Activation of Chi recombinational hotspots by RecBCD-like enzymes from enteric bacteria

Chi sites, 5'G-C-T-G-G-T-G-G-3', enhance homologous recombination in Escherichia coli and are activated by the RecBCD enzyme. To test the ability of Chi to be activated by analogous enzymes from other bacteria, we cloned recBCD-like genes from diverse bacteria into an E. coli recBCD deletion mutant. Clones from seven species of enteric bacteria conferred to this deletion mutant recombination proficiency, Chi hotspot activity in lambda Red- Gam- vegetative crosses, and RecBCD enzyme activities, including Chi-dependent DNA strand cleavage. Three clones from Pseudomonas aeruginosa and Ps. putida conferred recombination proficiency and ATP-dependent nuclease activity, but neither Chi hotspot activity nor Chi-dependent DNA cleavage. These results imply that Chi has been conserved as a recombination-promoting signal for RecBCD-like enzymes in enteric bacteria but not in more distantly related bacteria such as Pseudomonas spp. We discuss the possibility that other, presently unknown, nucleotide sequences serve the same function as Chi in Pseudomonas spp.     

Chi-dependent DNA strand cleavage by RecBC enzyme

Chi sites enhance in their vicinity homologous recombination by the E. coli RecBC pathway. We report here that RecBC enzyme catalyzes Chi-dependent cleavage of one DNA strand, that containing the Chi sequence 5'G-C-T-G-G-T-G-G3'. Chi-specific cleavage is greatly reduced by single base pair changes within the Chi sequence and by mutations within the E. coli recC gene, coding for a RecBC enzyme subunit. Although cleavage occurs preferentially with double-stranded DNA, the product of the reaction is single-stranded DNA. These results demonstrate the direct interaction of RecBC enzyme with Chi sites that was inferred from the genetic properties of Chi and recBC, and they support models of recombination in which Chi acts before the initiation of strand exchange.     

Homologous pairing in vitro stimulated by the recombination hotspot, Chi.

Genetic recombination in Escherichia coli is stimulated at DNA sequences known as Chi sites, 5'-GCT-GGTGG-3'. We describe the in vitro formation of homologously paired joint molecules that is dependent upon this recombination hotspot. Chi-dependent joint molecule formation requires RecA, RecBCD, and SSB proteins and a Chi site in the donor linear dsDNA. The donor dsDNA is unwound by RecBCD enzyme, and the invasive strand is generated by nicking at Chi. This Chi-dependent invading strand must contain homology to the recipient supercoiled DNA substrate at its newly formed 3' end for efficient joint molecule formation. Action at Chi generates invasive ssDNA from the 5' but not the 3' side of Chi, suggesting that the nuclease activity of RecBCD enzyme is attenuated upon encountering a Chi site. These results support the view that RecBCD enzyme action can precede RecA protein action and reconcile the seemingly opposing degradative and recombination functions of RecBCD enzyme.     

Genetic Dissection of the Biochemical Activities of Recbcd Enzyme

RecBCD enzyme of Escherichia coli is required for the major pathway of homologous recombination following conjugation. The enzyme has an ATP-dependent DNA unwinding activity, ATP-dependent single-stranded (ss) and double-stranded (ds) DNA exonuclease activities, and an activity that makes a ss DNA endonucleolytic cut near Chi sites. We have isolated and characterized ten mutations that reduced recombination proficiency and inactivated some, but not all, activities of RecBCD enzyme. One class of mutants had weak ds DNA exonuclease activity and lacked Chi-dependent DNA cleavage activity, a second class lacked only Chi-dependent DNA cleavage activity, and a third class retained all activities tested. The properties of these mutants indicate that the DNA unwinding and ss DNA exonuclease activities of the RecBCD enzyme are not sufficient for recombination. Furthermore, they suggest that the Chi-dependent DNA cleavage activity or another, as yet unidentified activity or both are required for recombination. The roles of the RecBCD enzymatic activities in recombination and exclusion of foreign DNA are discussed in light of the properties of these and other recBCD mutations.     

Chi-dependent intramolecular recombination in Escherichia coli.

Homologous recombination in Escherichia coli is enhanced by a cis-acting octamer sequence named Chi (5''-GCTGGTGG-3'') that interacts with RecBCD. To gain insight into the mechanism of Chi-enhanced recombination, we recruited an experimental system that permits physical monitoring of intramolecular recombination by linear substrates released by in vivo restriction from infecting chimera phage. Recombination of the released substrates depended on recA, recBCD and cis-acting Chi octamers. Recombination proficiency was lowered by a xonA mutation and by mutations that inactivated the RuvABC and RecG resolution enzymes. Activity of Chi sites was influenced by their locations and by the number of Chi octamers at each site. A single Chi site stimulated recombination, but a combination of Chi sites on the two homologs was synergistic. These data suggest a role for Chi at both ends of the linear substrate. Chi was lost in all recombinational exchanges stimulated by a single Chi site. Exchanges in substrates with Chi sites on both homologs occurred in the interval between the sites as well as in the flanking interval. These observations suggest that the generation of circular products by intramolecular recombination involves Chi-dependent processing of one end by RecBCD and pairing of the processed end with its duplex homolog.     

dGTP triphosphohydrolase, a unique enzyme confined to members of the family Enterobacteriaceae.

The enzyme dGTP triphosphohydrolase (dGTPase; EC 3.1.5.1) was assayed in partially purified extracts of several genera of bacteria, and it was found to be strictly confined to members of the family Enterobacteriaceae. Whereas 11 of 12 enteric bacteria had comparable activity for this enzyme, 8 of 8 nonenteric bacteria, including species in the very closely related genera Vibrio and Aeromonas, did not assay positively for this enzyme. When challenged with Escherichia coli anti-dGTPase antiserum, the active enzymes fell into three groups, retaining 0, approximately 50, or 100% of their original activity. A computer search has revealed an amino acid sequence in the E. coli enzyme which matches well with the single-stranded-DNA binding motif of Prasad and Chiu (J. Mol. Biol. 193:579-584, 1987) and may account for the enzyme's observed interaction with DNA. As far as we are aware, this is the only enzymatic activity so far reported to be present solely in the enteric bacteria.     

Chi hotspots trigger a conformational change in the helicase-like domain of AddAB to activate homologous recombination

In bacteria, the repair of double-stranded DNA breaks is modulated by Chi sequences. These are recognised by helicase-nuclease complexes that process DNA ends for homologous recombination. Chi activates recombination by changing the biochemical properties of the helicase-nuclease, transforming it from a destructive exonuclease into a recombination-promoting repair enzyme. This transition is thought to be controlled by the Chi-dependent opening of a molecular latch, which enables part of the DNA substrate to evade degradation beyond Chi. Here, we show that disruption of the latch improves Chi recognition efficiency and stabilizes the interaction of AddAB with Chi, even in mutants that are impaired for Chi binding. Chi recognition elicits a structural change in AddAB that maps to a region of AddB which resembles a helicase domain, and which harbours both the Chi recognition locus and the latch. Mutation of the latch potentiates the change and moderately reduces the duration of a translocation pause at Chi. However, this mutant displays properties of Chi-modified AddAB even in the complete absence of bona fide hotspot sequences. The results are used to develop a model for AddAB regulation in which allosteric communication between Chi binding and latch opening ensures quality control during recombination hotspot recognition.     

How RecBCD Enzyme and Chi Promote DNA Break Repair and Recombination: a Molecular Biologist's View

Summary: The repair of DNA double-strand breaks (DSBs) is essential for cell viability and important for homologous genetic recombination. In enteric bacteria such as Escherichia coli, the major pathway of DSB repair requires the RecBCD enzyme, a complex helicase-nuclease regulated by a simple unique DNA sequence called Chi. How Chi regulates RecBCD has been extensively studied by both genetics and biochemistry, and two contrasting mechanisms to generate a recombinogenic single-stranded DNA tail have been proposed: the nicking of one DNA strand at Chi versus the switching of degradation from one strand to the other at Chi. Which of these reactions occurs in cells has remained unproven because of the inability to detect intracellular DNA intermediates in bacterial recombination and DNA break repair. Here, I discuss evidence from a combination of genetics and biochemistry indicating that nicking at Chi is the intracellular (in vivo) reaction. This example illustrates the need for both types of analysis (i.e., molecular biology) to uncover the mechanism and control of complex processes in living cells.     

Unexpected DNA context-dependence identifies a new determinant of Chi recombination hotspots

Homologous recombination occurs especially frequently near special chromosomal sites called hotspots. In Escherichia coli, Chi hotspots control RecBCD enzyme, a protein machine essential for the major pathway of DNA break-repair and recombination. RecBCD generates recombinogenic single-stranded DNA ends by unwinding DNA and cutting it a few nucleotides to the 3′ side of 5′ GCTGGTGG 3′, the sequence historically equated with Chi. To test if sequence context affects Chi activity, we deep-sequenced the products of a DNA library containing 10 random base-pairs on each side of the Chi sequence and cut by purified RecBCD. We found strongly enhanced cutting at Chi with certain preferred sequences, such as A or G at nucleotides 4–7, on the 3′ flank of the Chi octamer. These sequences also strongly increased Chi hotspot activity in E. coli cells. Our combined enzymatic and genetic results redefine the Chi hotspot sequence, implicate the nuclease domain in Chi recognition, indicate that nicking of one strand at Chi is RecBCD''s biologically important reaction in living cells, and enable more precise analysis of Chi''s role in recombination and genome evolution.     

The Evolution of Insertion Sequences within Enteric Bacteria

To identify mechanisms that influence the evolution of bacterial transposons, DNA sequence variation was evaluated among homologs of insertion sequences IS1, IS3 and IS30 from natural strains of Escherichia coli and related enteric bacteria. The nucleotide sequences within each class of IS were highly conserved among E. coli strains, over 99.7% similar to a consensus sequence. When compared to the range of nucleotide divergence among chromosomal genes, these data indicate high turnover and rapid movement of the transposons among clonal lineages of E. coli. In addition, length polymorphism among IS appears to be far less frequent than in eukaryotic transposons, indicating that nonfunctional elements comprise a smaller fraction of bacterial transposon populations than found in eukaryotes. IS present in other species of enteric bacteria are substantially divergent from E. coli elements, indicating that IS are mobilized among bacterial species at a reduced rate. However, homologs of IS1 and IS3 from diverse species provide evidence that recombination events and horizontal transfer of IS among species have both played major roles in the evolution of these elements. IS3 elements from E. coli and Shigella show multiple, nested, intragenic recombinations with a distantly related transposon, and IS1 homologs from diverse taxa reveal a mosaic structure indicative of multiple recombination and horizontal transfer events.     

Distribution of Chi-Stimulated Recombinational Exchanges and Heteroduplex Endpoints in Phage Lambda

The recombination hotspot Chi, 5' G-C-T-G-G-T-G-G 3', stimulates the RecBCD recombination pathway of Escherichia coli. We have determined, with precision greater than previously reported, the distribution of Chi-stimulated exchanges around a Chi site in phage lambda. Crosses of lambda phages with single base-pair mutations surrounding a Chi site were conducted in and analyzed on mismatch correction-impaired hosts to preserve heteroduplex mismatches for analysis. Among phages recombinant for flanking markers, Chi stimulated exchanges most intensely in the intervals immediately adjacent to the Chi site, both to its right and to its left. Stimulation fell off abruptly to the right but gradually to the left (with respect to the orientation of the Chi sequence written above). We have also determined that Chi stimulated the formation of heteroduplex DNA, which frequently had one endpoint to the right of Chi and the other endpoint to the left. These data support a model of Chi-stimulated recombination in which RecBCD enzyme cuts DNA immediately to the right of Chi and unwinds DNA to the left of Chi; segments of unwound single-stranded DNA are sometimes, but not always, degraded before synapsis with homologous DNA.     

Over-representation of Chi sequences caused by di-codon increase in Escherichia coli K-12

Chi sequences (5'-GCTGGTGG-3') are cis-acting 8 bp sequence elements that enhance homologous recombination promoted by the RecBCD pathway in Escherichia coli. The genome of E. coli K-12 MG1655 contains 1009 Chi sequences and this frequency far exceeds the expected value for occurrence of an 8 bp sequence in a genome of this size. It is generally thought that the over-representation of Chi sequences indicates that they have been selected for during evolution because of their function in recombination. The genes from three E. coli strains (K-12, O157 and CFT) were classified into three categories (island, match to other E. coli, and backbone). Island genes have a different base composition and codon usage in comparison with those in the backbone genes, therefore they were relatively new and not yet adapted to the base composition patterns and codon usage typical of the recipient genome. The over-representation of Chi sequences was examined by comparing Chi frequencies and codon frequencies between island and backbone genes. The difference in the CTGGTG di-codon frequency between the backbone and island genes was correlated with the frequency of Chi sequences which were translated in the Leu-Val (-G/CTG/GTG/G-) reading frame in the K-12 strain. These results suggest that the main reading frame of Chi sequences increased as a result of the di-codon CTG-GTG increasing under a genome-wide pressure for adapting to the codon usage and base composition of the E. coli K-12 strain, and that the RecBCD recombinase might adjust its recognition sequence to a frequently occurring oligomer such as G-CTG-GTG-G.     

Sequence heterogeneities among 16S ribosomal RNA sequences, and their effect on phylogenetic analyses at the species level

We have analyzed what phylogenetic signal can be derived by small subunit rRNA comparison for bacteria of different but closely related genera (enterobacteria) and for different species or strains within a single genus (Escherichia or Salmonella), and finally how similar are the ribosomal operons within a single organism (Escherichia coli). These sequences have been analyzed by neighbor-joining, maximum likelihood, and parsimony. The robustness of each topology was assessed by bootstrap. Sequences were obtained for the seven rrn operons of E. coli strain PK3. These data demonstrated differences located in three highly variable domains. Their nature and localization suggest that since the divergence of E. coli and Salmonella typhimurium, most point mutations that occurred within each gene have been propagated among the gene family by conversions involving short domains, and that homogenization by conversions may not have affected the entire sequence of each gene. We show that the differences that exist between the different operons are ignored when sequences are obtained either after cloning of a single operon or directly from polymerase chain reaction (PCR) products. Direct sequencing of PCR products produces a mean sequence in which mutations present in the most variable domains become hidden. Cloning a single operon results in a sequence that differs from that of the other operons and of the mean sequence by several point mutations. For identification of unknown bacteria at the species level or below, a mean sequence or the sequence of a single nonidentified operon should therefore be avoided. Taking into account the seven operons and therefore mutations that accumulate in the most variable domains would perhaps increase tree resolution. However, if gene conversions that homogenize the rRNA multigene family are rare events, some nodes in phylogenetic trees will reflect these recombination events and these trees may therefore be gene trees rather than organismal trees.      

Global versus local regulatory roles for Lrp-related proteins: Haemophilus influenzae as a case study

Lrp (leucine-responsive regulatory protein) plays a global regulatory role in Escherichia coli, affecting expression of dozens of operons. Numerous lrp-related genes have been identified in different bacteria and archaea, including asnC, an E. coli gene that was the first reported member of this family. Pairwise comparisons of amino acid sequences of the corresponding proteins shows an average sequence identity of only 29% for the vast majority of comparisons. By contrast, Lrp-related proteins from enteric bacteria show more than 97% amino acid identity. Is the global regulatory role associated with E. coli Lrp limited to enteric bacteria? To probe this question we investigated LrfB, an Lrp-related protein from Haemophilus influenzae that shares 75% sequence identity with E. coli Lrp (highest sequence identity among 42 sequences compared). A strain of H. influenzae having an lrfB null allele grew at the wild-type growth rate but with a filamentous morphology. A comparison of two-dimensional (2D) electrophoretic patterns of proteins from parent and mutant strains showed only two differences (comparable studies with lrp(+) and lrp E. coli strains by others showed 20 differences). The abundance of LrfB in H. influenzae, estimated by Western blotting experiments, was about 130 dimers per cell (compared to 3,000 dimers per E. coli cell). LrfB expressed in E. coli replaced Lrp as a repressor of the lrp gene but acted only to a limited extent as an activator of the ilvIH operon. Thus, although LrfB resembles Lrp sufficiently to perform some of its functions, its low abundance is consonant with a more local role in regulating but a few genes, a view consistent with the results of the 2D electrophoretic analysis. We speculate that an Lrp having a global regulatory role evolved to help enteric bacteria adapt to their ecological niches and that it is unlikely that Lrp-related proteins in other organisms have a broad regulatory function.     

Occurrence and characteristics of the cytolysin A gene in Shigella strains and other members of the family Enterobacteriaceae

Cytolysin A (ClyA, HlyE, SheA) is a hemolytic pore-forming toxin found in Escherichia coli and Salmonella enterica serovars Typhi and Paratyphi A. In the present study, analysis of several Shigella strains revealed that they harbor only nonfunctional clyA gene copies that have been inactivated either by the integration of insertion sequence (IS) elements (Shigella dysenteriae, Shigella boydii, and Shigella sonnei strains) or by a frameshift mutation (Shigella flexneri). Shigella dysenteriae and S. boydii strains also exhibited IS-associated deletions at the clyA locus. PCR and Southern blot analyses as well as database searches indicated that clyA-related DNA sequences are completely absent in strains belonging to various other genera of the family Enterobacteriaceae. According to these data, ClyA may play a role only for a rather small subset of the enteric bacteria.     

Control of RecBCD Enzyme Activity by DNA Binding- and Chi Hotspot-Dependent Conformational Changes

Faithful repair of DNA double-strand breaks by homologous recombination is crucial to maintain functional genomes. The major Escherichia coli pathway of DNA break repair requires RecBCD enzyme, a complex protein machine with multiple activities. Upon encountering a Chi recombination hotspot (5′ GCTGGTGG 3′) during DNA unwinding, RecBCD's unwinding, nuclease, and RecA-loading activities change dramatically, but the physical basis for these changes is unknown. Here, we identify, during RecBCD's DNA unwinding, two Chi-stimulated conformational changes involving RecC. One produced a marked, long-lasting, Chi-dependent increase in protease sensitivity of a small patch, near the Chi recognition domain, on the solvent-exposed RecC surface. The other change was identified by crosslinking of an artificial amino acid inserted in this RecC patch to RecB. Small-angle X-ray scattering analysis confirmed a major conformational change upon binding of DNA to the enzyme and is consistent with these two changes. We propose that, upon DNA binding, the RecB nuclease domain swings from one side of RecC to the other; when RecBCD encounters Chi, the nuclease domain returns to its initial position determined by crystallography, where it nicks DNA exiting from RecC and loads RecA onto the newly generated 3′-ended single-stranded DNA during continued unwinding; a crevice between RecB and RecC increasingly narrows during these steps. This model provides a physical basis for the intramolecular “signal transduction” from Chi to RecC to RecD to RecB inferred previously from genetic and enzymatic analyses, and it accounts for the enzymatic changes that accompany Chi's stimulation of recombination.     

Identification of enteric pathogens by heat shock protein 60 kDa (HSP60) gene sequences

A highly specific and reproducible approach for the simultaneous detection of enteric pathogenic bacteria was developed using bacterial hsp60 gene and molecular biological tools. A single pair of universal primers was derived from the highly conserved sequence of hsp60 genes encompassing a 600-bp hypervariable region. PCR amplification followed by either dot blot hybridization or restriction enzyme digestion performed on 38 enteric bacteria indicated that this approach could differentiate not only different genera such as Campylobacter, Yersinia and Vibrio, but also species that are closely related genetically, such as between C. jejuni and C. coli, or between Salmonella and Shigella or Escherichia coli.     

Generalized recombination: nucleotide sequence homology between Chi recombinational hotspots

Chi sites stimulate generalized recombination catalyzed by the RecA-RecBC-dependent system of E. coli. This stimulation occurs over a region of several thousand base pairs surrounding the Chi site. These sites arise by mutation at four distinct loci in bacteriophage lambda. We report here the nucleotide sequence surrounding one of these loci, chi B, located between the xis and reda genes. Alteration of a single GC base pair, by deletion or by transversion to a CG base pair, creates the Chi recombinational hotspot chi + B. In a section of 30 bp, the chi + B sequence has 23 bp in common with the chi + C sequence determined previously. We presume that some part of this common sequence is the recognition sequence for a protein which acts at a rate-limiting step of generalized recombination.