Selecting representative model micro-organisms

Background  

Micro-biological research relies on the use of model organisms that act as representatives of their species or subspecies, these are frequently well-characterized laboratory strains. However, it has often become apparent that the model strain initially chosen does not represent important features of the species. For micro-organisms, the diversity of their genomes is such that even the best possible choice of initial strain for sequencing may not assure that the genome obtained adequately represents the species. To acquire information about a species' genome as efficiently as possible, we require a method to choose strains for analysis on the basis of how well they represent the species.     

Mycobacterium tuberculosis ClpX Interacts with FtsZ and Interferes with FtsZ Assembly

FtsZ assembly at the midcell division site in the form of a Z-ring is crucial for initiation of the cell division process in eubacteria. It is largely unknown how this process is regulated in the human pathogen Mycobacterium tuberculosis. Here we show that the expression of clpX was upregulated upon macrophage infection and exposure to cephalexin antibiotic, the conditions where FtsZ-ring assembly is delayed. Independently, we show using pull-down, solid-phase binding, bacterial two-hybrid and mycobacterial protein fragment complementation assays, that M. tuberculosis FtsZ interacts with ClpX, the substrate recognition domain of the ClpXP protease. Incubation of FtsZ with ClpX increased the critical concentration of GTP-dependent polymerization of FtsZ. Immunoblotting revealed that the intracellular ratio of ClpX to FtsZ in wild type M. tuberculosis is approximately 1∶2. Overproduction of ClpX increased cell length and modulated the localization of FtsZ at midcell sites; however, intracellular FtsZ levels were unaffected. A ClpX-CFP fusion protein localized to the cell poles and midcell sites and colocalized with the FtsZ-YFP protein. ClpX also interacted with FtsZ mutant proteins defective for binding to and hydrolyzing GTP and possibly for interactions with other proteins. Taken together, our results suggest that M. tuberculosis ClpX interacts stoichiometrically with FtsZ protomers, independent of its nucleotide-bound state and negatively regulates FtsZ activities, hence cell division.     

The expression of transthyretin mRNA in the developing rat brain

Specific cDNA and oligonucleotide probes were used to study the appearance of transthyretin mRNA in developing rat brain using Northern gel analysis, cytoplasmic dot hybridization, and in situ hybridization. Transthyretin mRNA in embryonic rat brain was found to be confined to the epithelial layer of the choroid plexus primordia appearing first in the fourth ventricle, followed by appearance in the lateral ventricles, and subsequently in the third ventricle. Transthyretin mRNA was localized in these cells from early stages of neuroepithelium differentiation, showing that it is a sensitive marker for the differentiation of the choroid plexus within the fetal brain.     

ParA of Mycobacterium smegmatis co‐ordinates chromosome segregation with the cell cycle and interacts with the polar growth determinant DivIVA

Mycobacteria are among the clinically most important pathogens, but still not much is known about the mechanisms of their cell cycle control. Previous studies suggested that the genes encoding ParA and ParB (ATPase and DNA binding protein, respectively, required for active chromosome segregation) may be essential in Mycobacterium tuberculosis. Further research has demonstrated that a Mycobacterium smegmatis parB deletion mutant was viable but exhibited a chromosome segregation defect. Here, we address the question if ParA is required for the growth of M. smegmatis, and which cell cycle processes it affects. Our data show that parA may be deleted, but its deletion leads to growth inhibition and severe disturbances of chromosome segregation and septum positioning. Similar defects are also caused by ParA overproduction. EGFP–ParA localizes as pole‐associated complexes connected with a patch of fluorescence accompanying two ParB complexes. Observed aberrations in the number and positioning of ParB complexes in the parA deletion mutant indicate that ParA is required for the proper localization of the ParB complexes. Furthermore, it is shown that ParA colocalizes and interacts with the polar growth determinant Wag31 (DivIVA homologue). Our results demonstrate that mycobacterial ParA mediates chromosome segregation and co‐ordinates it with cell division and elongation.     

Modelling expected trout ranges under current and future water temperature regimes in the Eastern Cape,South Africa

Different values have resulted in conflicts between anglers and conservation lobbies in the management of trout in South Africa. Key to the conflict is the demarcation of boundaries to areas in which brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss currently occur, or are likely to establish following stocking for angling. To provide a longer-term perspective on these areas, we developed models to link salmonid biological thermal thresholds to elevation. These, when applied spatially using a digital elevation model with a probability of occurrence model, provided the basis for estimating potentially available thermal habitat for these two cold water species. Here, we acknowledge that other variables (stocking history; river connectivity) also play a role in understanding trout distributions. Using a simple scenario of an increase in mean daily water temperatures of 2 °C, we demonstrated that both brown and rainbow trout are likely to exhibit considerable range reductions in the future. Because it is possible that these range restrictions will result in an increasing desire to introduce trout into areas above their current distribution limits for the maintenance of angling opportunities, conservation managers should prioritise these areas, with management interventions seeking to understand what will help to limit introductions.     

RNase HI Is Essential for Survival of Mycobacterium smegmatis

RNases H are involved in the removal of RNA from RNA/DNA hybrids. Type I RNases H are thought to recognize and cleave the RNA/DNA duplex when at least four ribonucleotides are present. Here we investigated the importance of RNase H type I encoding genes for model organism Mycobacterium smegmatis. By performing gene replacement through homologous recombination, we demonstrate that each of the two presumable RNase H type I encoding genes, rnhA and MSMEG4305, can be removed from M. smegmatis genome without affecting the growth rate of the mutant. Further, we demonstrate that deletion of both RNases H type I encoding genes in M. smegmatis leads to synthetic lethality. Finally, we question the possibility of existence of RNase HI related alternative mode of initiation of DNA replication in M. smegmatis, the process initially discovered in Escherichia coli. We suspect that synthetic lethality of double mutant lacking RNases H type I is caused by formation of R-loops leading to collapse of replication forks. We report Mycobacterium smegmatis as the first bacterial species, where function of RNase H type I has been found essential.     

Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

The septal association of Mycobacterium tuberculosis MtrB, the kinase partner of the MtrAB two-component signal transduction system, is necessary for the optimal expression of the MtrA regulon targets, including ripA, fbpB, and ftsI, which are involved in cell division and cell wall synthesis. Here, we show that MtrB, irrespective of its phosphorylation status, interacts with Wag31, whereas only phosphorylation-competent MtrB interacts with FtsI. We provide evidence that FtsI depletion compromises the MtrB septal assembly and MtrA regulon expression; likewise, the absence of MtrB compromises FtsI localization and, possibly, FtsI activity. We conclude from these results that FtsI and MtrB are codependent for their activities and that FtsI functions as a positive modulator of MtrB activation and MtrA regulon expression. In contrast to FtsI, Wag31 depletion does not affect MtrB septal assembly and MtrA regulon expression, whereas the loss of MtrB increased Wag31 localization and the levels of PknA/PknB (PknA/B) serine-threonine protein kinase-mediated Wag31 phosphorylation. Interestingly, we found that FtsI decreased levels of phosphorylated Wag31 (Wag31∼P) and that MtrB interacted with PknA/B. Overall, our results indicate that MtrB interactions with FtsI, Wag31, and PknA/B are required for its optimal localization, MtrA regulon expression, and phosphorylation of Wag31. Our results emphasize a new role for MtrB in cell division and cell wall synthesis distinct from that regulating the MtrA phosphorylation activities.     

Characterization of a novel calcium-binding 90-kDa glycoprotein (BM-90) shared by basement membranes and serum

The protein BM-90 was solubilized from the mouse Engelbreth-Holm-Swarm tumor with neutral buffers in molar yields lower (15-30%) than found for other basement membrane proteins (e.g. laminin, BM-40). The purified protein was shown to be rich in cysteine (5 mol%) and to change in SDS electrophoresis from an 84-kDa position to a 95-kDa one upon reduction. BM-90 was also shown to be a calcium-binding protein. The N-terminal sequence of BM-90, as well as those of several internal peptides, showed no identity with any known protein sequences, indicating that it is a new protein. Specific radioimmunoassays showed no or only minor cross-reactions with other known basement membrane proteins. Immunological assays demonstrated BM-90 to be present in neutral salt extracts from mouse heart and kidney, in serum (20-40 micrograms/ml) and in the medium of various cultured cells (0.1-1 microgram/ml). The protein in these samples was identical in size to BM-90 purified from the tumor, indicating that negligible degradation occurs during purification. An extracellular matrix localization of BM-90 was shown by immunofluorescence for Reichert's membrane, lens capsules and other basement membranes. Thus, BM-90 appears to be a novel basement membrane protein whose functions remain to be studied.     

Predicted warming and browning affect timing and magnitude of plankton phenological events in lakes: a mesocosm study

1. Aquatic ecosystems in Northern Europe are expected to face increases in temperature and water colour (TB) in future. While effects of these factors have been studied separately, it is unknown whether and how a combination of them might affect phenological events and trophic interactions. 2. In a mesocosm study, we combined both factors to create conditions expected to arise during the coming century. We focused on quantifying effects on timing and magnitude of plankton spring phenological events and identifying possible mismatches between resources (phytoplankton) and consumers (zooplankton). 3. We found that the increases in TB had important effects on timing and abundance of different plankton groups. While increased temperature led to an earlier peak in phytoplankton and zooplankton and a change in the relative timing of different zooplankton groups, increased water colour reduced chlorophyll‐a concentrations. 4. Increased TB together benefitted cladocerans and calanoid copepods and led to stronger top‐down control of algae by zooplankton. There was no sign of a mismatch between primary producers and grazers as reported from other studies. 5. Our results point towards an earlier onset of plankton spring growth in shallow lakes in future with a stronger top‐down control of phytoplankton by zooplankton grazers.     

Cholesterol oxidase is required for virulence of Mycobacterium tuberculosis

Recent reports have indicated that cholesterol plays a crucial role during the uptake of mycobacteria by macrophages. However, the significance of cholesterol modification enzymes encoded by Mycobacterium tuberculosis for bacterial pathogenicity remains unknown. Here, the authors explored whether the well-known cholesterol modification enzyme, cholesterol oxidase (ChoD), is important for virulence of the tubercle bacillus. Homologous recombination was used to replace the choD gene from the M. tuberculosis genome with a nonfunctional copy. The resultant mutant (delta choD) was attenuated in peritoneal macrophages. No attenuation in macrophages was observed when the same strain was complemented with an intact choD gene controlled by a heat shock promoter (delta choDP(hsp)choD). The mice infection experiments confirm the significance of ChoD in the pathogenesis of M. tuberculosis.