Identification of nucleotide sequences for the specific and rapid detection of Yersinia pestis.

Suppression subtractive hybridization, a cost-effective approach for targeting unique DNA, was used to identify a 41.7-kb Yersinia pestis-specific region. One primer pair designed from this region amplified PCR products from natural isolates of Y. pestis and produced no false positives for near neighbors, an important criterion for unambiguous bacterial identification.

A plasmid partition system of the P1-P7par family from the pMT1 virulence plasmid of Yersinia pestis.

The complete sequence of the virulence plasmid pMT1 of Yersinia pestis KIM5 revealed a region homologous to the plasmid partition (par) region of the P7 plasmid prophage of Escherichia coli. The essential genes parA and parB and the downstream partition site gene, parS, are highly conserved in sequence and organization. The pMT1parS site and the parA-parB operon were separately inserted into vectors that could be maintained in E. coli. A mini-P1 vector containing pMT1parS was stably maintained when the pMT1 ParA and ParB proteins were supplied in trans, showing that the pMT1par system is fully functional for plasmid partition in E. coli. The pMT1par system exerted a plasmid silencing activity similar to, but weaker than those of P7par and P1par. In spite of the high degree of similarity, especially to P7par, it showed unique specificities with respect to the interactions of key components. Neither the P7 nor P1 Par proteins could support partition via the pMT1parS site, and the pMT1 Par proteins failed to support partition with P1parS or P7parS. Typical of other partition sites, supernumerary copies of pMT1parS exerted incompatibility toward plasmids supported by pMT1par. However, no interspecies incompatibility effect was observed between pMT1par, P7par, and P1par.

The stability region of the large virulence plasmid of Shigella flexneri encodes an efficient postsegregational killing system.

The large virulence plasmid pMYSH6000 of Shigella flexneri contains a determinant that is highly effective in stabilizing otherwise unstable plasmids in Escherichia coli. Expression of two small contiguous genes, mvpA and mvpT (formerly termed STBORF1 and STBORF2), was shown to be sufficient for stability. Mutations in mvpT abolished plasmid stability, and plasmids expressing only mvpT killed the cells unless mvpA was supplied from a separate plasmid or from the host chromosome. When replication of a plasmid carrying the minimal mvp region was blocked, growth of the culture stopped after a short lag and virtually all of the surviving cells retained the plasmid. Thus, the mvp system stabilizes by a highly efficient postsegregational killing (PSK) mechanism, with mvpT encoding a cell toxin and mvpA encoding an antidote. The regions that surround the mvp genes in their original context have an inhibitory effect that attenuates plasmid stabilization and PSK. The region encompassing the mvp genes also appears to contain an additional element that can aid propagation of a pSC101-based plasmid under conditions where replication initiation is marginal. However, this appears to be a relatively nonspecific effect of DNA insertion into the plasmid vector.

Probing the structure of complex macromolecular interactions by homolog specificity scanning: the P1 and P7 plasmid partition systems.

The P1 plasmid partition locus, P1 par, actively distributes plasmid copies to Escherichia coli daughter cells. It encodes two DNA sites and two proteins, ParA and ParB. Plasmid P7 uses a similar system, but the key macromolecular interactions are species specific. Homolog specificity scanning (HSS) exploits such specificities to map critical contact points between component macromolecules. The ParA protein contacts the par operon operator for operon autoregulation, and the ParB contacts the parS partition site during partition. Here, we refine the mapping of these contacts and extend the use of HSS to map protein-protein contacts. We found that ParB participates in autoregulation at the operator site by making a specific contact with ParA. Similarly, ParA acts in partition by making a specific contact with ParB bound at parS. Both these interactions involve contacts between a C-terminal region of ParA and the extreme N-terminus of ParB. As a single type of ParA-ParB complex appears to be involved in recognizing both DNA sites, the operator and the parS sites may both be occupied by a single protein complex during partition. The general HSS strategy may aid in solving the three-dimensional structures of large complexes of macromolecules.

Plasmid maintenance functions of the large virulence plasmid of Shigella flexneri.

The large virulence plasmid pMYSH6000 of Shigella flexneri contains a replicon and a plasmid maintenance stability determinant (Stb) on adjacent SalI fragments. The presence of a RepFIIA replicon on the SalI C fragment was confirmed, and the complete sequence of the adjacent SalI O fragment was determined. It shows homology to part of the transfer (tra) operon of the F plasmid. Stb stabilizes a partition-defective P1 miniplasmid in Escherichia coli. A 1.1-kb region containing a homolog of the F trbH gene was sufficient to confer stability. However, the trbH open reading frame could be interrupted without impairing stability. Deletion analysis implicated the involvement of two small open reading frames, STBORF1 and STBORF2, that fully overlap trbH in the opposite direction. These open reading frames are closely related to the vagC and vagD genes of the Salmonella dublin virulence plasmid and to open reading frame pairs in the F trbH region and in the chromosomes of Dichelobacter nodosus and Haemophilus influenzae. Stb appears to promote better-than-random distribution of plasmid copies and is a plasmid incompatibility determinant. The F homolog does not itself confer stability but exerts incompatibility against the activity of the Stb system. Stb is likely to encode either an active partition system or a postsegregational killing system. It shows little similarity to previously studied plasmid stability loci, but the genetic organization of STBORF1 and STBORF2 resembles that of postsegregational killing mechanisms.

The P1 ParA protein and its ATPase activity play a direct role in the segregation of plasmid copies to daughter cells.

The P1 ParA protein is an ATPase that recognizes the parA promoter region where it acts to autoregulate the P1 parA-parB operon. The ParB protein is essential for plasmid partition and recognizes the cis-acting partition site parS. The regulatory role of ParA is also essential because a controlled level of ParB protein is critical for partition. However, we show that this regulatory activity is not the only role for ParA in partition. Efficient partition can be achieved without autoregulation as long as Par protein levels are kept within a range of low values. The properties of ParA mutants in these conditions showed that ParA is essential for some critical step in the partition process that is independent of par operon regulation. The putative nucleotide-binding site for the ParA ATPase was identified and disrupted by mutation. The resulting mutant was substantially defective for autoregulation and completely inactive for partition in a system in which the need for autoregulation is abolished. Thus, the ParA nucleotide-binding site appears to be necessary both for the repressor activity of ParA and for some essential step in the partition process itself. We propose that the nucleotide-bound form of the enzyme adopts a configuration that favours binding to the operator, but that the ATPase activity of ParA is required for some energetic step in partition of the plasmid copies to daughter cells.

P1 and P7 plasmid partition: ParB protein bound to its partition site makes a separate discriminator contact with the DNA that determines species specificity.

The cis-acting P1 and P7 parS sites are responsible for active partition of P1 and P7 plasmids to daughter cells. The two sites are similar but function only with ParB proteins from the correct species. Using hybrid ParB proteins and hybrid parS sites, we show that specificity is determined by contacts between bases that lie within two parS hexamer boxes and a region in the ParB C-terminus. We refer to these contacts as discriminator contacts. The P7 discriminator contacts were mapped to 3 and 2 bp respectively within the two parS hexamer boxes, and a 10 amino acid region of P7 ParB. Similarly placed residues of different sequence are responsible for the P1 discriminator contact. The discriminator contacts are distinct from previously identified DNA binding contacts which involve different ParB and parS regions. Deletion of the ParB C-terminus that makes the discriminator contact does not diminish in vitro binding but renders it species independent. The discriminator contact is therefore a negative function, interfering with binding of the wrong ParB, but not providing energy for the binding of the correct one. Similar discriminator contacts might be responsible for specificities seen among families of eukaryotic DNA binding proteins that share common binding motifs.

The homologous operons for P1 and P7 plasmid partition are autoregulated from dissimilar operator sites.

The plasmid-partition regions of the P1 and P7 plasmid prophages in Escherichia coli are homologues which each encode two partition proteins, ParA and ParB. The equivalent P1 and P7 proteins are closely related. In each case, the proteins are encoded by an operon that is autoregulated by the ParA and ParB proteins in concert. This regulation is species-specific, as the P1 proteins are unable to repress the P7 par operon and vice versa. The homologous ParA proteins are primarily responsible for repression and bind to regions that overlap the operon promoter in both cases. The DNA-binding domain of the P7 autorepressor lies in the amino-terminal end of the P7 ParA protein. This region includes a helix-turn-helix motif that has a clear counterpart in the P1 ParA sequence. However, despite the common regulatory mechanism and the similarity of the proteins involved in repression, the promoter-operator sequences of these two operons are very different in sequence and organization. The operator is located downstream of the promoter in P1 and upstream of it in P7, and the two regions show little, if any, homology. How these differences may have arisen from a common ancestral form is discussed.

Analysis of the site for second-strand initiation during replication of the Streptomyces plasmid pIJ101.

The indigenous plasmid pIJ101 is the parent of many cloning vectors used in Streptomyces. One early pIJ101 derivative, pIJ702, has been particularly widely used. pIJ702 lacks sti:cop/korB and accumulates single-stranded DNA (ssDNA). The 1.2 kb BclI-BclI sti:cop/korB and 0.7 kb SpeI-BclI sti regions were isolated from pIJ101 and cloned into pIJ702 at the PstI site in both orientations. No ssDNA was detected in constructs containing sti present in its correct orientation with respect to the basic replicon, with or without cop/korB. Constructs which contained sti in the reverse orientation did accumulate ssDNA. Thus, sti is only active as the site for second-strand synthesis in its natural orientation. Furthermore, sti inserted in either orientation into the structurally unstable pIJ702-pUC8 shuttle vectors prevented them from rearranging in S. lividans. The sti function was defined to a 0.53 kb SpeI-SacII fragment and the probable site for second-strand initiation (ssi) was identified.

Post-UV survival and mutagenesis in DNA repair-proficient and -deficient strains of Escherichia coli K-12 grown in 5-azacytidine to inhibit DNA cytosine methylation: evidence for mutagenic excision repair.

Inhibition of cytosine methylation by growth in 5-azacytidine (5-azaC), did not affect the sensitivities to DNA damage induced by exposure to ultraviolet light (UV) of Escherichia coli K-12 strains AB1157 dcm+, which is fully DNA repair-proficient, LR68 (a dcm derivative of AB1157), JC3890 dcm+ uvrB, deficient in error-free excision repair, TK702 dcm+ umuC, deficient in error-prone repair, or TK501 dcm+ uvrB umuC, which lacks both excision repair and error-prone repair. However, growth in 5-azaC increased the post-UV survival of strains AB2463 recA(Def), AB2470 recB and AB2494 lexA(Ind-), which are deficient in the induction or expression of recombination repair or error-prone repair of DNA. Spontaneous mutation frequencies were increased in strains LR68, AB2463, AB2470 and AB2494 by growth in 5-azaC, but remained unaltered in strains AB1157, JC3890, TK702 or TK501. Growth in 5-azaC significantly increased UV-induced mutation frequencies in strains AB2463 and AB2470, significantly reduced UV-induced mutation in strain JC3890, but had little effect on UV-induced mutation in the other strains. The results suggest that 5-azaC may induce a normally error-free DNA repair pathway to become error-prone and therefore genotoxic.

Increase in susceptibility to EcoRII restriction of bacteriophage lambda produced by propagation on host cells growing in 5-azacytidine: a new in-vivo method for demonstration of DNA-methylation inhibition.

The efficiency of plating on EcoRII-restricting cells of bacteriophage lambda vir propagated on an Escherichia coli K-12 dcm+ host decreased with increase in concentration of 5-azacytidine (5-azaC) in the propagating medium. This illustrates, in-vivo, the inhibition of DNA-cytosine methylation induced by 5-azaC and provides a simple system for the detection of DNA-methylation inhibitors.

Ultraviolet light induction of lambda from dcm host strains alleviates EcoRII restriction of phage.

A new form of restriction alleviation is demonstrated for phage induced by ultraviolet light from dcm strains of Escherichia coli K-12. EcoRII restriction of the induced phage is alleviated, which is the first report of Type II restriction alleviation. Unlike previously reported restriction alleviation, the increase in phage-plating efficiency is not dependent upon irradiation of the plating host for its induction.