Programmed cell death in the life cycle of Streptomyces

Audio recording of this discussion

The life cycle of Streptomyces

Like the many filamentous cyanobacteria, the Streptomycetes are clearly multicellular Bacteria. A colony of a streptomycete is composed of at least 3 very distinct cell types: vegetative hyphae, aerial hyphae, and spores. Generally speaking, these develop in this order; spores germinate into vegetative hyphae, from which come aerial hyphae during a round of programmed cell death of the vegetative hypahe, and from the aerial hyphae develop spores during another round of programmed cell death of the aerial hyphae. These cells types are structurally and functional distinct, and strongly resemble filamentous fungi; this is a case of convergent evolution, apparently caused (as usual) by the common constraints and solutions of a shared lifestyle. Another aspect of multicellularity apparent in the streptomycetes is senescence; they are mortal. A colony will eventually "go to seed" (spores, of course) even if nutrients remain available, and die. Only the spores will carry on.

Miguélez EM, Hardisson C, and Manzanal MB. 1999 Hyphal death during colony development in Streptomyces antibioticus: Morphological evidence for the existence of a process of cell deletion in a multicellular prokaryote. J. Cell Biol. 145:515-525.

Question : Is cell death in Streptomyces colonies a controlled, developmental process?

In this paper, the authors focus on one aspect of multicellularity, in attempt to confirm it's presence (at least in some form) in the multicellular bacterium Streptomyces antibioticus. That aspect of multicellularity is programmed cell death, the organized self-destruction of some cells for the benefit of others in the organism. They examined cultures of S. antibioticus throughout it's life cycle using scanning (3D) and transmission (2D slices) electron microscopy. What they see, as hyphae get older and older, is that they die in a specific sequence of events. First, the nucleoids get disrupted and dispersed - this can also be seen as degradation of the DNA (see Fig. 9). As the nucleoid disperses, the cytoplasm gets progressively emptied - this can be seen in the EM's (Figs 1 and 3), and also as the disappearence of both RNA and protein (Fig. 9). This is not "autolysis"; the cytoplasmic membrane remains intact. This is senescence and programmed cell death. Only when the cell is utterly defunct does the membrane collapse, but even then the cell wall remains intact (although shriveled). There are 2 waves of such cell death in these cultures; the first seems to correspond to the self-sacrifice of the vegetative mycelia when the aerial mycelia emerge, and the second when the aerial mycelia die when the spores are produced. Notice that there is no loss of total dry cell mass as these cells die - the contents of the dying cells are recycled to the growing cells.

Manteca A, Fernandez M, Sanchez J. 2005 BMC Microbiol. 5:51 Mycelium development in Streptomyces antibioticus ATCC11891 occurs in an orderly pattern which determines multiphase growth curves.
This paper is also available in an online version at: http://www.biomedcentral.com/1471-2180/5/5

Purpose : to observe the morphology of Streptomyces during the main developmental stages of surface growth, using dyes that allow them to distinguish living and non-living cells, with particular attention to horizontal organization.

Abstract

Background
The current model for the developmental cycle of Streptomyces confluent cultures on agar surface is based on the assumption that the only differentiation takes place along the transverse axis (bottom-up): a vegetative (substrate) mycelium grows completely live and viable on the surface and inside the agar until it undergoes a death process and differentiates to a reproductive (aerial) mycelium which grows into the air. Hence, this vertical description assumes that the development in the pre-sporulating phases is more or less homogeneous in all zones of the plate surface.

Results
The work presents a detailed analysis of the differentiation cycle in Streptomyces antibioticus ATCC11891 considering a different spatial dimension: the longitudinal axes, represented by the plate surface. A previously unsuspected complexity during the substrate mycelial phase was detected. We have demonstrated that the young substrate hyphae suffer an early death round that has not been previously described. Subsequently, the remaining mycelium grows in successive waves which vary according to the density of the spore inoculum. In the presence of dense inocula (1.5 × 106 spores per plate), the hyphae develop in regular circles, approximately 0.5 cm in diameter. By contrast, with highly diluted inocula (6 × 103 spores per plate), aerial mycelium develops initially in the form of islands measuring 0.9 mm in diameter. Further mycelial development occurs between the circles or islands until the plate surface is totally covered. This pattern persists throughout the entire developmental cycle including the sporulation phases.

Conclusion
An early death round during the substrate mycelial phase of Streptomyces antibioticus ATCC11891 takes place prior to successive growth periods in surface cultures. These developmental periods in turn, determine the shape of the complex multiphase growth curves observed. As shown here, these results also apply to other Streptomyces strains and species. Understanding these peculiarities of the Streptomyces developmental cycle is essential in order to properly interpret the morphological/biochemical data obtained from solid cultures and will expand the number of potential phenotypes subject to study.

The authors grow Streptomyces on agar plates from spores, stain them with propidium iodide (which stains dead cells red) and SYTO 9 (which stains live cells green), and capture images using confocal laser-scanning fluorescent microscopy.

Figure 6, below, is the "take-home" message of the paaper, the model that describes everything they see.

Phase A : 0-7hr
During this phase, some of the spores germinate and send out hyphae. These are unusual hyphae; they are septated, separated into distinct individual cells, rather than syncytial, continuous hyphae without being divided into individual cells. All of the other hyphae in the rest of the cell cycle, until spores form, are syncytial. These septated hyphae grow out to cover the surface of the plate, then most of them undergo a previously unknown round of programmed cell death, in which alternating cells die. The hyphae appear variegated in these images, with alternating red (dead) and green (live) cells.

Phase B : 7-10hr
Within the background of variegated hyphae, the last spores to germinate produce syncytial hyphae that do not beecome variegated. These grow in the form of rings about 5mm in diameter. Within these rings, and between them, the hyphae remain variegated.

Phase C : 10-14hr
The non-variegated hyphae in the outer edges of the rings grow rapidly.

Phase D : 14-16hr
The circles of non-variegated hyphae stop growing, and from some of the living cells in the variegated hyphae underlying these circles come new non-variegated hyphae that grow inward toward the center of the circle. These 'islands' of re-growth along the edge of the circle fuse as they grow inward to form concentric rings of new growth. In later papers, the authors make clear that these are aerial hyphae, and the previous circle-forming vegetative hyphae have undergone a round of programmed cell death.

Phase E : 16-20hr
These islands of aerial hyphae grow rapidly within the circles, both inwardly and from the bottom up, creating dense disks of hyphae where the earlier circles were.

Phase F : 20-30hr
Aerial hyphae grow out to fill in the areas between circles, although these remain less dense than the growth in the circles.

Phase G : 30-45hr
The aerial hyphae undergo programmed cell death, except the ends of the hyphae, that will become spores in the next phase. Sporulation begins in the circles, although most also undergo preogrammed cell death for some reason.

Phase H : 45-96hr
Sporoulation in earnest, both within and between circles. Remaining hyphae between circles die.

Confocal laser-scanning fluorescence microscopy analysis of the development-linked cell death processes of Streptomyces antibioticus ATCC11891 in confluent surface cultures. Developmental phases (A-H) and culture times (hours) are indicated. Picture D2 was obtained under the phase contrast microscope. The other images correspond to culture sections stained with SYTO 9 and propidium iodide. E2 is a cross section view; the other images correspond to longitudinal sections (see methods). Arrows in E2 indicate the eccentric circles of live mycelium developing from the bottom upwards, forming distinct layers with well-defined boundaries. Arrows in the rest of the images indicate circle edges.

They also run the same observations with plates innoculated with a lower density of spores. The biggest differences they see are:

1) The initial septated germination hyphae are mostly viable, until they grow to a certain density, then they undergo death of alternating cells to form variegated hyphae.

2) No circles are formed by live filaments in stage B or C, but grow instead as fast-growing islands.

Longitudinal sections of Streptomyces antibioticus ATCC11891 surface cultures obtained using a diluted inoculum (6 × 103 spores per plate). The developmental phases (A'-H') and culture times are indicated. Samples were stained with SYTO 9 and propidium iodide. Arrow in picture A' indicates a group of hyphae in transition from presenting uniform green fluorescence to a variegated appearance, in which live (green) and dead (red) segments alternate in the same hypha.

To make sure these observations weren't some oddity of this specie, they ddid the same obserbations with 3 other species of the genus Streptomyces, and saw the same thing:

Longitudinal sections of different Streptomyces strains growing in confluent surface cultures. (a) S. coelicolor A3(2). (b) S. glaucescens ETH22794. (c) S. antibioticus ETH7451. Culture times are indicated.

Lastly, to make sure what they saw wasn't something unusual because of the growth media, they checked 3 other kinds of plaate media, and saw no real difference:

Cross sections of S. antibioticus ATCC11891 cultures in different solid media. Developmental time points and culture media are indicated. Casαα is GAE plus 2% casamino acids.

So, Streptomyces have have a complex life cycle, with development occuring in three-dimensions and time; up/down and time (vegetative hyphae -> aerial hyphae ->spores), and across the surface (circles, islands, and waves of growth). They undergo several rounds of programmed cell death that is controlled in both time and space. It may be poorly understood, and even what we know difficult to sort out, but it is clear that this is a programmed multicellular developmental cycle, even though there is no differentiation between germ and somatic cell lines.