Biomedical subjects

S K Highlander

Publications and source records attributed to S K Highlander.

24 records · Page 2Linked to original sources

PubMed1987

Plasmid repopulation kinetics in Staphylococcus aureus.

We have analyzed the kinetic route by which the indirectly controlled Staphylococcus aureus plasmid, pT181, responds to and corrects fluctuations in copy number. The kinetics of copy number correction from low to steady-state levels (termed repopulation) were determined using two different methods of copy number reduction. Thermosensitive replication (Tsr) mutants of pT181 were grown at nonpermissive temperatures to lower copy number and then shifted to a permissive temperature to allow repopulation. After the downshift, both wild-type and copy mutant plasmids, with active inhibitors, exhibited a burst of exponential replication that resulted in a two- to threefold overshoot of normal steady-state copy numbers. This was followed by inhibition of replication and eventual reestablishment of the steady-state replication rate. Similar replication kinetics were observed when these plasmids were introduced into naive cells by high-frequency transduction. By contrast, a pT181 copy mutant with a nonfunctional inhibitor-target regulation did not overshoot its steady-state copy number, but instead repopulated asymptotically. These results suggest that at low copy numbers, pT181 and its derivatives replicate at near-maximal rates and overshoot prior to the establishment of an inhibitory concentration of repressor. The maximal replication rate is independent of the plasmid's cop genotype. As the copy number increases, inhibitor accumulates and eventually reduces the replication rate. In the absence of an active inhibitor, the steady-state copy number is established at a level that must be limited by some other invariant factor.

DNA Replication↗

PubMed1985

Replication control for pT181, an indirectly regulated plasmid.

PT181 is a fully sequenced Staphylococcus aureus plasmid whose size is 4,437 bp. It specifies tetracycline resistance and has a copy number of about 22 per cell in exponentially growing cultures. The functional organization of the pT181 replicon is centered around the coding sequence for a 35-kd protein, RepC, that is absolutely required for replication of the plasmid. The replication origin is contained within the repC coding sequence and the region immediately 5' to the RepC start is involved in control of the plasmid replication rate. PT181 replication is controlled at the level of RepC synthesis by a negative regulatory system that is functionally similar to that of the Co1E1 and IncFII plasmids of Escherichia coli. The pT181 control circuit involves 2 short transcripts, RNA I and RNA II, that are transcribed from the region specifying the 5' end of the untranslated repC mRNA leader and in the opposite direction. These are referred to as countertranscripts. The countertranscripts regulate RepC synthesis by a mechanism that probably involves interaction with the repC mRNA leader in a manner that interferes with translation. Both of the countertranscripts seem to be necessary for normal replication control; their separate roles remain unclear. Unlike plasmids of the Co1E1 and IncFII groups, plasmids such as Co1E1 are considered to have direct regulation of replication because the inhibitory element of the copy control circuit directly inhibits the initiation of replication. Plasmids such as pT181 are considered to have indirect regulation of replication because the product of the regulated step, RepC, is trans-active. Plasmids of the IncFII type are considered to have direct regulation of replication because the product of the regulated step, RepA is cis-active The analysis of pT181 replication physiology has illustrated 2 important differences between directly and indirectly regulated plasmids: a) for directly regulated plasmids, copy mutants specifying a normal inhibitor substance but an inactive target site exclude the wild-type or recessive mutants by directly interfering with their replication. Analogous mutants of indirectly regulated plasmids coexist readily with the wild-type and all mutants (although they do manifest segregational incompatibility) because the Rep protein is always shared by all plasmids in the cell, regardless of its source. b) Mutations of directly regulated plasmids in the region where target transcript and countertranscript overlap may give rise to totally new incompatibility groups because they engender independently self-correcting copy pools.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1984

Control of pT181 replication I. The pT181 copy control function acts by inhibiting the synthesis of a replication protein.

pT181 is a fully sequenced 4.4-kb 20 copy Tcr plasmid from Staphylococcus aureus. Its replication system involves a unique unidirectional origin embedded in the coding sequence for a plasmid-determined protein, RepC, that is required for initiation. When joined to a 55 copy carrier plasmid, pE194, pT181 excludes autonomous isologous replicons by inhibiting their replication. Two types of spontaneous pT181 copy mutants have been isolated, one that eliminates sensitivity to this inhibition and another that does not. A spontaneous 180-bp deletion, delta 144, eliminates both the inhibitory activity and sensitivity to it. This deletion increases copy number by 50-fold and RepC production by at least 10-fold. It is located directly upstream from the repC coding sequence and the deletion-bearing plasmid supports the replication of inhibitor-sensitive plasmids in cells containing active inhibitor. This effect is probably due to the overproduction of RepC by the delta 144 plasmid. On the basis of these results, it is suggested that RepC synthesis is negatively controlled by an inhibitor that is encoded directly upstream from the repC coding sequence and acts as a tareget set in the same region. It is likely, therefore, that pT181 replication rate is determined by the level of RepC.

Bacterial Proteins↗

PubMed1984

Control of pT181 replication II. Mutational analysis.

We describe the isolation and analysis of mutations affecting the regulation of Staphylococcus aureus plasmid pT181 replication. Previous results suggested that regulation is achieved by control of the synthesis of RepC, a plasmid-coded replication protein and that the primary negative control element is CopA RNA, which consists of two transcripts that are complementary to the 5' region of the repC mRNA leader. CopA inhibition probably involves a base pairing interaction with the complementary region of the RepC mRNA leader which would facilitate the formation of a downstream stem-loop in the leader that occludes the repC ribosome binding site. RepC is freely diffusible so that regulation of pT181 replication is indirect. Both CopA RNA-sensitive (recessive) and -insensitive (dominant) mutants were isolated. The recessives have defects in CopA RNA structure or activity, the dominants have defects in the site of action (target) of the inhibitor. Some dominants were located within the copA coding sequence. These therefore affect the structure of CopA RNA as well as that of its target. Other dominant mutations mapped outside of the copA gene and therefore produced wild-type CopA RNA. In contrast to directly regulated plasmids, pT181 copy mutants producing wild-type inhibitor could be co-maintained with the wild-type plasmid and mutational changes in inhibitor-target specificity did not change incompatibility specificity.

Bacillus subtilis↗

PubMed1982

Partial characterization of a small, multiple-copy plasmid from Streptomyces espinosus and the derivation of a high copy-number deletion mutant.

An organism classified as Streptomyces espinosus was found to carry an approx. 9.2-kb plasmid. This plasmid, designated pUC6, has a copy number of 30-40 per host genome equivalent. Plasmid pUC1061, a copy-number mutant of pUC6, was isolated after in vitro deletion of an approx. 2.0-kb XhoI restriction fragment. Plasmid pUC1061 has a copy number of 500-600. Plasmid pUC1061 appears to be incompatible with pUC6 and will transform a pUC6-containing culture at a frequency of approx. 1%. The sizes, restriction maps and copy numbers of plasmids pUC6 and pUC1061 indicate these may be valuable vectors for gene cloning Streptomyces.

Chromosome Deletion↗

PubMedDate not supplied

DNA sequence of the Pasteurella haemolytica leukotoxin gene cluster.

Bovine serum was used to identify a recombinant phage clone carrying the Pasteurella haemolytica leukotoxin gene. This fragment produced the 102-kD leukotoxin and several smaller P. haemolytica-specific protein antigens in Escherichia coli. An additional contiguous fragment, containing sequences upstream from the leukotoxin gene. Using these clones, we determined the nucleotide sequence of a 7745-bp region that included four open reading frames: an upstream gene, lktC; the leukotoxin gene, lktA; and two downstream genes, lktB, and lktD. The predicted molecular weights of the proteins encoded by these genes were 19.9, 102, 79.6, and 54.7 kD, respectively. These genes and their predicted proteins were similar in organization and in sequence to the corresponding elements of the gene cluster that encodes an E. coli alpha-hemolysin and its activation and secretion functions. Expression of the leukotoxin was enhanced in E. coli, by fusing the gene to the lac promoter. Under these conditions the leukotoxin was not secreted into the medium, as it is in P. haemolytica. However, in the presence of the alpha-hemolysin genes, the leukotoxin was secreted into the medium, demonstrating functional complementation by the hemolysin secretory system.

Amino Acid Sequence↗