Biomedical subjects

Elizabeth J Harry

Publications and source records attributed to Elizabeth J Harry.

9 recordsLinked to original sources

PubMed2006

Improved 2-DE of microorganisms after acidic extraction.

2-DE separations of protein extracts sometimes have problems with poor resolution and streaking. This problem is particularly apparent with microorganisms, most notably those with a large cell wall. Here we describe a novel, rapid protocol for the extraction of microorganisms in acidic conditions, leading to increased resolution and 2-D gel quality. The efficiency of the protocol is demonstrated with extracts of bacteria, Escherichia coli and Bacillus subtilis; fungus, Trichoderma harzianum and yeast, Saccharomyces cerevisiae. We also demonstrate using a membrane centrifugal filtration, that large acidic molecules in excess of 100 kDa, probably including cell wall material, are responsible for the separation difficulties. A range of acidic extraction conditions were investigated, and it was found that optimal extraction is achieved using an extraction solution acidified to pH 3 by 80 mM citric acid. These findings have significant implications for the proteomic study of many medically, agriculturally and environmentally significant microorganisms, as the cell walls of these organisms are often considerably more complex than many commonly studied laboratory strains.

Acids↗

PubMed2006

Trapping of a spiral-like intermediate of the bacterial cytokinetic protein FtsZ.

The earliest stage in bacterial cell division is the formation of a ring, composed of the tubulin-like protein FtsZ, at the division site. Tight spatial and temporal regulation of Z-ring formation is required to ensure that division occurs precisely at midcell between two replicated chromosomes. However, the mechanism of Z-ring formation and its regulation in vivo remain unresolved. Here we identify the defect of an interesting temperature-sensitive ftsZ mutant (ts1) of Bacillus subtilis. At the nonpermissive temperature, the mutant protein, FtsZ(Ts1), assembles into spiral-like structures between chromosomes. When shifted back down to the permissive temperature, functional Z rings form and division resumes. Our observations support a model in which Z-ring formation at the division site arises from reorganization of a long cytoskeletal spiral form of FtsZ and suggest that the FtsZ(Ts1) protein is captured as a shorter spiral-forming intermediate that is unable to complete this reorganization step. The ts1 mutant is likely to be very valuable in revealing how FtsZ assembles into a ring and how this occurs precisely at the division site.

Amino Acid Sequence↗

PubMed2005

Bacillus subtilis YabA is involved in determining the timing and synchrony of replication initiation.

It is shown here by flow cytometry that Bacillus subtilis YabA negatively regulates the timing of replication initiation. When the level of YabA was reduced, replication began at a decreased cell mass and when the level was increased, initiation was delayed. Synchrony of replication initiation was also disrupted at low levels of YabA. Yfp-YabA localized as foci in cells. Since YabA was reported to interact with DnaN (beta subunit of DNA polymerase III), co-localization of Yfp-YabA with the polymerase was examined using a Cfp fusion with DnaX (tau subunit of DNA polymerase III). It is reported that YabA appears to localize at the replication forks only at a late stage of DNA replication.

Bacillus subtilis↗

PubMed2005

Cell division protein DivIB influences the Spo0J/Soj system of chromosome segregation in Bacillus subtilis.

The initiation of the developmental process of sporulation in the rod-shaped bacterium Bacillus subtilis involves the activation of the Spo0A response regulator. Spo0A then drives the switch in the site of division septum formation from midcell to a polar position. Activated Spo0A is required for the transcription of key sporulation loci such as spoIIG, which are negatively regulated by the Soj protein. The transcriptional repressing activity of Soj is antagonized by Spo0J, and both proteins belong to the well-conserved Par family of partitioning proteins. Soj has been shown to jump from nucleoid to nucleoid via the cell pole. The dynamic behaviour of Soj is somehow controlled by Spo0J, which prevents the static association of Soj with the nucleoid, and presumably its transcriptional repression activity. Soj in turn is required for the proper condensation of Spo0J foci around the oriC region. The asymmetric partitioning of the sporangial cell requires DivIB and other proteins involved in vegetative (medial) division. We describe an allele of the cell division gene divIB (divIB80) that reduces the cellular levels of DivIB, and affects nucleoid structure and segregation in growing cells, yet has no major impact on cell division. In divIB80 cells Spo0J foci are not correctly condensed and Soj associates statically with the nucleoid. The divIB80 allele prevents transcription of spoIIG, and arrests sporulation prior to the formation of the asymmetric division septum. The defect in Spo0A-dependent gene expression, and the Spo- phenotype can be suppressed by expression of divIB in trans or by deletion of the soj-spo0J locus. However, deletion of the spo0J-soj region does not restore the normal cellular levels of DivIB. Therefore, the reduced levels of DivIB in the divIB80 mutant are sufficient for efficient cell division, but not to sustain a second, earlier function of DivIB related to the activity of the Spo0J/Soj system of chromosome segregation.

Bacillus subtilis↗

PubMed2005

A streamlined approach to high-throughput proteomics.

Proteomics has rapidly become an important tool for life science research, allowing the integrated analysis of global protein expression from a single experiment. To accommodate the complexity and dynamic nature of any proteome, researchers must use a combination of disparate protein biochemistry techniques, often a highly involved and time-consuming process. Whilst highly sophisticated, individual technologies for each step in studying a proteome are available, true high-throughput proteomics that provides a high degree of reproducibility and sensitivity has been difficult to achieve. The development of high-throughput proteomic platforms, encompassing all aspects of proteome analysis and integrated with genomics and bioinformatics technology, therefore represents a crucial step for the advancement of proteomics research. ProteomIQ (Proteome Systems) is the first fully integrated, start-to-finish proteomics platform to enter the market. Sample preparation and tracking, centralized data acquisition and instrument control, and direct interfacing with genomics and bioinformatics databases are combined into a single suite of integrated hardware and software tools, facilitating high reproducibility and rapid turnaround times. This review will highlight some features of ProteomIQ, with particular emphasis on the analysis of proteins separated by 2D polyacrylamide gel electrophoresis.

Automation↗

PubMed2004

The midcell replication factory in Bacillus subtilis is highly mobile: implications for coordinating chromosome replication with other cell cycle events.

During vegetative growth, rod-shaped bacterial cells such as Escherichia coli and Bacillus subtilis divide precisely at midcell. It is the Z ring that defines the position of the division site. We previously demonstrated that the early stages of chromosome replication are linked to midcell Z ring assembly in B. subtilis and proposed a direct role for the centrally located replication factory in masking and subsequently unmasking the midcell site for Z ring assembly. We now show that the replication factory is significantly more scattered about the cell centre than the Z ring in both vegetative cells and outgrown spores of B. subtilis. This finding is inconsistent with the midcell replication factory acting as a direct physical block to Z ring assembly. Time-lapse experiments demonstrated that the lower precision of replication factory positioning results from its high mobility around the cell centre. Various aspects of this mobility are presented and the results are discussed in the light of current views on the determinants of positional information required for accurate chromosome segregation and cell division.

Bacillus subtilis↗

PubMed2003

Early targeting of Min proteins to the cell poles in germinated spores of Bacillus subtilis: evidence for division apparatus-independent recruitment of Min proteins to the division site.

The earliest event in bacterial cell division is the assembly of a tubulin-like protein, FtsZ, at mid-cell to form a ring. In rod-shaped bacteria, the Min system plays an important role in division site placement by inhibiting FtsZ ring formation specifically at the polar regions of the cell. The Min system comprises MinD and MinC, which form an inhibitor complex and, in Bacillus subtilis, DivIVA, which ensures that division is inhibited only in the polar regions. All three proteins localize to the division site at mid-cell and to cell poles. Their recruitment to the division site is dependent on localization of both 'early' and 'late' division proteins. We have examined the temporal and spatial localization of DivIVA relative to that of FtsZ during the first and second cell division after germination and outgrowth of B. subtilis spores. We show that, although the FtsZ ring assembles at mid-cell about halfway through the cell cycle, DivIVA assembles at this site immediately before cell division and persists there during Z-ring constriction and completion of division. We also show that both DivIVA and MinD localize to the cell poles immediately upon spore germination, well before a Z ring forms at mid-cell. Furthermore, these proteins were found to be present in mature, dormant spores. These results suggest that targeting of Min proteins to division sites does not depend directly on the assembly of the division apparatus, as suggested previously, and that potential polar division sites are blocked at the earliest possible stage in the cell cycle in germinated spores as a mechanism to ensure that equal-sized daughter cells are produced upon cell division.

Bacillus subtilis↗

PubMed2003

Increasing the ratio of Soj to Spo0J promotes replication initiation in Bacillus subtilis.

The ParA and ParB protein families are well conserved in bacteria. However, their functions are still unclear. In Bacillus subtilis, Soj and Spo0J are members of these two protein families, respectively. A previous report revealed that replication initiated early and asynchronously in spo0J null mutant cells, as determined by flow cytometry. In this study, we examined the cause of this promotion of replication initiation. Deletion of both the soj and spo0J genes restored the frequency of replication initiation to almost the wild-type level, suggesting that production of Soj in the absence of Spo0J leads to early and asynchronous initiation of replication. Consistent with this suggestion, overproduction of Soj in wild-type cells had the same effect on replication initiation as in the spo0J null mutant, and overproduction of both Soj and Spo0J did not. These results indicate that when the ratio of Soj to Spo0J increases, Soj interferes with tight control of replication initiation and causes early and asynchronous initiation. Whereas replication initiation also occurred significantly earlier in the two spo0J mutants, spo0J14 and spo0J17, it occurred only slightly early in the sojK16Q mutant and was delayed in the sojG12V mutant. Although Soj localized to nucleoids in the spo0J mutants, the two Soj mutant proteins were distributed throughout the cell or localized to cell poles. Thus, interestingly, the promotion of replication initiation seems to correlate with localization of Soj to nucleoids. This may suggest that Soj inhibits transcription of some cell cycle genes and leads to early and asynchronous initiation of replication. In wild-type cells Spo0J counteracts this Soj function.

Bacillus subtilis↗

PubMed2002

The Min system is not required for precise placement of the midcell Z ring in Bacillus subtilis.

In bacteria, the Min system plays a role in positioning the midcell division site by inhibiting the formation of the earliest precursor of cell division, the Z ring, at the cell poles. However, whether the Min system also contributes to establishing the precise placement of the midcell Z ring is unresolved. We show that the Z ring is positioned at midcell with a high degree of precision in Bacillus subtilis, and this is completely maintained in the absence of the Min system. Min is therefore not required for correct midcell Z ring placement in B. subtilis. Our results strongly support the idea that the primary role of the Min system is to block Z ring formation at the cell poles and that a separate mechanism must exist to ensure cell division occurs precisely at midcell.

Bacillus subtilis↗