Mycobacteria are members of the actinomycetes that grow by tip extension and lack apparent homologues of the known cell division regulators found in other rod-shaped bacteria. Previous work using static microscopy on dividing mycobacteria led to the hypothesis that these cells can grow and divide asymmetrically, and at a wide range of sizes, in contrast to the cell growth and division patterns observed in the model rod-shaped organisms. In this study, we test this hypothesis using live-cell time-lapse imaging of dividing Mycobacterium smegmatis labelled with fluorescent PBP1a, to probe peptidoglycan synthesis and label the cell septum. We demonstrate that the new septum is placed accurately at mid-cell, and that the asymmetric division observed is a result of differential growth from the cell tips, with a more than 2-fold difference in growth rate between fast and slow growing poles. We also show that the division site is not selected at a characteristic cell length, suggesting this is not an important cue during the mycobacterial cell cycle.

We have previously reported on the development and assessment of the tetracycline inducible vector pMIND [1]. Here we report the development of improved pMIND vectors that exhibit both reduced basal transcription in the absence of inducer and increased fold induction in the presence of inducer. An amino acid change in the repressor protein, TetR(Z), produced a 6-fold reduction in basal transcription compared to the original pMIND-Lx and a 100-fold induction of LuxAB in the presence of tetracycline. An integration version of the improved vector (pMEND-Lx) was constructed which resulted in a 9-fold reduction in basal transcription compared to pMIND-Lx and a 17-fold induction of LuxAB in the presence of tetracycline. Further improvements were obtained by cloning the pMEND TetRO promoter into an alternative vector backbone. The resulting vector, pKW08-Lx, exhibited a 70-fold reduction in background compared to pMIND-Lx and a 230-fold induction of LuxAB in the presence of tetracycline. An integration version of pKW08-Lx was constructed and the basal transcription for this vector was zero; an 11-fold induction of LuxAB was observed in the presence of tetracycline. The construction of these improved mycobacterial vectors will prove extremely useful for genetic studies.

BackgroundTwo general approaches to prokaryotic live-cell imaging have been employed to date, growing bacteria on thin agar pads or growing bacteria in micro-channels. The methods using agar pads 'sandwich' the cells between the agar pad on the bottom and a glass cover slip on top, before sealing the cover slip. The advantages of this technique are that it is simple and relatively inexpensive to set up. However, once the cover slip is sealed, the environmental conditions cannot be manipulated. Furthermore, desiccation of the agar pad, and the growth of cells in a sealed environment where the oxygen concentration will be in gradual decline, may not permit longer term studies such as those required for the slower growing mycobacteria.FindingsWe report here a modified agar pad method where the cells are sandwiched between a cover slip on the bottom and an agar pad on top of the cover slip (rather than the reverse) and the cells viewed from below using an inverted microscope. This critical modification overcomes some of the current limitations with agar pad methods and was used to produce time-lapse images and movies of cell growth for Mycobacterium smegmatis and Mycobacterium bovis BCG.ConclusionsThis method offers improvement on the current agar pad methods in that long term live cell imaging studies can be performed and modification of the media during the experiment is permitted.

Correct chromosomal segregation, coordinated with cell division, is crucial for bacterial survival, but despite extensive studies, the mechanisms underlying this remain incompletely understood in mycobacteria. We report a detailed investigation of the dynamic interactions between ParA and ParB partitioning proteins in Mycobacterium smegmatis using microfluidics and time-lapse fluorescence microscopy to observe both proteins simultaneously. During growth and division, ParB presents as a focused fluorescent spot that subsequently splits in two. One focus moves towards a higher concentration of ParA at the new pole, while the other moves towards the old pole. We show ParB movement is in part an active process that does not rely on passive movement associated with cell growth. In some cells, another round of ParB segregation starts before cell division is complete, consistent with initiation of a second round of chromosome replication. ParA fluorescence distribution correlates with cell size, and in sister cells, the larger cell inherits a local peak of concentrated ParA, while the smaller sister inherits more homogeneously distributed protein. Cells which inherit more ParA grow faster than their sister cell, raising the question of whether inheritance of a local concentration of ParA provides a growth advantage. Alterations in levels of ParA and ParB were also found to disturb cell growth.

Catholic Health Initiatives (CHI) set a goal of reducing birth injuries throughout the system. Although the number of injuries was small, clinical and risk leadership agreed that even one injury is too many. Through a multidisciplinary partnership to initiate perinatal bundles, the CHI Perinatal Care Collaborative was able to continue the reduction of injuries each year.

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