Thymine-starvation-induced chromosomal fragmentation is not required for thymineless death in Escherichia coli.

Thymine or thymidine starvation induces robust chromosomal fragmentation in Escherichia coli thyA deoCABD mutants and is proposed to be the cause of thymineless death (TLD). However, fragmentation kinetics challenges the idea that fragmentation causes TLD, by peaking before the onset of TLD and disappearing by the time TLD accelerates. Quantity and kinetics of fragmentation also stay unchanged in hyper-TLD-exhibiting recBCD mutant, making its faster and deeper TLD independent of fragmentation as well. Elimination of fragmentation without affecting cellular metabolism did not abolish TLD in the thyA mutant, but reduced early TLD in the thyA recBCD mutant, suggesting replication-dependent, but undetectable by pulsed-field gel, double-strand breaks contributed to TLD. Chromosomal fragmentation, but not TLD, was eliminated in both the thyA and thyA recBCD mutants harboring deoCABD operon. The expression of a single gene, deoA, encoding thymidine phosphorylase, was sufficient to abolish fragmentation, suggesting thymidine-to-thymine interconversion during T-starvation being a key factor. Overall, this study reveals that chromosomal fragmentation, a direct consequence of T-starvation, is either dispensable or redundant for the overall TLD pathology, including hyper-TLD in the recBCD mutant. Replication forks, unlike chromosomal fragmentation, may provide a minor contribution to TLD, but only in the repair-deficient thyA deoCABD recBCD mutant.

Thymineless Death in Escherichia coli Is Unaffected by Chromosomal Replication Complexity.

Thymineless death (TLD) is a rapid loss of viability of unclear mechanism in cultures of thyA mutants starved for thymine/thymidine (T starvation). It is accepted that T starvation repeatedly breaks replication forks, while recombinational repair restores them, but when the resulting futile breakage-repair cycle affects the small replication bubbles at oriC, the origin is degraded, killing the cell. Indeed, cells with increased chromosomal replication complexity (CRC), expressed as an elevated origin/terminus (ori/ter) ratio, die more extensively during TLD. Here we tested this logic by elevating the CRC in Escherichia colithyA mutants before T starvation, anticipating exaggerated TLD. Unexpectedly, TLD remained unaffected by a CRC increase to either the natural limit (ori/ter ratio, ∼6) or the functional limit (ori/ter ratio, ∼16). Moreover, when we forced the CRC over the functional limit (ori/ter ratio, ∼30), TLD lessened. Thus, prior overinitiation does not sensitize cells to TLD. In contradiction with the published results, even blocking new replication initiations by the dnaA(Ts) defect at 42°C fails to prevent TLD. Using the thyA dnaA(Ts) mutant in a new T starvation protocol that excludes new initiations, we show that at 42°C, the same degree of TLD still occurs when chromosomes are demonstrably nonreplicating. Remarkably, 80% of the chromosomal DNA in these nonreplicating T-starved cells is still lost, by an unclear mechanism.IMPORTANCE Thymineless death kills cells of any type and is used in anticancer and antimicrobial treatments. We tested the idea that the more replication forks there are in the chromosome during growth, the more extensive the resulting thymineless death. We varied the number of replication forks in the Escherichia coli chromosome, as measured by the origin-to-terminus ratio, ranging it from the normal 2 to 60, and even completely eliminated replication forks in the nonreplicating chromosomes (ori/ter ratio = 1). Unexpectedly, we found that thymineless death is unaffected by the intensity of replication or by its complete absence; we also found that even nonreplicating chromosomes still disappear during thymine starvation. We conclude that thymineless death can kill E. coli independently of chromosomal replication.

Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways.

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome.

The nucleoid of Escherichia coli comprises DNA, nucleoid associated proteins (NAPs) and RNA, whose role is unclear. We found that lysing bacterial cells embedded in agarose plugs in the presence of RNases caused massive fragmentation of the chromosomal DNA. This RNase-induced chromosomal fragmentation (RiCF) was completely dependent on the presence of RNase around lysing cells, while the maximal chromosomal breakage required fast cell lysis. Cell lysis in plugs without RNAse made the chromosomal DNA resistant to subsequent RNAse treatment. RiCF was not influenced by changes in the DNA supercoiling, but was influenced by growth temperature or age of the culture. RiCF was partially dependent on H-NS, histone-like nucleoid structuring- and global transcription regulator protein. The hupAB deletion of heat-unstable nucleoid protein (HU) caused increase in spontaneous fragmentation that was further increased when combined with deletions in two non-coding RNAs, nc1 and nc5. RiCF was completely dependent upon endonuclease I, a periplasmic deoxyribonuclease that is normally found inhibited by cellular RNA. Unlike RiCF, the spontaneous fragmentation in hupAB nc1 nc5 quadruple mutant was resistant to deletion of endonuclease I. RiCF-like phenomenon was observed without addition of RNase to agarose plugs if EDTA was significantly reduced during cell lysis. Addition of RNase under this condition was synergistic, breaking chromosomes into pieces too small to be retained by the pulsed field gels. RNase-independent fragmentation was qualitatively and quantitatively comparable to RiCF and was partially mediated by endonuclease I.

Pulsed-field gel electrophoresis does not break E. coli chromosome undergoing excision repair after UV irradiation.

We showed before that long linear DNA molecules containing single-strand interruptions and undergoing pulsed-field gel electrophoresis (PFGE) tend to break into subfragments (electrophoretic nick instability). Here we show that circular chromosomal DNA with single-strand interruptions remains in the wells during PFGE. This means that the presence of nicks in immobile circular DNA is not enough to break this DNA during PFGE. In other words, under the conditions of our study, the artifactual conversion of nicks into double-strand breaks that we detect in linear DNA does not contribute to the overall level of chromosomal fragmentation, as measured by PFGE.

Static and Dynamic Factors Limit Chromosomal Replication Complexity in Escherichia coli, Avoiding Dangers of Runaway Overreplication.

We define chromosomal replication complexity (CRC) as the ratio of the copy number of the most replicated regions to that of unreplicated regions on the same chromosome. Although a typical CRC of eukaryotic or bacterial chromosomes is 2, rapidly growing Escherichia coli cells induce an extra round of replication in their chromosomes (CRC = 4). There are also E. coli mutants with stable CRC∼6. We have investigated the limits and consequences of elevated CRC in E. coli and found three limits: the "natural" CRC limit of ∼8 (cells divide more slowly); the "functional" CRC limit of ∼22 (cells divide extremely slowly); and the "tolerance" CRC limit of ∼64 (cells stop dividing). While the natural limit is likely maintained by the eclipse system spacing replication initiations, the functional limit might reflect the capacity of the chromosome segregation system, rather than dedicated mechanisms, and the tolerance limit may result from titration of limiting replication factors. Whereas recombinational repair is beneficial for cells at the natural and functional CRC limits, we show that it becomes detrimental at the tolerance CRC limit, suggesting recombinational misrepair during the runaway overreplication and giving a rationale for avoidance of the latter.

Replication fork inhibition in seqA mutants of Escherichia coli triggers replication fork breakage.

SeqA protein negatively regulates replication initiation in Escherichia coli and is also proposed to organize maturation and segregation of the newly replicated DNA. The seqA mutants suffer from chromosomal fragmentation; since this fragmentation is attributed to defective segregation or nucleoid compaction, two-ended breaks are expected. Instead, we show that, in SeqA's absence, chromosomes mostly suffer one-ended DNA breaks, indicating disintegration of replication forks. We further show that replication forks are unexpectedly slow in seqA mutants. Quantitative kinetics of origin and terminus replication from aligned chromosomes not only confirm origin overinitiation in seqA mutants, but also reveal terminus under-replication, indicating inhibition of replication forks. Pre-/post-labelling studies of the chromosomal fragmentation in seqA mutants suggest events involving single forks, rather than pairs of forks from consecutive rounds rear-ending into each other. We suggest that, in the absence of SeqA, the sister-chromatid cohesion 'safety spacer' is destabilized and completely disappears if the replication fork is inhibited, leading to the segregation fork running into the inhibited replication fork and snapping the latter at single-stranded DNA regions.

Trapping and breaking of in vivo nicked DNA during pulsed field gel electrophoresis.

Pulsed field gel electrophoresis (PFGE) offers a high-resolution approach to quantify chromosomal fragmentation in bacteria, measured as percentage of chromosomal DNA entering the gel. The degree of separation in pulsed field gel (PFG) depends on the size of DNA as well as various conditions of electrophoresis such as electric field strength, time of electrophoresis, switch time, and buffer composition. Here we describe a new parameter, the structural integrity of the sample DNA itself, that influences its migration through PFGs. We show that subchromosomal fragments containing both spontaneous and DNA damage-induced nicks are prone to breakage during PFGE. Such breakage at single-strand interruptions results in artifactual decrease in molecular weight of linear DNA making accurate determination of the number of double-strand breaks difficult. Although breakage of nicked subchromosomal fragments is field strength independent, some high-molecular-weight subchromosomal fragments are also trapped within wells under the standard PFGE conditions. This trapping can be minimized by lowering the field strength and increasing the time of electrophoresis. We discuss how breakage of nicked DNA may be mechanistically linked to trapping. Our results suggest how to optimize conditions for PFGE when quantifying chromosomal fragmentation induced by DNA damage.

Replication forks stalled at ultraviolet lesions are rescued via RecA and RuvABC protein-catalyzed disintegration in Escherichia coli.

Ultraviolet (UV) irradiation is not known to induce chromosomal fragmentation in sublethal doses, and yet UV irradiation causes genetic instability and cancer, suggesting that chromosomes are fragmented. Here we show that UV irradiation induces fragmentation in sublethal doses, but the broken chromosomes are repaired or degraded by RecBCD; therefore, to observe full fragmentation, RecBCD enzyme needs to be inactivated. Using quantitative pulsed field gel electrophoresis and sensitive DNA synthesis measurements, we investigated the mechanisms of UV radiation-induced chromosomal fragmentation in recBC mutants, comparing five existing models of DNA damage-induced fragmentation. We found that fragmentation depends on active DNA synthesis before, but not after, UV irradiation. At low UV irradiation doses, fragmentation does not need excision repair or daughter strand gap repair. Fragmentation absolutely depends on both RecA-catalyzed homologous strand exchange and RuvABC-catalyzed Holliday junction resolution. Thus, chromosomes fragment when replication forks stall at UV lesions and regress, generating Holliday junctions. Remarkably, cells specifically utilize fork breakage to rescue stalled replication and avoid lethality.

The BlcC (AttM) lactonase of Agrobacterium tumefaciens does not quench the quorum-sensing system that regulates Ti plasmid conjugative transfer.

The conjugative transfer of Agrobacterium plasmids is controlled by a quorum-sensing system consisting of TraR and its acyl-homoserine lactone (HSL) ligand. The acyl-HSL is essential for the TraR-mediated activation of the Ti plasmid Tra genes. Strains A6 and C58 of Agrobacterium tumefaciens produce a lactonase, BlcC (AttM), that can degrade the quormone, leading some to conclude that the enzyme quenches the quorum-sensing system. We tested this hypothesis by examining the effects of the mutation, induction, or mutational derepression of blcC on the accumulation of acyl-HSL and on the conjugative competence of strain C58. The induction of blc resulted in an 8- to 10-fold decrease in levels of extracellular acyl-HSL but in only a twofold decrease in intracellular quormone levels, a measure of the amount of active intracellular TraR. The induction or mutational derepression of blc as well as a null mutation in blcC had no significant effect on the induction of or continued transfer of pTiC58 from donors in any stage of growth, including stationary phase. In matings performed in developing tumors, wild-type C58 transferred the Ti plasmid to recipients, yielding transconjugants by 14 to 21 days following infection. blcC-null donors yielded transconjugants 1 week earlier, but by the following week, transconjugants were recovered at numbers indistinguishable from those of the wild type. Donors mutationally derepressed for blcC yielded transconjugants in planta at numbers 10-fold lower than those for the wild type at weeks 2 and 3, but by week 4, the two donors showed no difference in recoverable transconjugants. We conclude that BlcC has no biologically significant effect on Ti plasmid transfer or its regulatory system.

An insecticidal GroEL protein with chitin binding activity from Xenorhabdus nematophila.

Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an approximately 58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of approximately 3.6 microg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed alpha-chitin binding activity with Kd approximately 0.64 microm and Bmax approximately 4.68 micromol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an approximately 8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity.

Broad-host-range expression vectors with tightly regulated promoters and their use to examine the influence of TraR and TraM expression on Ti plasmid quorum sensing.

Experiments requiring strong repression and precise control of cloned genes can be difficult to conduct because of the relatively high basal level of expression of currently employed promoters. We report the construction of a family of vectors that contain a reengineered lacI(q)-lac promoter-operator complex in which cloned genes are strongly repressed in the absence of inducer. The vectors, all based on the broad-host-range plasmid pBBR1, are mobilizable and stably replicate at moderate copy number in representatives of the alpha- and gammaproteobacteria. Each vector contains a versatile multiple cloning site that includes an NdeI site allowing fusion of the cloned gene to the initiation codon of lacZalpha. In each tested bacterium, a uidA reporter fused to the promoter was not expressed at a detectable level in the absence of induction but was inducible by 10- to 100-fold, depending on the bacterium. The degree of induction was controllable by varying the concentration of inducer. When the vector was tested in Agrobacterium tumefaciens, a cloned copy of the traR gene, the product of which is needed at only a few copies per cell, did not confer activity under noninducing conditions. We used this attribute of very tight and variably regulatable control to assess the relative amounts of TraR required to activate the Ti plasmid conjugative transfer system. We identified levels of induction that gave wild-type transfer frequencies, as well as levels that induced correspondingly lower frequencies of transfer. We also used this system to show that the antiactivator TraM sets the level of intracellular TraR required for tra gene activation.

Induction and loss of Ti plasmid conjugative competence in response to the acyl-homoserine lactone quorum-sensing signal.

Conjugative transfer of the Ti plasmids of Agrobacterium tumefaciens is controlled by a quorum-sensing system composed of TraR and its signal N-(3-oxo-octanoyl)-L-homoserine lactone. This system is, in turn, controlled by the conjugative opines produced by crown gall tumors induced on plants by the bacteria. Using nonpolar traI mutants, we examined the kinetics of induction of conjugative transfer in response to exogenous acyl-homoserine lactone. In the absence of the antiactivator TraM, onset of induction of transfer requires about 30 min, 15 to 20 min of which is needed for expression and construction of the conjugative apparatus. TraM delays the onset of conjugation by 30 min. While the rate of development of conjugative competence was not significantly affected by levels of TraR, maximum efficiencies of transfer were correlated with amounts of the activator in the donors. Donors harboring Ti plasmids lacking TraM were fully induced by the quormone at concentrations as low as 100 pM. TraM raised the concentration of signal required for maximum activity to 1 nM. Donors grown in batch culture retained conjugative competence following signal removal, even when in stationary phase. However, donors kept in balanced growth rapidly lost transfer ability following signal removal. Loss of transfer was mirrored by a decrease in levels of active TraR. Decreases in TraR activity and conjugative competence could be accounted for by dilution associated with cell division, suggesting that while induction of Ti plasmid conjugation is an active process, the cells lack a mechanism for disassembling the conjugative apparatus when signals become limiting.

N-(3-hydroxyhexanoyl)-l-homoserine lactone is the biologically relevant quormone that regulates the phz operon of Pseudomonas chlororaphis strain 30-84.

Phenazine production by Pseudomonas fluorescens 2-79 and P. chlororaphis isolates 30-84 and PCL1391 is regulated by quorum sensing through the activator PhzR and acyl-homoserine lactones (acyl-HSLs) synthesized by PhzI. PhzI from P. fluorescens 2-79 produces five acyl-HSLs that include four 3-hydroxy species. Of these, N-(3-hydroxyhexanoyl)-HSL is the biologically relevant ligand for PhzR. The quorum-sensing systems of P. chlororaphis strains 30-84 and PCL1391 have been reported to produce and respond to N-(hexanoyl)-HSL. These differences were of interest since PhzI and PhzR of strain 2-79 share almost 90% sequence identity with orthologs from strains 30-84 and PCL1391. In this study, as assessed by thin-layer chromatography, the three strains produce almost identical complements of acyl-HSLs. The major species produced by P. chlororaphis 30-84 were identified by mass spectrometry as 3-OH-acyl-HSLs with chain lengths of 6, 8, and 10 carbons. Heterologous bacteria expressing cloned phzI from strain 30-84 produced the four 3-OH acyl-HSLs in amounts similar to those seen for the wild type. Strain 30-84, but not strain 2-79, also produced N-(butanoyl)-HSL. A second acyl-HSL synthase of strain 30-84, CsaI, is responsible for the synthesis of this short-chain signal. Strain 30-84 accumulated N-(3-OH-hexanoyl)-HSL to the highest levels, more than 100-fold greater than that of N-(hexanoyl)-HSL. In titration assays, PhzR(30-84) responded to both N-(3-OH-hexanoyl)- and N-(hexanoyl)-HSL with equal sensitivities. However, only the 3-OH-hexanoyl signal is produced by strain 30-84 at levels high enough to activate PhzR. We conclude that strains 2-79, 30-84, and PCL1391 use N-(3-OH-hexanoyl)-HSL to activate PhzR.

Activation of the phz operon of Pseudomonas fluorescens 2-79 requires the LuxR homolog PhzR, N-(3-OH-Hexanoyl)-L-homoserine lactone produced by the LuxI homolog PhzI, and a cis-acting phz box.

The phz operon of Pseudomonas fluorescens 2-79, which produces phenazine-1-carboxylate, is preceded by two genes, phzR and phzI, that are homologs of quorum-sensing gene pairs of the luxR-luxI family. Deleting phzR and phzI from strain 2-79 led to loss of production of the antibiotics, as well as a suite of six acyl-homoserine lactones (acyl-HSLs) that includes four 3-hydroxy- derivatives and two alkanoyl-HSLs. Strain 2-79 accumulates N-(3-hydroxy-hexanoyl)-L-HSL to levels 20 and 30 times those of N-(hexanoyl)-L-HSL and N-(3-hydroxy-octanoyl)-HSL, the next most abundant species produced by this isolate. Expression of a clone of phzI in Escherichia coli and P. fluorescens 1855 resulted in the synthesis of all six acyl-HSLs. Maximal activation of phzA and phzR fused to lacZ and uidA reporters, respectively, required PhzR and the acyl-HSL signals. PhzR-mediated expression of the phzA::lacZ fusion responded with highest sensitivity and greatest magnitude to pure N-(3-hydroxy-hexanoyl)-L-HSL. When exposed to organic extracts of culture supernatants containing the six acyl-HSLs at their normal levels, the reporter responded strongly to N-(3-hydroxy-hexanoyl)-L-HSL but did not respond to any of the other five acyl-HSLs. The transcriptional start sites for the divergently oriented phzA and phzR genes were mapped by primer extension analysis. An 18-bp almost perfect inverted repeat, the phz box, is located between the phzI and phzR promoters. Disrupting this repeat abolished PhzR-dependent activation of phzA and phzR. We conclude that PhzI of strain 2-79 synthesizes 3-OH acyl-HSLs and that P. fluorescens 2-79 uses N-(3-hydroxy-hexanoyl)-HSL as its quorum-sensing signal. We also conclude that PhzR, with its quormone, activates expression of phzA and phzR and that this activation requires an intact phz box sequence located in the divergent promoter region.

Loss of catabolite repression function of HPr, the phosphocarrier protein of the bacterial phosphotransferase system, affects expression of the cry4A toxin gene in Bacillus thuringiensis subsp. israelensis.

HPr, the phosphocarrier protein of the bacterial phosphotransferase system, mediates catabolite repression of a number of operons in gram-positive bacteria. In order to participate in the regulatory process, HPr is activated by phosphorylation of a conserved serine-46 residue. To study the potential role of HPr in the regulation of Cry4A protoxin synthesis in Bacillus thuringiensis subsp. israelensis, we produced a catabolite repression-negative mutant by replacing the wild-type copy of the ptsH gene with a mutated copy in which the conserved serine residue of HPr was replaced with an alanine. HPr isolated from the mutant strain was not phosphorylated at Ser-45 by HPr kinase, but phosphorylation at His-14 was found to occur normally. The enzyme I and HPr kinase activities of the mutant were not affected. Analysis of the B. thuringiensis subsp. israelensis mutant harboring ptsH-S45A in the chromosome showed that cry4A expression was derepressed from the inhibitory effect of glucose. The mutant strain produced both cry4A and sigma(35) gene transcripts 4 h ahead of the parent strain, but there was no effect on sigma(28) synthesis. In wild-type B. thuringiensis subsp. israelensis cells, cry4A mRNA was observed from 12 h onwards, while in the mutant it appeared at 8 h and was produced for a longer period. The total amount of cry4A transcripts produced by the mutant was higher than by the parent strain. There was a 60 to 70% reduction in the sporulation efficiency of the mutant B. thuringiensis subsp. israelensis strain compared to the wild-type strain.