Escherichia coli XerC recombinase is required for chromosomal segregation at cell division

XerC is a site-specific recombinase of the bacteriophage lambda integrase family that is encoded by xerC at 3700 kbp on the genetic map of Escherichia coli. The protein was originally identified through its role in converting multimers of plasmid ColE1 to monomers; only monomers are stably inherited. Here we demonstrate that XerC also has a role in the segregation of replicated chromosomes at cell division. xerC mutants form filaments with aberrant nucleotides that appear unable to partition correctly. A DNA segment (dif) from the replication terminus region of the E. coli chromosome binds XerC and acts as a substrate for XerC-mediated site-specific recombination when inserted into multicopy plasmids. This dif segment contains a region of 28 bp with sequence similarity to the crossover region of ColE1 cer. The cell division phenotype of xerC mutants is suppressed in strains deficient in homologous recombination, suggesting that the role of XerC/dif in chromosomal metabolism is to convert any chromosomal multimers (arising through homologous recombination) to monomers.     

The XerC recombinase of Proteus mirabilis: characterization and interaction with other tyrosine recombinases

XerC and XerD are two site-specific recombinases, which act on different sites to maintain replicons in a monomeric state. This system, which was first discovered and studied in Escherichia coli, is present in several species including Proteus mirabilis, where the XerD recombinase was previously characterized by our laboratory. In this paper, we report the presence of the xerC gene in P. mirabilis. Using in vitro reactions, we show that the two P. mirabilis recombinases display binding and cleavage activity on the E. coli dif site and the ColE1 cer site, together or in collaboration with E. coli recombinases. In vivo, P. mirabilis XerC and XerD are able to resolve and monomerize a plasmid containing two cer sites, increasing its stability. However, P. mirabilis XerC, in combination with E. coli XerD, is unable to perform these functions.     

Detection of XerC and XerD recombinases in gram-negative bacteria of the family Enterobacteriaceae.

XerC and XerD are site-specific recombinases of the lambda integrase family which resolve multimeric replicons to monomers by acting at specific sites such as cer, ckr, nmr, parB, and psi, which are found in plasmids, or at the dif site found in the Escherichia coli chromosome. By using Southern hybridizations to cloned E. coli xerC and xerD genes and a cer-nmr plasmid-based resolution assay, the presence of these genes in several species of Enterobacteriaceae is shown.     

Xer-mediated site-specific recombination at cer generates Holliday junctions in vivo.

Normal segregation of the Escherichia coli chromosome and stable inheritance of multicopy plasmids such as ColE1 requires the Xer site-specific recombination system. Two putative lambda integrase family recombinases, XerC and XerD, participate in the recombination reactions. We have constructed an E. coli strain in which the expression of xerC can be tightly regulated, thereby allowing the analysis of controlled recombination reactions in vivo. Xer-mediated recombination in this strain generates Holliday junction-containing DNA molecules in which a specific pair of strands has been exchanged in addition to complete recombinant products. This suggests that Xer site-specific recombination utilizes a strand exchange mechanism similar or identical to that of other members of the lambda integrase family of recombination systems. The controlled in vivo recombination reaction at cer requires recombinase and two accessory proteins, ArgR and PepA. Generation of Holliday junctions and recombinant products is equally efficient in RuvC- and RuvC+ cells, and in cells containing a multicopy RuvC+ plasmid. Controlled XerC expression is also used to analyse the efficiency of recombination between variant cer sites containing sequence alterations and heterologies within their central regions.     

Derivatives of ColE1 cer show altered topological specificity in site-specific recombination.

ColE1 contains a 250-bp sequence (cer) which is required in cis for the conversion of plasmid dimers to monomers. Recombination between cer and parB (a dimer resolution site from plasmid CloDF13) occurs in vivo at low frequency. The properties of the resulting hybrid sites have been studied. The type I hybrid closely resembles wild-type cer. It supports intramolecular recombination and requires the products of the chromosomal xerA, xerB and xerC genes together with the 250-bp site. In contrast, the type II hybrid (although differing from type I by only 2 bp) functions independently of the topological relationship of the participating sites, supporting both inter- and intramolecular recombination. Furthermore, recombination between type II sites is independent of the products of the xerA and xerB genes and requires a site of less than 50 bp.     

The arginine repressor is essential for plasmid-stabilizing site-specific recombination at the ColE1 cer locus.

The heritable stability in Escherichia coli of the multicopy plasmid ColE1 and its natural relatives requires that the plasmids be maintained in the monomeric state. Plasmid multimers, that arise through recA-dependent homologous recombination, are normally converted to monomers by a site-specific recombination system that acts at a specific plasmid site (cer in ColE1). No plasmid functions that act at this site have been identified. In contrast, two unlinked E.coli genes that encode functions required for cer-mediated site-specific recombination have been identified. Here we describe the isolation and characterization of one such gene (xerA) and show it to be identical to the gene encoding the repressor of the arginine biosynthetic genes (argR). The argR protein binds to cer DNA both in vivo and in vitro in the presence of arginine. We believe this binding is required to generate a higher order protein-DNA complex within the recombinational synapse. The argR gene of Bacillus subtilis complements an E.coli argR deficiency for cer-mediated recombination despite the two proteins having only 27% amino acid identity.     

Genetic characterization of gram-positive homologs of the XerCD site-specific recombinases

Homologs of the XerCD enzymes, which in Escherichia coli have been shown to be responsible for resolving chromosomal multimers prior to chromosome segregation, were identified in the genomes of Staphylococcus aureus and Streptococcus pneumoniae. Phylogenetic and conservation pattern analysis suggests that the S. aureus gene products are orthologs of XerC and D. A S. aureus xerC null mutant displayed in vitro characteristics consistent with the segregation defect reported for E. coli xer mutants, and was found to be attenuated in a murine infection model. Strikingly, the S. aureus xerD gene appears to be absolutely required for viability, and may therefore be the first example of an essential gene of the lambda integrase family. In contrast, phylogenetic and conservation pattern analysis show that the S. pneumoniae gene products are more closely related to phage integrases than to XerCD. S. pneumoniae xer1, 2 and 3 null mutants were each found to be attenuated in a murine infection model, suggesting that they may control processes which affect virulence.     

DNA degradation in the terminus region of resolvase mutants of Escherichia coli, and suppression of this degradation and the Dif phenotype by recD

We recently proposed that guillotining of dimer chromosomes occurs at cell division in resolvase mutants of Escherichia coli. This was based on the abnormal pattern of cell division observed in 10-14% of the cells in microcolonies of xerC, xerD and dif mutants. A prediction of this guillotining is that DNA degradation should occur in the terminus region, in the vicinity of the dif locus. We have tested this by DNA-DNA hybridization and have observed that dif was absent in about 22% of the chromosomes in exponentially growing xerC mutants. A locus 206 kb from dif was not affected by this degradation. We have also observed that degradation did not occur in xerC recD mutants, and that the low efficiency of plating associated with the Dif phenotype was suppressed in this strain. A model is proposed in which rapid degradation of the terminus region does not occur in recD mutants following guillotining, and that this permits the initiation of repair of broken dimer chromosomes prior to completion of cell division.     

xerB, an Escherichia coli gene required for plasmid ColE1 site-specific recombination, is identical to pepA, encoding aminopeptidase A, a protein with substantial similarity to bovine lens leucine aminopeptidase.

The heritable stability of ColE1 is dependent on a site-specific recombination system which acts to resolve plasmid multimers into monomers. This plasmid stabilizing recombination system requires the presence in cis of the ColE1 cer region, plus at least two trans-acting factors encoded by the xerA and xerB genes of Escherichia coli. The xerB gene has been cloned and sequenced and found to encode a polypeptide with a calculated mol. wt of 55.3 kd. The predicted amino acid sequence of this protein exhibits striking similarity to that of bovine lens leucine aminopeptidase (53 kd). The biological significance of this similarity is corroborated by genetic and biochemical evidence which suggests that xerB is identical to the E.coli and S.typhimurium pepA genes that encode aminopeptidase A.     

Linker mutagenesis in the gene encoding the periplasmic maltose-binding protein of E. coli

A plasmid carrying the malE gene, coding for the periplasmic maltose-binding protein of E. coli, was submitted to random mutagenesis by the insertion of a BamHI linker. About 25% of the clones recovered had acquired a BamHI site in the gene malE. Most of the linker insertions were accompanied by small deletions with an average size of 30 base pairs. Among 21 mutants synthesizing a stable maltose binding protein, 8 were still able to grow on maltose. A preliminary analysis of these mutants indicates that certain regions of the protein may not be essential for maltose transport.     

Characterization of the Rickettsia prowazekii pepA gene encoding leucine aminopeptidase.

The pepA gene, encoding a protein with leucine aminopeptidase activity, was isolated from Rickettsia prowazekii, an obligate intracellular parasitic bacterium. Nucleotide sequence analysis revealed an open reading frame of 1,502 bp that would encode a protein of 499 amino acids with a calculated molecular weight of 53,892, a size comparable to that of the protein produced in Escherichia coli minicells containing the rickettsial gene. Also, heat-stable leucine aminopeptidase activity was demonstrable in an E. coli peptidase-deficient strain containing R. prowazekii pepA. Comparison of the amino acid sequence of the R. prowazekii PepA with the characterized leucine aminopeptidases from E. coli, Arabidopsis thaliana, and bovine eye lens revealed that 39.8, 34.9, and 34.0% of the residues were identical, respectively. Residues proposed to be part of the active site or involved in the binding of metal ions in the bovine metalloenzyme were all conserved in R. prowazekii PepA. However, despite the structural and enzymatic similarity to E. coli PepA, the R. prowazekii protein was unable to complement the cer site-specific, PepA-dependent recombination system found in E. coli that resolves ColE1-type plasmid multimers into their monomeric forms.     

Purification and characterization of the gene 1.2 protein of bacteriophage T7

Gene 1.2 of bacteriophage T7, located near the primary origin of DNA replication at position 15.37 on the T7 chromosome, encodes a 10,059-dalton protein that is essential for growth on Escherichia coli optA1 strains (Saito, H., and Richardson, C. C. (1981) J. Virol. 37, 343-351). In the absence of the T7 1.2 and E. coli optA gene products, the degradation of E. coli DNA proceeds normally, and T7 DNA synthesis is initiated at the primary origin. However, T7 DNA synthesis ceases prematurely and the newly synthesized DNA is degraded; no viable phage particles are released. The gene 1.2 protein has been purified to apparent homogeneity from cells in which the cloned 1.2 gene is overexpressed. Purification of the [35S] methionine-labeled protein was followed by monitoring the radioactivity of the protein and by gel electrophoresis. The purified protein has been identified as the product of gene 1.2 on the basis of molecular weight and partial amino acid sequence. We have found that extracts of E. coli optA1 cells infected with T7 gene 1.2 mutants are defective in packaging exogenous T7 DNA when such extracts are prepared late in infection. Purified gene 1.2 protein restores packaging activity to these defective extracts, thus providing a biological assay for gene 1.2 protein. No specific enzymatic activity has been found associated with the purified gene 1.2 protein.     

Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta

Bacillus thuringiensis subsp. kurstaki HD-73 produces a crystal protein which is lethal to many lepidopteran larvae. The gene encoding this crystal protein has been isolated from a 75-kb plasmid and engineered into a recombinant Escherichia coli plasmid for analysis. The complete nucleotide sequences of the coding region and 387-bp 5' and 376-bp 3' to the coding region have been determined. The 3537-bp of the coding region specify a protein of Mr 133 330. The full-length gene and several 3' -truncated derivatives of the gene were examined in both E. coli and in an E. coli minicell-expression system to determine if the carboxy end of the protein is essential for toxicity. The results presented here provide the primary structure of the crystal protein gene and show that the N-terminal 68-kDal peptide is toxic, but at a lower level than the full-length gene product.     

Cloning and structure of the hem A gene of Escherichia coli K-12

An Escherichia coli gene, which complements two independent hemA mutants of E. coli, has been cloned onto a multi-copy plasmid and both its strands have been sequenced. Both complemented mutants produce 5-aminolevulinic acid (ALA) and display fluorescence after 24h. The cloned sequence appears to encode a 46-kDa protein, which when produced in the maxicell procedure is processed to a 41-kDa protein as determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. The amino acid sequence of the cloned gene product shows no significant homologies with any cloned ALA synthase, nor with any protein, in two E. coli databanks. A second cloned gene fragment, which has its coding region 34 bp away from the coding region of the gene that complements hemA, has been identified as part of protein release factor 1(RF1), thus confirming the location of hemA at min 26.7 and mapping it precisely near RF1. We have shown that E. coli utilizes the intact five-carbon chain of glutamate for the synthesis of ALA [Li et al., J Bacteriol. 171 (1989b) 2547-2552].     

Synthesis of a gene for the protein kinase domain of the epidermal growth factor receptor and its expression in Escherichia coli

A gene encoding the protein kinase domain of the epidermal growth factor receptor has been chemically synthesised, cloned and expressed in Escherichia coli. The 942-base-pair gene was constructed by enzymatic ligation of 56 oligonucleotides and cloned into an expression vector downstream of the E. coli trp promoter. Production of active gene product was confirmed by means of a protein kinase assay, demonstrating that the enzymatic activity of the protein kinase domain of the epidermal growth factor receptor is retained after expression in E. coli.     

Deletion of the penicillin-binding protein 5 gene of Escherichia coli.

A strain of Escherichia coli that has a deletion of the entire dacA gene has been constructed. The complete lack of penicillin-binding protein 5 in this strain establishes that the activity of this protein is not essential for the growth of E. coli.     

The CER3 wax biosynthetic gene from Arabidopsis thaliana is allelic to WAX2/YRE/FLP1

The cuticle coats the aerial organs of land plants and is composed of a cutin matrix embedded and overlayed with waxes. The Arabidopsis CER3 gene is important for cuticular wax biosynthesis and was reported to correspond to At5g02310 encoding an E3 ubiquitin ligase. Here, we demonstrate that CER3 is not At5g02310 and instead corresponds to WAX2/YRE/FLP1 (At5g57800), a gene of unknown function required for wax biosynthesis. CER3 protein has also been implicated in cutin production because strong cer3 alleles display organ fusions. Leaf cutin analysis of two cer3 alleles did not reveal significant differences in cutin load or composition, indicating that CER3 has no major role in leaf cutin formation.     

Structural gene for the phosphate-repressible phosphate-binding protein of Escherichia coli has its own promoter: complete nucleotide sequence of the phoS gene

The complete nucleotide sequence of the phoS gene, the structural gene for the phosphate-repressible, periplasmic phosphate-binding protein Escherichia coli K-12, was determined. The phosphate-binding protein is synthesized in a precursor form which includes an additional N-terminal segment containing 25 amino acid residues, with the general characteristics of a signal sequence. The amino acid sequence derived from the nucleotide sequence shows the mature protein to be composed of 321 amino acids with a calculated molecular weight of 34,427. The phoS gene is not part of an operon and is transcribed counterclockwise with respect to the E. coli genetic map. A promoter region has been identified on the basis of homology with the consensus sequence of other E. coli promoter regions. However, an alternative promoter region has been identified on the basis of homology with the promoter regions of the phoA and phoE genes, the structural genes for alkaline phosphatase and outer-membrane pore protein e, respectively.