Alain Levine

Inactivation of prophage λ repressor in Vivo

Jacob & Monod (1961) postulated that prophage A induction results from the inactivation of the λ repressor by a cellular inducer. Although it has been shown that the phage A repressor is inactivated by the recA gene product in vitro (Roberts et al., 1978), we wanted to determine the action of the “cellular inducer” in vivo. Our results have led to a new model, which defines the relationship between the “cellular inducer” and the recA gene product. n nIn order to quantitate the action of the cellular inducer on the λ repressor, we made use of bacteria with elevated cellular levels of the λ repressor (hyperimmune lysogens). We determined the kinetics of repressor inactivation promoted by three representative inducing treatments: ultraviolet light irradiation, thymine deprivation and temperature shift-up of tif-1 mutants. n nThe kinetics of repressor decay in wild-type monolysogens indicate that repressor inactivation is a relatively slow cellular process that takes a generation time to reach completion. Incomplete inactivation of the repressor without subsequent prophage development may occur in a cell. We call this phenomenon detected at the biochemical level “subinduction”. In hyperimmune lysogens. subinduction is always the case. n nA high cellular level of A repressor that prevents prophage λ induction does not prevent induction of a heteroimmune prophage such as 434 or 80. Although the cellular inducer does not seem specific for any inducible prophage, it does not inactivate two prophage repressors present in a cell in a random manner. We have called this finding “preferential repressor inactivation”. Preferential repressor inactivation may be accounted for by considering that the intracellular concentration of a repressor determines its susceptibility to the action of the inducer. n nIn bacteria with varying repressor levels, a fixed amount of repressor molecules is inactivated per unit of time irrespective of the initial repressor concentration. The rate of repressor inactivation depends on the catalytic capacity of the cellular inducer that behaves as a saturated enzyme. In wild-type bacteria the cellular inducer seems to be produced in a limited amount, to have a weak catalytic capacity and a relatively short half-life. The amount of the inducer formed after tif-1 expression is increased in STS bacteria overproducing a tif-1-modified RecA protein. This result is an indication that a modified form of the RecA protein causes repressor inactivation in vivo. n nFrom the results obtained we propose a model concerning the formation of the cellular inducer. We postulate that the cellular inducer is formed in a two-step reaction. The is model visualises how the RecA protein can be induced to high cellular concentrations, even though the RecAp protease molecules remain at a low concentration. The latter accounts for the limited proteolytic activity found in vivo.

A checkpoint involving RTP, the replication terminator protein, arrests replication downstream of the origin during the Stringent Response in Bacillus subtilis

Regulation of DNA replication in Bacillus subtilis involves a post‐initiation mechanism which is subject to control by the Stringent System, an essential regulatory network, mediated by the alarmone, ppGpp. In detailed studies using DNA‐DNA hybridization procedures, we have now shown that, following the induction of the Stringent Response, replication is blocked downstream of the origin, on the left, close to the hut marker (‐175 kb) and on the right, beyond the soft10 marker (+199 kb). In addition, we provide evidence that inhibition of replication under these conditions requires the replication terminator protein (RTP). In a mutant lacking RTP, a protein normally involved in termination of chromosomal replication through recognition of specific terminator sequences, replication continues past the sites normally blocked by the Stringent Response. These data strengthen the argument that this second level of control of DNA replication occurs at specific sites, the Stringent Terminus (STer) sites, either side of oriC Such sites are presumably related to the sequence involved in RTP recognition at the terminus, terC. We propose that the binding of RTP must be modulated, perhaps through the action of ppGpp, to recognize post‐initiation control sequences during the Stringent Response, in order to block replisome movement. This, therefore, acts as a checkpoint in chromosome elongation.

Cellular levels of the prophage λ and 434 repressors

As a prerequisite to a quantitative study of the inactivation of phage repressors in vivo (Bailone et al., 1979), the cellular concentrations of the bacteriophage λ and 434 repressors have been measured in bacteria with varying repressor levels. n nUsing the DNA-binding assay we have determined the conditions for optimal repressor titration. The sensitivity of the λ repressor assay was increased by adding magnesium ions to the binding mixture; this procedure was without effect on the titration of the 434 repressor. The measures of the cellular repressor concentrations varied with the method of cell disruption. n nThe cellular concentration of λ repressor, about 140 active repressor molecules per monolysogen, was relatively constant under specific cultural conditions. The repressor concentration increased with the number of cI gene copies but not in direct proportion. n nThe 434 repressor concentration, hardly detectable in extracts of lysogens carrying an imm434 prophage, was greatly enhanced in bacteria carrying the newly constructed plasmid pGY101, that encodes the 434 cI gene. n nThe cellular repressor level produced by 434 is lower than that produced by λ: this indicates that the maintenance of the prophage state is ensured by a relatively small number of repressor molecules binding tightly to the operator sites.

The replication checkpoint control in Bacillus subtilis: identification of a novel RTP-binding sequence essential for the replication fork arrest after induction of the stringent response

We have shown previously that induction of the stringent response in Bacillus subtilis resulted in the arrest of chromosomal replication between 100 and 200u2003kb either side of oriC at distinct stop sites, designated LSTer and RSTer, left and right stringent terminators respectively. This replication checkpoint was also shown to involve the RTP protein, normally active at the chromosomal terminus. In this study, we show that the replication block is absolutely dependent upon RelA, correlated with high levels of ppGpp, but that efficient arrest at STer sites also requires RTP. DNA–DNA hybridization data indicated that one or more such LSTer sites mapped to gene yxcC (−128u2003kb from oriCu200a). A 7.75u2003kb fragment containing this gene was cloned into a theta replicating plasmid, and plasmid replication arrest, requiring both RelA and RTP, was demonstrated. This effect was polar, with plasmid arrest only detected when the fragment was orientated in the same direction with respect to replication, as in the chromosome. This LSTer2 site was further mapped to a 3.65u2003kb fragment overlapping the next40 probe. Remarkably, this fragment contains a 17u2003bp sequence (B′‐1) showing 76% identity with an RTP binding site (B sequence) present at the chromosomal terminus. This B′‐1 sequence, located in the gene yxcC, efficiently binds RTP in vitro, as shown by DNA gel retardation studies and DNase I footprinting. Importantly, precise deletion of this sequence abolished the replication arrest. We propose that this modified B site is an essential constituent of the LSTer2 site. The differences between arrest at the normal chromosomal terminus and arrest at LSTer site are discussed.

Induction of recA+-protein synthesis in Escherichia coli

SummaryEscherichia coli was infected with λprecA+to determine the genetic and physiological factors controlling recA+gene expression. When λprecA+replication was prevented by superinfection immunity, recA+protein synthesis was induced by UV radiation. The recA+gene is negatively controlled by the lexA+gene product because i) a dominant lexA mutation, lexA3, prevented induction of recA+protein synthesis ii) a recessive lexA mutation, tsl-1, caused induction of recA+protein synthesis. Conversely positive control of recA+gene expression requires recA+protein because i) a co-dominant tif-1 mutation (a recA mutation) caused induction of recA+protein synthesis ii) a recessive mutation, recA1, prevented cis-induction of recA protein synthesis. recA+protein and Protein X of UV irradiated bacteria co-migrated and were subject to the same physiological and genetic controls. It is concluded that Protein X is recA+protein. λ lysogenic induction was prevented by TPCK, a protease inhibitor. However TPCK did not prevent induction of recA+protein synthesis, indicating that induction of the two processes occurs in different ways. It is suggested that the lexA+and recA+proteins normally combine to repress the recA+gene. Derepression might occur after DNA damaging treatments because the amount of this complex would be reduced by recA+protein i) binding to single-stranded DNA and/or ii) being activated to function proteolytically towards regulatory molecules such as λ repressor.

Cell cycle checkpoints in bacteria

When DNA replication is interrupted in bacteria, a specific inhibitor (SfiA), a component of the SOS system, is synthesised which transiently blocks cell division. This is the prototype, dispensable, cell cycle checkpoint, essential for maximal survival under a particular stress. In contrast, no process specifically signalling the termination of chromosomal replication to activate the subsequent division event, which might be termed an essential checkpoint, has yet been demonstrated. In E coli, a specific mechanism is apparently required to reactivate replication forks blocked by damage, but its molecular basis is unclear. Induction of the stringent response, mediated by RelA via the level of ppGpp, presumably to optimise macromolecular synthesis according to the availability of nutrients, activates a control system which inhibits DNA replication in both E coli and B subtilis. In E coli, this blocks new rounds of initiation at oriC, although the mechanism is not clear. Conversely, initiation is not blocked in B subtilis, but replication is blocked apparently at a number of distinct sites 100-200 kb downstream and either side of oriC. This nutrient-dependent replicating checkpoint specifically requires RTP, the chromosomal terminator protein, and new evidence indicates that specific RTP binding sites may be involved in this post-initiation control mechanism. A similar post-initiation control mechanism appears to block replication reversibly after premature initiation in B subtilis, indicating that this system may have a dual function, limiting replication in starvation conditions and as a mechanism to compensate for premature initiations.

Chromosomal initiation in Bacillus subtilis may involve two closely linked origins

SummaryWhen the dnaB37 initiation mutant of Bacillus subtilis is returned to a permissive temperature following a period at 45° C, a synchronous round of DNA replication immediately ensues. Using this system we have been able to analyse the first fragments to be replicated while avoiding the use of thymine starvation or inhibitors of DNA replication. Such treatments are necessary to achieve even modest synchrony in germinating spores. Our results showed that the first fragment to be replicated was a 4kb BamHI-SalI restriction fragment, BS6. In contrast, when the analysis was performed out in the presence of novobiocin, an inhibitor of DNA gyrase, replication from BS6 was inhibited and the first fragment to be replicated was BS5, a 5.6 kb fragment located 1.7 kb to the right of BS 6. Replication from both putative origins was suppressed by rifamycin and was dependent upon dnaB. The results are discussed in relation to previous attempts to identify the first replicating fragment in germinating spores. We also discuss the possibility that B. subtilis contains two origins and suggest that either can act as the primary origin under certain conditions, or alternatively that both origins may act in concert in normal bidirectional replication, each site being required for the leading strand in each direction.

Post-initiation control of chromosomal replication in Bacillus subtilis : a mechanism for limiting over-replication or for duplicating key growth and sporulation genes ?

We used the Bacillus subtilis dnaB37 mutant, which is defective in initiation, to synchronize DNA replication in order to identify the first fragments to be replicated following initiation and to study the control of this process under various conditions. We show by DNA/DNA hybridization analysis that, after returning the mutant from 45 degrees C to the permissive temperature (30 degrees C), the origin region relative to other sequences is over-replicated (approximately 2-fold) during the first round. This was confirmed by autoradiographic analysis. The over-replicated region is however limited to about 190 kb on the left and right arms. Replication apparently resumes from these positions during the following round of replication. We propose that, in B. subtilis, in addition to the first level of control at the origin, there is a second level or post-initiation control downstream of the origin which limits DNA replication resulting from premature initiation. We believe that these two levels of control are tightly coupled under conditions of balanced growth. Using the same system, we have now shown that DNA replication is subject to stringent control, an important regulatory network in bacteria. These studies demonstrate that the inhibition of replication induced during the stringent response does not occur at the primary origin. In fact, by DNA/DNA hybridization, replication forks were found to be blocked at similar positions to the post-initiation control sites described above. Moreover, replication appears to resume from regions close to the stalled replisomes upon removal of the stringent response.(ABSTRACT TRUNCATED AT 250 WORDS)

Régulation de la transcription de l'ADN du bactériophage λ infectant un système membranaire subcellulaire dérivé de sphéroplastes d'Escherichia coli

SummaryIn vitro transcription of DNA isolated from mature bacteriophage λ CI857 was studied by infecting a membranous cell-free system obtained from lysates of E. coli spheroplasts. The messenger RNA of lambda was characterized by hybridization with the AT-rich and GC-rich halves of λ DNA molecules and with the two separated strands of the λ genome, as well as by the competition method with λ messenger RNA extracted from induced lysogenic E. coli cells.The results indicate that the transfer of genetic information by the system is a specifically regulated process leading to formation of “early” and “late” messengers. Transcription is selective and asymmetric.Chloramphenicol and cytosine arabinoside added at the start of the incubation suppress the synthesis of late mRNA and slightly affect the synthesis of early mRNA.Synthesis of λ DNA is observed.DNAase added after 10 minutes of incubation, does not prevent an important synthesis of late mRNA.Synthesis of λ DNA and proteins appears to be a prerequisite for the transcription of late cistrons.The prominent role of a complex between λ DNA and membranes in the production of mRNA and particularly in the selective and asymmetric transcription of λ DNA into late messenger is emphasized.

Synthèse in vitro d'endolysine de bactériophage lambda dans un système membranaire d'Escherichia coli

SummaryWe have previously shown that a membranous cell-free system derived from uninfected penicillin spheroplasts of E. coli transcribes early and late messenger RNAs from λ DNA.This in vitro system will also transcribe and translate the endolysin gene R of λ DNA. The enzyme activity that results from in vitro synthesis corresponds to λ endolysin (a typical late protein) by several criteria.DNA from λ CI857 sus R5 ts 9B and λ CI857 sus S7 pgal, mutants carrying nonsense mutations in genes involved in the host lysis, are inactive in the synthesis of endolysin with an extract of non permissive cells, although they are fully active with an extract of permissive cells. Furthermore, suppression of these mutations is entirely dependent on addition of supernatant from suppressor strains.The endolysin synthesis from a thermosensitive λ CI mutant is observed at 40°C and not at 30°C. This suggests that the product of CI gene is formed and acts in the in vitro system at 30°C.Enzymatic activity is detected after a 15 min lag period.Membranes and double stranded λ DNA are absolutely required for the enzyme synthesis. Ribosomes and supernatant highly stimulate the in vitro system.Inhibitors of RNA, DNA and protein synthesis (actinomycine D; cytosine arabinoside; DNA-ase; and chloramphenicol respectively) will prevent endolysin production when added at zero time. If DNA-ase or actinomycine D are added after 20 min of incubation, only partial inhibition of endolysin synthesis occurs. It is therefore concluded, according to our previous observations, that messengers are stable enough to allow enzyme synthesis after delayed addition of the inhibitors in the in vitro system.It appears that there is a complete regulation in the membranous system like in vivo and which starts with the early and late messenger formation and leads to active late protein synthesis.