Nitric oxide-induced homologous recombination in Escherichia coli is promoted by DNA glycosylases

Nitric oxide (NO*) is involved in neurotransmission, inflammation, and many other biological processes. Exposure of cells to NO* leads to DNA damage, including formation of deaminated and oxidized bases. Apurinic/apyrimidinic (AP) endonuclease-deficient cells are sensitive to NO* toxicity, which indicates that base excision repair (BER) intermediates are being generated. Here, we show that AP endonuclease-deficient cells can be protected from NO* toxicity by inactivation of the uracil (Ung) or formamidopyrimidine (Fpg) DNA glycosylases but not by inactivation of a 3-methyladenine (AlkA) DNA glycosylase. These results suggest that Ung and Fpg remove nontoxic NO*-induced base damage to create BER intermediates that are toxic if they are not processed by AP endonucleases. Our next goal was to learn how Ung and Fpg affect susceptibility to homologous recombination. The RecBCD complex is critical for repair of double-strand breaks via homologous recombination. When both Ung and Fpg were inactivated in recBCD cells, survival was significantly enhanced. We infer that both Ung and Fpg create substrates for recombinational repair, which is consistent with the observation that disrupting ung and fpg suppressed NO*-induced recombination. Taken together, a picture emerges in which the action of DNA glycosylases on NO*-induced base damage results in the accumulation of BER intermediates, which in turn can induce homologous recombination. These studies shed light on the underlying mechanism of NO*-induced homologous recombination.     

Homologous recombination promoted by Chi sites and RecBC enzyme of Escherichia coli

Chi sites are examples of special sites enhancing homologous recombination in their region of the chromosome. Chi, 5′ G-C-T-G-G-T-G-G3′, is a recognition site for the RecBC enzyme, which nicks DNA near Chi as it unwinds DNA. A molecular model of genetic recombination incorporating these features is reviewed.     

Evidence from terminal recombination gradients that FtsK uses replichore polarity to control chromosome terminus positioning at division in Escherichia coli

Chromosome dimers in Escherichia coli are resolved at the dif locus by two recombinases, XerC and XerD, and the septum-anchored FtsK protein. Chromosome dimer resolution (CDR) is subject to strong spatiotemporal control: it takes place at the time of cell division, and it requires the dif resolution site to be located at the junction between the two polarized chromosome arms or replichores. Failure of CDR results in trapping of DNA by the septum and RecABCD recombination (terminal recombination). We had proposed that dif sites of a dimer are first moved to the septum by mechanisms based on local polarity and that normally CDR then occurs as the septum closes. To determine whether FtsK plays a role in the mobilization process, as well as in the recombination reaction, we characterized terminal recombination in an ftsK mutant. The frequency of recombination at various points in the terminus region of the chromosome was measured and compared with the recombination frequency on a xerC mutant chromosome with respect to intensity, the region affected, and response to polarity distortion. The use of a prophage excision assay, which allows variation of the site of recombination and interference with local polarity, allowed us to find that cooperating FtsK-dependent and -independent processes localize dif at the septum and that DNA mobilization by FtsK is oriented by the polarity probably due to skewed sequence motifs of the mobilized material.     

Hotspots for generalized recombination in the Escherichia coli chromosome.

A naturally occurring hotspot for Rec recombination of Escherichia coli was located in the biotin operon. The phenotypes of the bio hotspot as observed in λbio transducing phage were identical to those of Chi mutations in phage λ. In addition to recA⁺ function, the site-specific stimulation of recombination required recB⁺ function. The stimulation took place when the hotspot was present in only one parent of the cross and even when present opposite a region of heterology.The demonstration of a Chi element in E. coli provoked us to measure the density of Chi elements on the chromosome. E. coli DNA sampled in λ transducing phage (either obtained by induction of secondary site lysogens or made in vitro from EcoRI cleavage fragments) showed one hotspot per 5 to 15 × 10³ bases. The high density and the fact that Chi stimulation of recombination can span the inter-Chi distance suggest that Chi might be important in Rec recombination in the absence of λ.     

Evidence that late-endosomal SNARE multimerization complex is promoted by transmembrane segments

Assembly of SNARE proteins into quaternary complexes is a critical step in membrane docking and fusion. Here, we have studied the influence of the transmembrane segments on formation of the late endosomal SNARE complex. The complex was assembled in vitro from full-length recombinant SNAREs and from mutants, where the transmembrane segments were either deleted or replaced by oligo-alanine sequences. We show that endobrevin, syntaxin 7, syntaxin 8, and vti1b readily form a complex. This complex forms a dimer as well as multimeric structures. Interestingly, the natural transmembrane segments accelerate the conversion of the quaternary complex to the dimeric form and are essential for multimerization. These in vitro results suggest that the transmembrane segments are responsible for supramolecular assembly of the endosomal SNARE complex.     

Evidence that late-endosomal SNARE multimerization complex is promoted by transmembrane segments

Assembly of SNARE proteins into quaternary complexes is a critical step in membrane docking and fusion. Here, we have studied the influence of the transmembrane segments on formation of the late endosomal SNARE complex. The complex was assembled in vitro from full-length recombinant SNAREs and from mutants, where the transmembrane segments were either deleted or replaced by oligo-alanine sequences. We show that endobrevin, syntaxin 7, syntaxin 8, and vti1b readily form a complex. This complex forms a dimer as well as multimeric structures. Interestingly, the natural transmembrane segments accelerate the conversion of the quaternary complex to the dimeric form and are essential for multimerization. These in vitro results suggest that the transmembrane segments are responsible for supramolecular assembly of the endosomal SNARE complex.     

Reciprocal recombination of chromosome and F. merogenote in Escherichia coli

Herman, Robert K. (Lawrence University, Appleton, Wis.). Reciprocal recombination of chromosome and F-merogenote in Escherichia coli. J. Bacteriol. 90:1664-1668. 1965.-Mitotic recombination of an F-merogenote with the bacterial chromosome was observed under conditions where both recombinant episome and reciprocally recombinant chromosome, if formed and if passed on to the same progeny, could be detected. Recombinant strains selected on the basis of having a recombinant F-merogenote were found frequently to contain a recombinant chromosome of the reciprocal type.     

Preferential unequal recombination in the glyS region of the Escherichia coli chromosome.

Duplications of the Escherichia coli chromosomal region carrying the glyS and xylloci can be selected by deoxyadenosine treatment of trpA36 glyS_LglyTsu_AGA or (glyUsu_AGA) cultures. The deoxyadenosine lowers the suppression efficiency of these missense suppressors, and growth is severely limited by the resulting tryptophan starvation. Prolonged growth in the presence of 250 μg deoxyadenosine/ml leads to the accumulation of mutants with two (or more) copies of the allele for glycyl-transfer RNA synthetase, glyS_L. The same duplication is obtained each time the selective pressure is applied. This was shown by physically isolating the duplicated region in the form of circular DNA excised from the duplication by recombination. In repeated experiments, a circular species 140,000 base-pairs in size was isolated. These results are interpreted as showing that there are two loci, one on each side of the glyS locus, and spaced 140,000 base-pairs apart, which are prone to recombining with each other in a manner leading to a genetic duplication.     

Evidence that the variable fluorescence in Chlorella is recombination luminescence

The fluorescence lifetime of oxygen-forming photosynthetic systems as a function of closed traps has been studied by several groups using light and poisons (usually 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)) to fix the closed trap state during the experiment. These measurements have now been carried out using light alone, by means of pump and probe laser pulses and a very efficient fast photomultiplier-digitizing system. It is found that the absolute amplitude of fast fluorescence (mean tau, approx. 0.3 ns) remains constant until over half the traps are filled. The amplitude of the slow fluorescence (tau approximately equal to 1.2 ns) increases with pump energy, and its response is best fit with a lag or finite rise-time of approx. 200 ps. This novel result is consistent with the hypothesis that the slow component of the fluorescence is actually recombination luminescence in the trap. Thus, the full trapping time, i.e., the time to form the P+I- state from an excitation in the O2 photosystem, is relatively slow.     

YqjD is an inner membrane protein associated with stationary-phase ribosomes in Escherichia coli

Here, we provide evidence that YqjD, a hypothetical protein of Escherichia coli, is an inner membrane and ribosome binding protein. This protein is expressed during the stationary growth phase, and expression is regulated by stress response sigma factor RpoS. YqjD possesses a transmembrane motif in the C-terminal region and associates with 70S and 100S ribosomes at the N-terminal region. Interestingly, E. coli possesses two paralogous proteins of YqjD, ElaB and YgaM, which are expressed and bind to ribosomes in a similar manner to YqjD. Overexpression of YqjD leads to inhibition of cell growth. It has been suggested that YqjD loses ribosomal activity and localizes ribosomes to the membrane during the stationary phase.     

Synthesis of penicillin-binding protein 6 by stationary-phase Escherichia coli.

The level of penicillin-binding protein 6, a D-alanine carboxypeptidase I, was found to be 2- to 10-fold higher in stationary-phase cells than in exponentially growing cells of Escherichia coli. This increase appeared to be due to de novo synthesis rather than to an unmasking of preexisting material. There was no comparable change in the amount of any of the other six penicillin-binding proteins.     

Production of phloroglucinol by Escherichia coli using a stationary-phase promoter

Escherichia coli was metabolically engineered using a new host-vector system to produce phloroglucinol. The key biosynthetic gene phlD (encoding a type III polyketide synthase) from Pseudomonas fluorescens was expressed in E. coli using the stationary-phase promoter of the fic gene and a high-copy plasmid. In shake-flasks, the engineered strain produced phloroglucinol up to 0.28 g/l with a productivity of 0.014 g/l h. About 9.2% of the glucose consumed was converted to phloroglucinol after 20 h. Compared with the widely used inducible T7 promoter system, this strain did not require IPTG induction and the final titer of phloroglucinol was 22% higher.     

Cin-mediated recombination at secondary crossover sites on the Escherichia coli chromosome.

The Cin recombinase is known to mediate DNA inversion between two wild-type cix sites flanking genetic determinants for the host range of bacteriophage P1. Cin can also act with low frequency at secondary (or quasi) sites (designated cixQ) that have lower homology to either wild-type site. An inversion tester sequence able to reveal novel operon fusions was integrated into the Escherichia coli chromosome, and the Cin recombinase was provided in trans. Among a total of 13 Cin-mediated inversions studied, three different cixQ sites had been used. In two rearranged chromosomes, the breakpoints of the inversions were mapped to cixQ sites in supB and ompA, representing inversions of 109 and 210 kb, respectively. In the third case, a 2.1-kb inversion was identified at a cixQ site within the integrated sequences. This derivative itself was a substrate for a second inversion of 1.5 kb between the remaining wild-type cix and still another cixQ site, thus resembling a reversion. In analogy to that which is known from DNA inversion on plasmids, homology of secondary cix sites to wild-type recombination sites is not a strict requirement for inversion to occur on the chromosome. The chromosomal rearrangements which resulted from these Cin-mediated inversions were quite stable and suffered no growth disadvantage compared with the noninverted parental strain. The mechanistic implications and evolutionary relevance of these findings are discussed.     

FtsK controls metastable recombination provoked by an extra Ter site in the Escherichia coli chromosome terminus

The FtsK protein is required for septum formation in Escherichia coli and as a DNA translocase for chromosome processing while the septum closes. Its domain of action on the chromosome overlaps the replication terminus region, which lies between replication pause sites TerA and TerC. An extra Ter site, PsrA*, has been inserted at a position common to the FtsK and terminus domains. It is well tolerated, although it compels replication forks travelling clockwise from oriC to stall and await arrival of counter-clockwise forks. Elevated recombination has been detected at the stalled fork. Analysis of PsrA*-induced homologous recombination by an excision test revealed unique features. (i) rates of excision near PsrA* may fluctuate widely from clone to clone, a phenomenon we term whimsicality, (ii) excision rates are nevertheless conserved for many generations, a phenomenon we term memorization; their metastability at the clone level is explainable by frequent shifting between three cellular states--high, medium and low probability of excision, (iii) PsrA*-induced excision is RecBC-independent and is strongly counteracted by FtsK, which in addition is involved in its whimsicality and (iv) whimsicality disappears as the distance from the pause site increases. Action of FtsK at a replication fork was unexpected because the factor was thought to act on the chromosome only at septation, i.e. after replication is completed. Idiosyncrasy of PsrA*-induced recombination is discussed with respect to possible intermingling of replication, repair and post-replication steps of bacterial chromosome processing during the cell cycle.     

Homologous recombination is promoted by translesion polymerase poleta

Two new studies provide in vivo ( [this issue of Molecular Cell]) and in vitro ( [this issue of Molecular Cell]) evidence that poleta functions to extend 3' strands exchanged during homologous recombination and raise the issue of how TLS polymerases are selected onto different substrates.     

Peptidoglycan biosynthesis in stationary-phase cells of Escherichia coli.

The ability of stationary-phase cells of Escherichia coli W7 to incorporate radioactive precursors into macromolecular murein has been studied. During the initial 6 h of the stationary phase, resting cells incorporated meso-[3H]diaminopimelic acid at a rate corresponding to the insertion of 1.3 X 10(4) disaccharide units min-1 cell-1. Afterwards, the rate of incorporation dropped drastically (90%) to a low but still detectable level. Incorporation during stationary phase did not result in an increased amount of total murein in the culture, suggesting that it was related to a turnover process. Analysis of the effects of a number of beta-lactam antibiotics indicated that incorporation of murein precursors in stationary-phase cells was mediated by penicillin-binding proteins, suggesting that the activity of penicillin-binding protein 2 was particularly relevant to this process.