination and RNA stability, three different point mutations tI1, tI2 , and tI3 were isolated and characterized. All the tI mutations map in the G + C-rich region of dyad symmetry in the terminator and decrease the transcriptional termination of tI in vivo from 99% for the wild type terminator to 81–93% as determined by galactokinase activity and in vitro from 80% for the wild type terminator to 8–12% using the E. coli RNA polymerase. Additionally, the tI mutations cause upstream transcript instability in vivo. This instability defect caused by tI mutations is compensated by the host mutant deficient in polynucleotide phosphorylase resulting in increased steady state levels of these mutant transcripts. The results show that the intact hairpin of tl is essential for efficient transcription termination and for maintaining mRNA stability by blocking the 3′ to 5′ exonucleolytic activity of polynucleotide phosphorylase.

The N gene product of coliphage gamma, with a number of host proteins (Nus factors), regulates phage gene expression by modifying RNA polymerase to a form that overrides transcription-termination signals. Mutations in host nus genes diminish this N-mediated antitermination. Here, we report the isolation and characterization of the rpoAD305E mutation, a single amino acid change in the carboxy terminal domain (CTD) of the alpha subunit of RNA polymerase, that enhances N-mediated antitermination. A deletion of the 3' terminus of rpoA, resulting in the expression of an alpha subunit missing the CTD, also enhances N-mediated antitermination and, similar to rpoAD305E, suppresses the effect of nus mutations. Thus, the N-Nus complex may be affected through contacts with the CTD of the alpha subunit of RNA polymerase, as is a group of regulatory proteins that influences initiation of transcription. What distinguishes our findings on the N-Nus complex from those of previous studies with transcription proteins is that all of the regulators characterized in those studies bind DNA and influence transcription initiation; whereas the N-Nus complex binds RNA and affects transcription elongation. A screen of some previously identified rpoA mutations that influence transcription activators revealed only one other amino acid change, L290H, in the CTD of the alpha subunit, that influences antitermination. Although our results provide evidence that interactions of the alpha subunit of RNA polymerase must be considered in forming models of transcription antitermination, they do not provide information as to whether the interactions of alpha that ultimately influence antitermination occur during initiation or during elongation of transcription.

Publisher Summary This chapter discusses the components of multiprotein-RNA complex that controls transcription elongation in Escherichia Coli phage λ. Studies of bacteriophage A led to the discovery of several basic mechanisms of transcriptional regulation. One of these is transcriptional antitermination, the process in which genes whose transcription is otherwise blocked by premature termination are expressed through termination suppression. In λ and related phages, genome-specific antiterminators convert RNA polymerase (RNAP) into a termination-resistant form during early phases of transcription elongation. The chapter describes the current understanding of how one such antiterminator, the λ N gene product, works. The chapter reviews the methods of overproduction, isolation, and assay of the N protein as well as several accessory factors that modulate transcription elongation in Escherichia coli in the form of a multiprotein-RNA complex. The availability of N and Nus factors in large scale should now make it possible not only to attempt to determine the structures of the individual protein components, and the various RNA-protein and protein-protein complexes by biophysical methods, but also to examine these interactions by conventional biochemical methods.

We have cloned the Francisella tularensis (Ft) grpE-dnaK-dnaJ heat-shock genes which are organized in that order. These genes allow heterologous genetic complementation of each respective mutant strain of Escherichia coli (Ec) for bacteriophage lambda growth. The nucleotide sequences of the Ft grpE-dnaK-dnaJ genes and the deduced amino-acid sequences share significant homologies with their respective Ec counterparts. The Ft DnaK and DnaJ proteins cross-react with polyclonal antibodies raised against the respective Ec proteins. The grpE-dnaK-dnaJ genes of Ft are organized in a fashion that is more characteristic of Gram+ bacteria.

Coliphage lambda employs systems of transcription termination and antitermination to regulate gene expression. Early gene expression is regulated by the phage-encoded N protein working with a series of Escherichia coli proteins, Nus, at RNA sites, NUT, to modify RNA polymerase to a termination-resistant form. Expression of lambda late genes is regulated by the phage-encoded Q antitermination protein. Q, which appears to use only one host factor, acts at a DNA site, qut, to modify RNA polymerase to a termination-resistant form. This review focuses on recent studies which show that: (i) N can mediate antitermination in vitro, independent of Nus proteins. (ii) Early genes in another lambdoid phage HK022 are also regulated by antitermination, where only an RNA signal appears necessary and sufficient to create a termination-resistant RNA polymerase. (iii) A part of the qut signal appears to be read from the non-template DNA strand. (iv) A host-encoded inhibitor of N antitermination appears to act through the NUT site as well as with the alpha subunit of RNA polymerase, and is antagonized by NusB protein.

The NusB and NusE (ribosomal protein S10) proteins function in transcription and translation. The two proteins form a complex that binds to the boxA sequence found in the leader RNA of rrn operons; boxA is required for transcription antitermination in rrn operons. Although binding of these two proteins to the boxA RNA of the bacteriophage lambda nut site has not been observed, both NusB and NusE as well as the RNA boxA sequence are required for lambda N-mediated antitermination. Studies identifying the amino acid changes caused by mutations in nusB and nusE and relating these changes to altered function are reported. It is concluded that boxA is essential for an effective NusB contribution to N-mediated antitermination and that by mutation NusB may be changed to allow more-effective binding to boxA variants.

A 6-kb EcoRl genomic DNA fragment of Coxiella burnetii, isolated from a recombinant bacteriophage λZapII library, allowed heterologous genetic complementation of Escherichia coli deleted for its dnaJ gene. The C. burnetii dnaJ gene was expressed in E. coli and identified by Western blot analysis using polyclonal antibodies raised against purified E. coli DnaJ protein. Deletion mapping and genetic complementation demonstrated that C. burnetii dnaJ is present on a 2-kb EcoRI-HindlII genomic DNA fragment, from which the nt sequence of the C. burnetii dnaJ gene was determined.

Summary A 3.2 kb EcoRI genomic DNA fragment of Coxiella burnetii was isolated by virtue of Its ability to suppress mucoidy in Eschertchia coli. Nucleotide sequence analysis revealed the presence of the genes homologous to rnc, era and recO of E. coli. Suppression of capsule synthesis, measured by β-galactosidase expression in Ion cps-lac fusion strains of E. coli, is caused by gene-dosage effects of the plasmid-borne rnc genes of either C. burnetii or E. coli. The rnc gene of C. burnetii complemented rn– E. coli hosts for lambda plaque morphology and stimulation of lambda N gene expression. We also demonstrated heterologous complementation of an E coli strain defective for the expression of Era, an essential protein in E. coli, using the plasmid-borne C. burnetii era. Under the control of the bacteriophage lambda PL promoter, this 3.2 kb EcoRI DNA fragment directed the synthesis in E. coli of three proteins with approximate molecular masses of 35,27 and 25 kDa. Antibodies against purified E. coli Era protein cross-reacted with the 35 kDa protein of C. burnetii on Western blots.

Publisher Summary This chapter discusses the recent developments in the study of Lambda (λ) phage gene expression showing that post-transcriptional control of phage genes by host functions provides highly sensitive regulatory circuits. The control of transcription initiation from the early promoters of bacteriophage λ is not sufficient to provide for subtle controls involved with the switch between lysogenic and lytic pathways. The ability of λ to choose between lytic and lysogenic development has evolved as a response to changes in the physiological state of the host cell. The frequency by which the phage enters the lytic or lysogenic pathway is determined in large measure by the nutritional state of the host cell. For example, starved cells lysogenize more efficiently than cells grown in a rich medium. In addition, the number of infecting phage particles per cell is also known to determine the rate of lysogenization: the higher the multiplicity of infection, the higher the rate of lysogenization. It is probable that the physiological state of the cell is signaled to the phage by host regulatory factors.

Intrinsic transcription terminators of prokaryotes are distinguished by a common RNA motif: a stem-loop structure high in guanine and cytosine content, followed by multiple uridine residues. Models explaining intrinsic terminators postulate that the stem-loop sequence is necessary only to form structure. In the tR2 terminator of coliphage lambda, single-nucleotide changes reducing potential RNA stem stability eliminated tR2 activity, and a compensatory change that restored the stem structure restored terminator activity. However, multiple changes in the stem sequence that should have either maintained or increased stability reduced terminator activity. These results suggest that the ability of the stem-loop structure to signal transcription termination depends on sequence specificity and secondary structure.

Two mutations in the ninR region of bacteriophage lambda that bypass a requirement for antitermination have been studied. One mutation, byp, has been cloned and mapped by marker rescue to a 417-base-pair segment in the ninR region of the genome. Analysis of the byp mutation by using promoter detection vectors, DNA sequencing, and S1 nuclease analysis showed that the byp mutation created a new promoter that transcribed gene Q. The second mutation analyzed was the deletion nin3. Sequence analysis revealed that 2,485 base pairs of the ninR region were removed, beginning within the ren gene and ending in an open reading frame termed ninG. The tR2 and tR3 terminators, and probably others, were removed by the nin3 deletion, thereby allowing the phage to be N independent and to grow in hosts defective for Nus antitermination factors.

The rap mutation in Escherichia coli prevents the growth of bacteriophage lambda. Phage mutations that overcome rap inhibition (bar) have been mapped to loci in the pL operon. We cloned and sequenced three mutations in two of these loci: barIa to the left arm of the lambda attachment site (attP) and barII in the ssb (ea10) gene. The mutations represent single base-pair changes within nearly identical 16-base-pair DNA segments. Each mutation disrupts a sequence of dyad symmetry within the segment. Plasmids carrying a bar+ sequence downstream to an active promoter are lethal to rap, but not rap+, bacteria. The bar sequences isolated from the lambda bar mutants are not lethal. We synthesized a minimal lambda barIa+ sequence, 5'-TATATTGATATTTATATCATT, and cloned it downstream to an inducible promoter. When transcribed, this sequence is sufficient to kill a rap strain.

The N gene product of phage λ is a positive regulatory factor for the transcription of other λ genes during phage development. It acts by modifying the RNA polymerase transcription complex. In the absence of N function, RNA polymerase initiates at the early λ promoters, p L and p R,but terminates transcription shortly after initiation at terminators, t L1 and t R1 respectively. The action of N prevents polymerase from terminating at t L1 and t R1 as well as at other more distant terminators in the p L and p R operon (see review by Friedman, 1988). In each of these operons, between the promoter and the first terminator, there is a target sequence which is also required for the N- antitermination process (Friedman et al., 1973; Rosenberg et al, 1978). These targets, nutL and nutR, have been defined genetically by mutations to block antitermination in the respective operons (Salstrom and Szybalski, 1978; Olson, et al., 1984). These mutations have defined two regions of nut, boxA and boxB (Friedman & Gottesman, 1983). The boxA site is conserved in sequence among other phages and E. coli whereas the box B site is unique for each phage type and is thought to be the N recognition site (Lazinski, et al., 1989) (See Figure 1).

Recombineering allows DNA cloned in Escherichia coli to be modified via lambda (lambda) Red-mediated homologous recombination, obviating the need for restriction enzymes and DNA ligases to modify DNA. Here, we describe the construction of three new recombineering strains (SW102, SW105 and SW106) that allow bacterial artificial chromosomes (BACs) to be modified using galK positive/negative selection. This two-step selection procedure allows DNA to be modified without introducing an unwanted selectable marker at the modification site. All three strains contain an otherwise complete galactose operon, except for a precise deletion of the galK gene, and a defective temperature-sensitive lambda prophage that makes recombineering possible. SW105 and SW106 cells in addition carry l-arabinose-inducible Cre or Flp genes, respectively. The galK function can be selected both for and against. This feature greatly reduces the background seen in other negative-selection schemes, and galK selection is considerably more efficient than other related selection methods published. We also show how galK selection can be used to rapidly introduce point mutations, deletions and loxP sites into BAC DNA and thus facilitate functional studies of SNP and/or disease-causing point mutations, the identification of long-range regulatory elements and the construction of conditional targeting vectors.

We report the construction of lambda galK promoter cloning vectors for cloning and characterization of strong promoters. This phage, which contains a unique Hind III cloning site, was applied to the cloning and analysis of transcription initiations of the regulatory region of the deo -operon of Escherichia coli and the P L promoter of bacteriophage lambda.

Polarity suppressor mutants that are conditional lethal for growth have been isolated in E. coli K12. The mutations map between the ilv and cya loci of the E. coli chromosome. Rho factor isolated from one of these ts mutants does not show transcription termination activity at any temperature tested; however, it is found to be temperature sensitive for its poly(C)-dependent ATPase activity. Unlike the previously known polarity suppressor mutants (suA and psu), the rho mutation suppresses all types of polarity. Other interesting properties of these mutants include ultraviolet sensitivity, recombination deficiency, and decreased ability to lysogenize temperate phages lambda and P1. Our results suggest that rho has an essential function in the growth and normal physiology of cells. The rho(ts) mutant allows the growth of phage lambda defective in the N gene. This result supports the model that N gene product prevents transcription termination by antagonizing rho activity.

Bacteriophage λ int gene expression is regulated differentially from transcripts originated at the p L and p I promoters. Transcripts initiated at p I terminate at the site t I and express int gene product efficiently. Polymerases starting at p L do not terminate at t I , due to the antiterminating activity of λ N protein. The p L transcripts are unable to express Int protein efficiently because sib , a control site overlapping t I in the unterminated RNA, is processed by host RNase III. We have isolated λsib − mutants by their inability to inhibit int expression from p L transcripts. sib mutations were genetically mapped to the left of the λ attachment site, and do not structurally alter this site for recombination. Several sib mutations do alter the nucleotide sequence of the overlapping sib and t I sites. The λsib − mutants tested prevent RNA processing but do not affect transcription termination in vivo .

Previous studies have attributed two activities to the NusA protein of Escherichia coli ; namely, termination and antitermination of transcription. To examine these activities, we isolated a temperature-sensitive mutant of the nusA gene ( nusA ts11). The mutant cells produce a thermolabile NusA protein and grow at 32 °C, but not at 42 °C. At 42 °C, nusA ts11 is recessive to nusA + and nusA 1, indicating the absence of its active gene product at that temperature. In the mutant, the efficiency of termination at the λt R1 terminator decreases, resulting in an increased expression of distal gene(s). On the other hand, the synthesis of the β-galactosidase and ββ′ subunits of RNA polymerase is reduced in the mutant. This mimics effects seen in vitro when NusA protein is removed from a coupled transcription-translation system. These results suggest that the NusA protein plays both negative and positive modulator roles in vivo . The mutation nusA ts11, unlike nusA 1, does not block λ phage growth at non-permissive temperatures, suggesting that NusA protein is not required for N antitermination in the mutant. Besides, the nusA ts11 allows λN am7 N am53 byp phage growth under sup 0 conditions, indicating that the N antitermination function is dispensable (at least partly) in this mutant.

We describe a cloning-expression vector system for selecting DNA fragments containing open reading frames (ORFs) and expressing them as beta-galactosidase (beta Gal) hybrid fusion proteins. The plasmid vector, pWS50, utilizes the very strong and easily regulated bacteriophage lambda promoter pL, and the efficient translation initiation signals of the N-terminal segment of the lambda cII gene. Fused distally to and out of translational phase with cII is the E. coli lacZ gene, lacking its own transcriptional and translational initiating signals. A unique restriction enzyme site (NruI) is located between the upstream regulatory sequences and the lacZ gene, which provides a cloning site for the insertion of blunt ended DNA fragments. In addition, there are two other unique restriction sites (NheI and BamHI) located in this region which can also be used as closing sites. If a DNA fragment does not contain any translation termination codons (i.e., an ORF), and is inserted correctly into the vector, the translational reading frame between cII and lacZ can be restored. Colonies containing these recombinants can be easily screened as LacZ+ on lactose indicator media. The beta-galactosidase fusion proteins produced from the LacZ+ recombinants are identified on sodium dodecyl sulfate polyacrylamide gels by their large size and high level of production. To test the ORF cloning-expression system, a segment of the human T-cell lymphotrophic virus type I envelope gene was cloned and expressed at high levels. The envelope-beta Gal fusion protein was recognized by antibodies in serum from a patient with adult T-cell leukemia.

A plasmid, pJL6, was constructed that contains a unique ClaI site twelve codons beyond the bacteriophage lambda cII gene initiation codon. This site allowed us to fuse the carboxy-terminal sequences of the avian myelocytomatosis virus (MC29) v-myc gene to the amino-terminal portion of the cII gene. Transcription of the hybrid gene is controlled from the phage lambda pL promoter. When this promoter is derepressed, Escherichia coli cells harboring the chimeric plasmid produce a level of cII-myc fusion protein greater than 5% of total cellular protein. Antibodies raised by this protein immunoprecipitate the MC29 gag-myc gene product, P110gag-myc.

Several laboratories have constructed expression vectors for the production of heterologous fusion proteins containing the N-terminal 13 amino acids of the bacteriophage lambda cII-coded protein in Escherichia coli. We have prepared a monoclonal antibody to a synthetic peptide having this CII amino acid sequence and have found that this antibody reacts with authentic CII protein in Western blot tests and with most CII peptide-containing fusion proteins in both radioimmunoprecipitation and Western blot assays. However, there are some CII-hybrid protein species with which the antibody does not react. Our findings indicate that this antibody is a valuable tool for detecting and purifying expressed proteins and in studying their structure and function.

We describe below the chemical synthesis of the right and left ends of bacteriophage Mu and characterize the activity of these synthetic ends in mini-Mu transposition. Mini-Mu plasmids were constructed which carry the synthetic Mu ends together with the Mu A and B genes under control of the bacteriophage λ p L promoter. Derepression of p L leads to a high frequency of mini-Mu transposition (5.6 × 10 −2 ) which is dependent on the presence of the Mu ends and the Mu A and B proteins. Five deletion mutants in the Mu ends were tested in the mini-Mu transposition system and their effects on transposition are described.

Ribonuclease III (RNase III) represents a family of double-stranded RNA (dsRNA) endonucleases. The simplest bacterial enzyme contains an endonuclease domain (endoND) and a dsRNA binding domain (dsRBD). RNase III can affect RNA structure and gene expression in either of two ways: as a dsRNA-processing enzyme that cleaves dsRNA, or as a dsRNA binding protein that binds but does not cleave dsRNA. We previously determined the endoND structure of Aquifex aeolicus RNase III (Aa-RNase III) and modeled a catalytic complex of full-length Aa-RNase III with dsRNA. Here, we present the crystal structure of Aa-RNase III in complex with dsRNA, revealing a noncatalytic assembly. The major differences between the two functional forms of RNase III·dsRNA are the conformation of the protein and the orientation and location of dsRNA. The flexibility of a 7 residue linker between the endoND and dsRBD enables the transition between these two forms.

The bacteriophage lambda int gene product, integrase, recombines the phage DNA with the host DNA at specific sites on each to accomplish lysogeny. The int gene is transcribed from two promoters, PL and PI, each regulated positively by lambda proteins. The expression of integrase is also controlled from a site, sib, in the b region of the phage genome. This is a unique regulatory site because it is located distal to the structural gene in relation to the promoters. The expression of int from the PL promoter is inhibited when sib is present. This effect appears to be specific for PL because sib does not cause inhibition of PI-dependent int synthesis. lambda mutants that contain alterations in the site have been isolated. Sequence analyses of the mutations reveal single base changes, spanning 37 base pairs (bp) in the b region, some 240 bp beyond the int gene. Another mutant, hef13, which has a phenotype similar to that of sib, introduces a nucleotide change within the same 37-bp region. The sib and hef mutations cluster within a region of dyad symmetry. Regulation of int synthesis by sib occurs after transcription of the int gene. There is no difference in the rate of PL-promoted int mRNA synthesis in either sib+ or sib- phage infections, yet int mRNA is less stable in the sib+ infection. Because RNase III host mutants are defective in sib regulation, processing of the PL mRNA at sib by this endoribonuclease may cause int mRNA decay and decrease int synthesis.

Transcription of the lambda repressor gene (cI) is positively regulated by the phage-encoded proteins cII and cIII. We have isolated and characterized the 5'-terminal region of this RNA and shown that it originates at a promoter (pE) located between genes cro and cII. The DNA sequence of this promoter shows little homology to other known promoters. Initiation of transcription from PE is abolished by the cis-dominant mutations cY; these mutations alter the "-10" and "-35" regions of the promoter. We propose that the "-35" region is the site of activation of PE, possibly via the direct interaction of protein cII.

Seven hybridomas producing murine monoclonal antibodies reactive against NusA protein of Escherichia coli were prepared. Antigenic determinants of these monoclonal antibodies have been mapped by immuno-blotting analyses using fusion proteins containing parts of NusA.The epitope of the N14 antibody maps in a hydrophobic amino acid (aa) cluster and consists of at least Ala-181 and Ser-183 residues. nusA 1 and nusA 11 mutations, which cause aa changes of these residues,abolish the antigenic reactivity to the N14 antibody.These antibodies react with intact NusA protein, indicating that the epitopes are exposed on the surface of NusA. Most of these epitopes cluster around the nusA 1 and nusA 11 mutation loci.

Deletions extending from the cro gene into boxA and nutR of the Rho-dependent tR1 terminator of bacteriophage lambda have been generated and cloned between promoters and the galK gene of Escherichia coli on a multicopy plasmid. Terminators placed between the promoters and galK restrict transcription and expression of galK on these plasmids. However, when lambda N protein is provided, and if a functional N interaction site, nutR, is intact, transcription antitermination occurs and galK expression increases. Deletions into the nutR region affect the ability to antiterminate. From the results obtained we conclude that: boxA, a site believed to bind host factors (Nus), is not required for transcription antitermination in this system; the host NusA function is required even in the absence of boxA; nutR is required for N antitermination; translation across the nutR sequence prevents N-dependent antitermination.