Lysogenic Growth Research Articles

Analysis of a regulator involved in the genetic switch between lysis and lysogeny of the temperate Lactococcus lactis phage phi LC3.

Sequencing of a 1.3-kb fragment of DNA from the temperate Lactococceus lactis subsp. cremoris phage phiLC3 revealed a pair of two divergently oriented ORFs, orf63 and orf286. The deduced amino acid sequence of the product of orf286 showed extensive homology to those of repressors of the temperate lactococcal phages rlt, Tuc2009 and BK5-T. A mutant with an amber mutation in orf286 gave rise to a clear plaque phenotype, indicating that this gene is involved in the lytic and lysogenic development of phiLC3. Gel mobility shift assays showed that the partially purified Orf286 protein bound specifically to the 224-bp intergenic region located between orf286 and orf63, and further characterization by DNase I footprinting analysis revealed that Orf286 protects two distinct sites within this region. Sequence analysis of the intergenic region revealed two putative, divergently oriented promoters, P1 and P2; orf286 and orf63 are probably transcribed from P1 and P2, respectively. In vivo analyses of P1 and P2 using beta-galactosidase as a reporter enzyme in L. lactis showed that transcription from P1 was repressed while transcription from P2 was stimulated in the presence of the Orf286 protein. These results suggest a complex role for the Orf286 protein in regulating the genetic switch between lytic and lysogenic growth of phiLC3.

The tetramerization domain of the Mnt repressor consists of two right-handed coiled coils.

The tetrameric Mnt repressor is involved in the genetic switch between the lysogenic and lytic growth of Salmonella bacteriophage P22. The solution structure of its C-terminal tetramerization domain, which holds together the two dimeric DNA-binding domains, has been determined by NMR spectroscopy. This structure reveals an assembly of four alpha-helical subunits, consisting of a dimer of two antiparallel coiled coils with a unique right-handed twist. The superhelical winding is considerably stronger and the interhelical separation closer than those found in the well-known left-handed coiled coils in fibrous proteins and leucine zippers. An unusual asymmetry arises between the two monomers that comprise one right-handed coiled coil. A difference in the packing to the adjacent monomer of the other coiled coil occurs with an offset of two helical turns. The two asymmetric monomers within each coiled coil interconvert on a time scale of seconds. Both with respect to symmetry and handedness of helical packing, the C2 symmetric four-helix bundle of Mnt differs from other oligomerization domains that assemble DNA-binding modules, such as that in the tumor suppressor p53 and the E. coli lac repressor.

Transcriptional silencing by the mycobacteriophage L5 repressor

The success of a temperate bacteriophage is dependent upon its ability to completely shut down expression of its lytic genes during lysogenic growth. Mycobacteriophage L5 accomplishes this by an atypical phage repressor, gp71, which binds to multiple asymmetric DNA sites. L5 gp71 regulates transcription initiation at an early lytic promoter, Pleft, but also affects downstream gene expression at 'stoperator' sites in the phage genome. The L5 genome is replete with stoperator sites located within short intergenic spaces in both the early and late lytic operons and oriented specifically with respect to transcription. Binding of gp71 to these sites results in a strong orientation-dependent polar effect on downstream gene expression and global silencing of prophage gene expression.

Reciprocal Regulation of the Early Promoter Region of Bacteriophage HP1 by the Cox and CI Proteins

We have identified a transcriptional switch at the early promoter region of bacteriophage HP1. This switch controls the transcription of the early lytic operon from the PR1and PR2promoters and the transcription of the lysogenic operon from the PLpromoter. The start sites of the three promoters were mapped, and using a chloramphenicol acetyl transferase assay, we have investigated the levels of transcription from the promoters in the absence or in the presence of two phage-encoded transcription factors: HP1 Cox and HP1 CI. The HP1 Cox protein repressed the production of PLtranscripts 30-fold, while the HP1 CI protein repressed lytic transcription at least 70-fold. Binding sites for HP1 Cox and CI were identified in the early promoter region; mutations of these sites eliminated transcriptional repression. In addition, a mutant CI protein was isolated which is temperature sensitive for repression. Taken together, these data demonstrate the reciprocal regulation of a transcriptional switch in which the actions of the two phage-encoded proteins at the phage early promoters determine the choice between lytic and lysogenic growth.

Gene expression in the cos region of the Streptomyces temperate actinophage phi C31.

A transcription map of a 5.12 kb region containing the cos ends of actinophage phi C31 was determined using RNA prepared from induced and uninduced cultures of the temperature-sensitive lysogen, Streptomyces coelicolor A3(2) (phi C31cts1). In induced cultures, RNA synthesis was detected only late in the lytic cycle. A late operon initiated downstream of the int gene on the right-hand end of the genome traversed the cos ends and extended at least 3.6 kb along the left-hand end. Shorter, possibly processed, mRNAs were also present. The map was superimposed on the DNA sequence of 2.8 kb of the region, part of which had been determined previously and part of which is presented here. The late-expressed transcripts contained a tRNA(Thr)-like gene and four ORFs (1, 2, 3 and 5) detected on the basis of codon bias. Analysis of the putative protein products showed that one of the ORFs could encode a lysis protein and at least one may be involved in DNA maturation. Transcription mapping of RNA from uninduced cultures demonstrated a 620 nt transcript, xRNA1, of ORF6. So far this is the only gene in phi C31 found to be expressed right to left with respect to the standard map of phi C31; its function during lysogenic growth could not be deduced from database searches.

KNOWLEDGE-BASED SIMULATION OF REGULATORY ACTION IN LAMBDA PHAGE

We have developed a knowledge-based but partially analytic simulation system. This system simulates regulatory action in lambda phage, a virus which infects E. coli. Specifically, we simulated the decision between its two developmental pathways, lytic and lysogenic growth. Our model is composed of two levels: roughly abstracted level and precisely abstracted level. The former level is discrete-event and knowledge-based. It covers overall regulations inside lambda phage in qualitative representation. On the other hand, the latter is based on quantitative chemical equations describing the sensitive bifurcation within pathways. In this way, qualitatively clear overview of regulatory action is efficiently simulated using knowledge base, and only the unpredictable part is analytically simulated in detail. This system can output not only input knowledge but also precise prediction by computational analysis, data which help molecular biologists find new theories of regulatory actions.

Isolation, Characterization, and Mapping of Temperature-Sensitive Mutations in the Genes Essential for Lysogenic and Lytic Growth of the Mycobacteriophage L1

Forty temperature-sensitive mutations affecting lytic growth and eight affecting both establishment and maintenance of lysogeny of the temperate mycobacteriophage L1 have been isolated. All of the latter mutations form one complementation group and map within a very short region around the 15% coordinate of the L1 genome; these affect a single gene, cl, coding for the L1 repressor. The former 40 mutations form 28 complementation groups, identifying 28 different genes, G1-G28, essential for the lytic growth of L1. These genes have been mapped using the Gts mutations. Of the 28 Gts mutants, 14 are defective in host lysis at 42° but not at 32° while the other 14 can lyse the host at both temperatures. Among the former 14 Gts mutants, 6 are also defective in L1 DNA synthesis at 42°, and they map in two different clusters, 4 around 65% and 2 around 84% of the L1 genome.

Bacteriophage P1 gene 10 is expressed from a promoter-operator sequence controlled by C1 and Bof proteins.

Gene 10 of bacteriophage P1 encodes a regulatory function required for the activation of P1 late promoter sequences. In this report cis and trans regulatory functions involved in the transcriptional control of gene 10 are identified. Plasmid-borne fusions of gene 10 to the indicator gene lacZ were constructed to monitor expression from the gene 10 promoter. Production of gp10-LacZ fusion protein became measurable at about 15 min after prophage induction, whereas no expression was observed during lysogenic growth. The activity of an Escherichia coli-like promoter, Pr94, upstream of gene 10, was confirmed by mapping the initiation site of transcription in primer extension reactions. Two phage-encoded proteins cooperate in the trans regulation of transcription from Pr94: C1 repressor and Bof modulator. Both proteins are necessary for complete repression of gene 10 expression during lysogeny. Under conditions that did not ensure repression by C1 and Bof, the expression of gp10-LacZ fusion proteins from Pr94 interfered with transformation efficiency and cell viability. Results of in vitro DNA-binding studies confirmed that C1 binds specifically to an operator sequence, Op94, which overlaps the -35 region of Pr94. Although Bof alone does not bind to DNA, together with C1 it increases the efficiency of the repressor-operator interaction. These results are in line with the idea that gp10 plays the role of mediator between early and late gene transcription during lytic growth of bacteriophage P1.

Stabilization of bacteriophage Mu repressor-operator complexes by the Escherichia coli integration host factor protein.

All of the previously described effects of integration host factor (IHF) on bacteriophage Mu development have supported the view that IHF favours transposition-replication over the alternative state of lysogenic phage growth. In this report we show that, consistent with a model in which Mu repressor binding to its operators requires a particular topology of the operator DNA, IHF stimulates repressor binding to the O1 and O2 operators and enhances Mu repression. IHF would thus be one of the keys, besides supercoiling and the H-NS protein, that lock the operator region into the appropriate topological conformation for high-affinity binding not only of the phage transposase but also of the phage repressor.

Introduction to Lambda Phages

The biology of lambda-derived vectors is extremely well understood. This introduction to the biology of lambda and related phages is included to help readers use the lambda vectors that are employed in the manual and to help readers understand new lambda vectors that are being developed. Specific topics include a discussion of lytic growth (i.e., requirements for DNA replication, packaging and lysis) and lysogenic growth (i.e., requirements for lysogenization and induction of a lysogen).

Identification of the DNA binding domain of the phage lambda cII transcriptional activator and the direct correlation of cII protein stability with its oligomeric forms.

The bacteriophage lambda transcriptional activator protein cII is a DNA-binding protein that coordinately regulates transcription from phage promoters important for lysogenic growth. We have genetically and structurally characterized more than 80 different single amino acid substitutions in this 97-amino-acid protein. A subset of 25 of these variant proteins was utilized for detailed biochemical analysis, which allows us to define specific domains critical for sequence-selective DNA recognition, nonspecific DNA binding, and protein oligomerization. The mutation studies also demonstrated the remarkable correlation of oligomeric structure of cII protein to its stability within the bacterial host. An Escherichia coli HtpR- strain has been identified that greatly stabilizes these highly unstable cII mutants.

Characterization of a third, cII-dependent, coordinately activated promoter on phage lambda involved in lysogenic development.

Lysogenic development by bacteriophage lambda is known to require the coordinate expression of two phage operons. Coordinate control is achieved by a positive regulatory mechanism which activates transcription from the promoters of these operons, PRE and PI. The positive effector is the phage regulatory protein cII. We now identify and characterize a third cII-dependent transcription unit on phage lambda, which is positioned in the middle of the Q regulatory gene and has an anti-Q orientation. We demonstrate the cII-dependent function of this promoter and precisely map its 5' transcription start-site both in vitro and in vivo. Most importantly, we show that cII binding and transcription activation at PaQ occur at essentially the same cII levels as those required for PRE and PI activation, and that all three promoters respond to cII at the same time following phage infection. In addition, DNase protection studies demonstrate that cII selectively interacts with the same TTGCN6TTGC DNA sequence repeat in the -35 region of PaQ which cII interacts with at both PRE and PI. We find that cII also binds other TTGCN6TTGC repeat sequences on lambda but binding at these sites does not lead to productive transcription. We conclude that PaQ functions in concert with PRE and PI to regulate the lysogenic growth response of phage lambda. We presume that the PaQ directed anti-sense Q RNA transcript functions to down-regulate the expression of the Q gene, which is needed for the expression of all phage late genes during lytic growth.

Knowledge-based simulation of genetic regulation in bacteriophage lambda.

We have developed a general-purpose computer program for the functional simulation of regulatory genetics. This simulator is knowledge-based and was developed using the Unit System, a software tool for the acquisition, representation, and manipulation of hierarchically organized knowledge. The advantages of a knowledge-based design are presented, and the simulator's architecture is described. Its performance on the decision between lytic and lysogenic growth in Bacteriophage Lambda is reported.

The relationship between function and DNA sequence in an intercistronic regulatory region in phage lambda.

rho factor-mediated transcription termination at the tr1 terminator site of bacteriophage lambda is examined. Mutations affecting the termination event are characterised. These mutations define features of the site which seem to be important to terminator function. In addition, other related transcriptional and translational regulatory elements are defined within the region surrounding the termination site. The potential molecular interactions and structural overlaps of these control signals apparently couple the regulation of the decision between lytic and lysogenic growth patterns by phage lambda.

Clear plaque mutants of phage P1

The effect of a shift in temperature on lysogens for temperature-sensitive clear mutants in each of the four P1 clear cistrons is described. Only gene c1 is induced to form phage by such a shift, so it is concluded that this gene is responsible for immunity repressor production. The growth of lysogens carrying a c2, c3, or c4 mutant stops when the temperature is shifted to that at which the clear gene cannot function, indicating that these clear plaque cistrons are also active in the prophage state. Gene c3, in addition to affecting the establishment of lysogeny, appears to be essential for vegetative phage development at early and late times after infection.