LIFE CYCLE OF MYXOBACTERIA1, 2, 3 The myxobacteria are one of natures early explorations of the survival benefits of a socially multicellular

style of life. They feed on particulate organic matter in the soil, like packs of microbial wolves, and on the colonies of other bacteria. After myxobacteria and other soil microbes have exhausted the particulate organics in their neighborhood, myxobacteria sense the fact and construct multicellular fruiting bodies within which they sporulate. Myxobacterial fruiting bodies have many shapes including mounds, spheres, cylinders, and combinations thereof (Figure 1).

Figure 1: Myxobacterial fruiting bodies. (a) Stigmatella aurantiaca, (b) Chondromyces catenulatus, (c) C. pediculatus, (d) C. apiculatus and (e) Myxococcus xanthus.

Since most of the studies till date are done on Myxococcus xanthus we will generalize the developmental pathway mainly based on this species. Under starvation myxobacteria undergo a complex developmental pattern (Figure 2) regulated by several signals. We now know that there are at least five such signals, referred to as Asg, Bsg, Csg, Dsg, and Esg (Table 1). Two of these signals, A and C, have been chemically characterized and have been shown to play roles in early aggregation and in fruiting body morphogenesis.

Figure 2: life cycle of Myxococcus xanthus.

Kaiser D (2004) Signaling in myxobacteria. Annu Rev Microbiol 58:75-98. Dworkin M (1996) Recent advances in the social and developmental biology of the myxobacteria. Microbiol Rev 60:70-102. 3 Jelsbak L, Sgaard-Andersen L (2003) Cell behavior and cellcell communication during fruiting body morphogenesis in Myxococcus xanthus. J Microbiol Methods 55:829-839.
2

Signaling group

B C D E

Table 1: Signals involved in development of Myxoccus xanthus. Signal Gene Gene product(s) (i) Transmitter domain of histidine protein kinase asgA Mixture of amino acids (ii) Receiver domain of response regulator and peptides generated DNA-binding transcription factor asgB by a mixture of Sigma factor asgC protease Histidine protein kinase asgD Amidohydrolase proteins asgE Unknown 90.4-kDa ATP-dependent protease bsgA Dimer of 17-kDa proteins Paracrine hormone csgA Short-chain alcohol dehydrogenase Sigma factor? Translation initiation dsg E1 decarboxylase of the branched-chain Fatty acids esg keto-acid dehydrogenase

Myxobacteria use soluble and cell-contact signals during their starvation-induced formation of fruiting bodies. These signals coordinate developmental gene expression with the cell movements that build fruiting bodies. Early in development, the quorum-sensing A-signal in Myxococcus xanthus helps to assess starvation and induce the first stage of aggregation. Later, the morphogenetic C-signal helps to pattern cell movement and shape the fruiting body. C-signal is a 17-kDa cell surface protein that signals by contact between the ends of two cells. The number of C-signal molecules per cell rises 100-fold from the beginning of fruiting body development to the end, when spores are formed. Traveling waves, streams, and sporulation have increasing thresholds for C-signal activity, and this progression ensures that spores form inside fruiting bodies. Other three signals namely B, D and E are not yet completely understood. A general time line in development is shown in Figure 3.

Figure 3: Developmental time line in Myxococcus xanthus.

A Signal Fruiting body development is induced by starvation, and the A-signal helps M. xanthus assess the state of its nutrition. A-signal-production is associated to five asg genes (Table 1). Myxococcus releases small quantities of A-signal, which are short 6 to 7 amino acid long

peptides, about 2 h into development, as shown in Figure 4. In addition, the extracellular concentration of A-signal is directly proportional to the density of M. xanthus cells. Thus A signal is a quorum sensing signal.

Figure 4: Starvation control of an A-signal-dependent gene (4521). Both A-signalproduction and Asignal-reception circuits are shown.

A-signal helps Myxococcus choose between alternative responses to nutrient limitation. One option is fruiting body development with differentiation of spores; the other is growth, slowed to a rate compatible with the level of nutrient remaining. Either alternative kills the majority of cells, but which is the better alternative depends on what the nutrient levels are likely to be in the future. If, on the one hand, nutrient is on its way to exhaustion, then slowing growth to match the level of residual nutrient will lead to slower and slower growth, and ultimately to death from starvation. Fruiting body development, on the other hand, kills the majority of cells because only 0.110% of cells will survive as differentiated myxospores. Because more than 30 new proteins are made during fruiting body development and sporulation, the capacity to synthesize proteins is needed during the sporulation phase. It follows that the cells must opt for development before any nutrient essential for protein synthesis is absent. M. xanthus uses its ribosomes and A-signal to predict whether nutrients are on their way to total exhaustion. Limitation for any amino acid, or starvation for carbon, energy, or phosphorus, induces fruiting body development. In M. xanthus the absence or shortage of any one of the charged tRNAs leads a ribosome, sensing with a codon that lacks its cognate amino-acylated tRNA, to synthesize guanosine tetra-(and penta-) phosphate [(p)ppGpp] by transferring di-phosphate from ATP to GTP. A rise in this highly phosphorylated nucleotide sets off a stringent response that stops the synthesis of new ribosomes, DNA, phospholipids, and peptidoglycan. Accumulation of (p)ppGpp is both necessary and sufficient for fruiting body development. The first component required for responding to the A-signal is the sensor HPKSasS (Figure 4). SasS senses the extracellular level of A-signal and triggers the expression of Asignal-dependent genes. A gene encoding an NtrC-like response regulator called SasR has been identified downstream of the sasS gene and is a likely candidate for the second member of a two-component sas system that would respond to A-signal. As a sigma-54 activator protein, SasR activate promoters of genes that are A-signal and starvation dependent. One 3

such is the promoter for 4521 that receives one input from the cells (p)ppGpp level and a second input from extracellular A-signal presumably via SasR (Figure 4). Importantly, the activator protein and the sigma-54 holoenzyme convey different pieces of information that together control the initiation of transcription of 4521. The (p)ppGpp concentration indicates the level of nutrient currently available to the cell; A-signal recalls the summary of an earlier assessment of starvation nutrient levels, which was made some two hours earlier (Figure 4). A maximum rate of 4521 expression requires both inputs; when either input is absent, the expression rate is low. Thus these inputs supply the cell two pieces of data from which it can extrapolate to a likely future nutrient level and thus decide whether to construct a fruiting body or enter stationary phase. B Signal bsg encodes one of the two lon proteases in M. xanthus. LonV is essential for growth, and LonD is essential for development. The lon recognizes and cleaves ribosomal proteins in starving cells if polyphosphate is available. Thus, lonD may be essential to make amino acids available for synthesis of new proteins during starvation-induced development. C Signal Intracellular (p)ppGpp and extracellular A-signal advance fruiting body development from the initial flat, roughly uniform distribution of cells to the first aggregates, which are small, asymmetric heaps of cells formed as traffic jams. Subsequent morphological development is initiated by an extracellular signal molecule, the C-signal. C-signaling requires end-to-end aligned contact between cells. C-signal manages cell movement to build the fruiting body, initiates the expression of many (C-signal-dependent) genes, and initiates sporulation. The Csignal coordinates these processes in time and space, using the transduction circuit shown in Figure 5. C-factor on the surface of the signal donor cell interacts with a receptive cell. That interaction requires direct contact between motile cells (Figure 6).

Figure 5: C-signal transduction circuit. Two cells are shown making end-to-end contact and signaling to each other. Both cells contain the same response circuit. p indicates proteolytic processing on the cell surface: p25 is cleaved to p17, which remains attached to the cell surface. p17 is then free to interact with the hypothetical C-signal sensor on the other cell. The reversal generator acts on both the A- and S-engines.

Figure 6: Model for C-signal induced aggregation. The model consists of three elements: (a) the basic event in the model is end-to-end contacts between cells with C-signal transmission followed by a change in cell behavior. (b) Scenarios 1 4 show how repeated end-to-end interactions with C-signal transmission between cells in a field are predicted to result in the formation of chains of cells. Consider cell A in scenario 1, which for simplicity is located at the edge of an initial aggregate and moving towards it. This cell is moving with high speed in longer periods and with a reduced number of stops and reversals as it is engaged in C-signaling with cells inside the aggregate. The bold, single-headed arrows next to the cell indicate this type of behavior. Cell not engaged in Csignaling (for example cell B in scenario 1) move with low speed and a high reversal frequency as marked by the double-headed arrows next to the cells. If cell B happens to establish end-toend contact with cell A (scenario 2), then the two cells will transmit the C-signal to each other. As a consequence, cell B is induced to move with high speed and low stop and reversal frequencies in the direction of cell A as long as it maintains the end-to-end contact. This sequence of events could be repeated sequentially, adding cell C, etc., thus creating a chain whose cells are moving in the direction determined by the leading cell A (scenarios 3 and 4). (c) Cell movement in chains is predicted to create alignment of neighboring cells. This will result in secondary chains of cells (marked by dark gray color). The cells in these secondary chains are associated with the initiating chain by lateral cell cell contacts and with other cells in the secondary chain by end-to-end contacts. Together, an initiating chain and its associated secondary chains will make up a stream. With time, most cells become organized in streams moving towards aggregation centers.

The precise function of C factor as a signal that mediates cell-cell communication is still unclear. Neither the mechanism of transmission of the C signal between cells nor the mechanism of its transduction to a cellular response is understood. Is it indeed a functioning dehydrogenase enzyme, or is it an enzymatic fossil acting now as a paracrine hormone? What is clear, however, is that it is a cell-bound signal that plays a major role in controlling the activities of more than 50 genes involved in rippling, aggregation, sporulation, and motility, and it functions not only to regulate the temporal expression of a variety of social and developmental activities but also to monitor the close spatial interactions among the cells. D Signal The exact nature of D signal is still unclear. It is thought to be some sigma factor which could be associated with transcriptions of genes required for protein synthesis during sporulation. E Signal esg gene codes for branchedchain keto acid dehydrogenase (BCKDH). BCKDH catalyzes the conversion of leucine to isovaleryl-CoA, which is a precursor for the synthesis of long-, isoand anti-isobranched fatty acids and for polyketide biosynthesis. These are essential while production of spores.