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The formation of spore-filled fruiting bodies by myxobacteria is a fascinating case of multicellular self-organization by bacteria. The organization of Myxococcus xanthus into fruiting bodies has long been studied not only as an important example of collective motion of bacteria, but also as a simplified model for developmental morphogenesis. Sporulation within the nascent fruiting body requires signaling between moving cells in order that the rod-shaped self-propelled cells differentiate into spores at the appropriate time. Probing the three-dimensional structure of myxobacteria fruiting bodies has previously presented a challenge due to limitations of different imaging methods. A new technique using Infrared Optical Coherence Tomography (OCT) revealed previously unknown details of the internal structure of M. xanthus fruiting bodies consisting of interconnected pockets of relative high and low spore density regions. To make sense of the experimentally observed structure, modeling and computer simulations were used to test a hypothesized mechanism that could produce high-density pockets of spores. The mechanism consists of self-propelled cells aligning with each other and signaling by end-to-end contact to coordinate the process of differentiation resulting in a pattern of clusters observed in the experiment. The integration of novel OCT experimental techniques with computational simulations can provide new insight into the mechanisms that can give rise to the pattern formation seen in other biological systems such as dictyostelids, social amoeba known to form multicellular aggregates observed as slugs under starvation conditions.

Plant ecologists have long been concerned with a seemingly paradoxical scenario in the relationship between plant growth and climate change: warming may actually increase the risk of plant frost damage. [...]

Mutations within the -12 and -24 elements provide evidence that the act promoter is recognized by sigma-54 RNA polymerase. Deletion of the -20 base pair, which lies between the two conserved elements of sigma-54 promoters, decreased expression by 90%. In addition, mutation of a potential enhancer sequence, around -120, led to an 80% reduction in act gene expression. actB, the second gene in the act operon, encodes a sigma-54 activator protein that is proposed to be an enhancer-binding protein for the act operon. All act genes, actA to actE, are expressed together and constitute an operon, because an in-frame deletion of actB decreased expression of actA and actE to the same extent. After an initially slow phase of act operon expression, which depends on FruA, there is a rapid phase. The rapid phase is shown to be due to the activation of the operon expression by ActB, which completes a positive feedback loop. That loop appears to be nested within a larger positive loop in which ActB is activated by the C signal via ActA, and the act operon activates transcription of the csgA gene. We propose that, as cells engage in more C signaling, positive feedback raises the number of C-signal molecules per cell and drives the process of fruiting body development forward.

Isovaleryl-coenzyme A (IV-CoA) is the starting unit for some secondary metabolites and iso-odd fatty acids in several bacteria. According to textbook biochemistry, IV-CoA is derived from leucine degradation, but recently an alternative pathway that branches from the well-known mevalonate-dependent isoprenoid biosynthesis has been described for myxobacteria. A double mutant was constructed in Myxococcus xanthus by deletion of genes involved in leucine degradation and disruption of mvaS encoding the 3-hydroxy-3-methylglutaryl-coenzyme A synthase. A dramatic decrease of IV-CoA-derived iso-odd fatty acids was observed for the mutant, confirming mvaS to be involved in the alternative pathway. Additional quantitative real-time reverse transcription-PCR experiments indicated that mvaS is transcriptionally regulated by isovalerate. Furthermore, feeding studies employing an intermediate specific for the alternative pathway revealed that this pathway is induced during fruiting body formation, which presumably increases the amount of IV-CoA available when leucine is limited.

A microbial geneticist recounts his experiences in order to encourage students of microbiology and genetics. He started using molecular genetics to understand the developmental biology of Myxococcus xanthus and has described, in part, the signal transduction pathway for C-signal that directs the morphogenesis of a fruiting body.

An isopropyI-[beta]-D-thiogalactopyranoside (IPTG)-inducible promoter was constructed in Myxococcus xanthus. The single-copy pilA gene encodes pilin, the monomer unit of M. xanthus type IV pili. To vary the level of pilA expression, we cloned its promoter in front of the lac operator, and a plasmid containing the construct was inserted into the chromosome of a [DELTA]pilA strain. Induction of pilin expression increased smoothly as the dose of IPTG added to the culture was increased. IPTG-induced pilin rescued S motility of the [DELTA]pilA strain to wild-type levels. The rate of S-motile swarming was round to be proportional to the number of pili (shear-sensitive pilin) produced rather than to the level of total pilin. In fact, S motility was not rescued until the total level of pilin was more than 50% of the wild-type level. This observation implies that a threshold concentration of pilin must be exceeded before the shear-sensitive material (pili) is polymerized in M. xanthus.

A search of the M1genome sequence, which includes 97% of the Myxococcus acanthus genes, identified 53 sequence homologs of [[sigma].sup.54]-dependent enhancer binding proteins (EBPs). A DNA microarray was constructed from the M1genome that includes those homologs and 318 other M. xanthus genes for comparison. To screen the developmental program with this array, an RNA extract from growing cells was compared with one prepared from developing cells at 12 h. Previous reporter studies had shown that M. xanthus has initiated development and has begun to express many developmentally regulated genes by 12 h. The comparison revealed substantial increases in the expression levels of 11 transcription factors that may respond to environmental stimuli. Six of the 53 EBP homologs were expressed at significantly higher levels at 12 h of development than during growth. Three were previously unknown genes, and they were inactivated to look for effects on fruiting body development. One knockout mutant produced fruiting bodies of abnormal shape that depended on the composition of the medium.

Myxobacteria build their species-specific fruiting bodies by cell movement and then differentiate spores in specific places within that multicellular structure. New steps in the developmental aggregation of Myxococcus xanthus were discovered through a frame-by-frame analysis of a motion picture. The formation and fate of 18 aggregates were captured in the time-lapse movie. Still photographs of 600 other aggregates were also analyzed. M. xanthus has two engines that propel the gliding of its rod-shaped cells: slime-secreting jets at the rear and retractile pili at the front. The earliest aggregates are stationary masses of cells that look like three-dimensional traffic jams. We propose a model in which both engines stall as the cells' forward progress is blocked by other cells in the traffic jam. We also propose that these blockades are eventually circumvented by the cell's capacity to turn, which is facilitated by the push of slime secretion at the rear of each cell and by the flexibility of the myxobacterial cell wall. Turning by many cells would transform a traffic jam into an elliptical mound, in which the cells are streaming in closed orbits. Pairs of adjacent mounds are observed to coalesce into single larger mounds, probably reflecting the fusion of orbits in the adjacent mounds. Although fruiting bodies are relatively large structures that contain [10.sup.5] cells, no long-range interactions between cells were evident. For aggregation, M. xanthus appears to use local interactions between its cells.

The soluble and cell-contact signals used during their starvation-induced formation of fruiting bodies by Myxobacteria are studied. The signals coordinate developmental gene expression with the cell movements that build fruiting bodies.

The fruiting body development and myxospore differentiation in Myxococcus xanthus is mediated by C-signal, which is regulated by the act operon. Research indicates that the specific activity of C-signal, in terms of its production and activity, influences the development of a fruiting body and spore differentiation.

Research has been conducted on the four fast-growing bacteria. The predicted highly expressed genes have been characterized for these bacteria via a method based on codon usage differences among gene classes.

Key Words cell-cell interaction, signal transduction, development, differentiation, dispersion * Abstract Multicellular organisms appear to have arisen from unicells numerous times. Multicellular cyanobacteria arose early in the history of life [...]

Results reveal that act1 gene encoding sigma(sup)54 activator proteins and insertional mutations lead to defective fruiting body development in Myxococcus xanthus. Data further indicate that the defect in sporulation is due to inadequate amounts of C signal protein produced in the mutant.

Research was conducted to examine the gliding mutants of Myxococcus xanthus with high reversal frequencies and small displacements. Quantitative, high-resolution single-cell motility assays were conducted which revealed that mutants that were defective in mg1AB or in cg1B reversed the direction of gliding at frequencies which were more than 1 order of magnitude higher than that of wild type cells. Results indicate that the Mg1A protein is essential in reversal frequency and gliding speed.

Starvation prompts the accumulation of (p)ppGpp in Myxococcus xanthus and starts the fruiting body development process. There is also the production of the A-factor. Tests on a mutant show that (p)ppGpp accumulation was stopped because of earlier developmental problems. There was no A-factor production either. Escherichia coli relA gene helped rescue the mutant.

Research was conducted to study the pilA expression in Myxococcus xanthus using a pilA-lacZ transcriptional fusion and antibody produced against PilA. M. xanthus cells on carbon-coated glow-discharged gris were treated with uranyl acetate and analyzed for pili using a transmission electron microscope. They were also purified by the surface pilin preparation technique. Results indicated that the expression of pilin can be regulated by a two-component regulatory system characterized by pilA and pilB.

The role of the tgl gene in stimulation, social motility and pilus production in Myxococcus xanthis cells is studied. The tgl locus was cloned with a linked insertion of transposon Tn5 using the kanamycin resistance encoded by the same transposon. It is shown that mutations in the tgl locus inactivate social gliding motility and prevent pili production. Social gliding and pili can also be restored to tgl mutant cells through stimulation.

The identification, location and role in pilus assembly of the Tgl protein are studied. It is shown that Tgl is a membrane protein needed for producing type IV pili, found at the pole of the Myxococcus xanthus cell, which is essential for social motility. It is also shown that Tgl is the only pilus protein that can be donated by other cells and is essential for the assembly of pilin subunits into filaments.

The RNA polymerase holoenzyme promoter of Myxococcus xanthus' 4521 gene, involved in starvation and A-signaling-dependent fruiting body development, compares favorably to the sigma54 family of enteric and other bacteria promoters. A shared holoenzyme with mbhA is likely but differences in their gene expression timing and conditions indicate upstream core promoter binding to different activator proteins.

The dsg mutants of Myxococcus xanthus have an amino acid sequence which is 50% and 51% similar to the amino acid sequence of translation initiation factor IF3 of Escherichia coli and Bacillus stearothermophilus, respectively. Translation of the Dsg protein starts at an AUC codon in E. coli cells. The missense mutation, which transforms Gly-134 to Glu in a region of amino acid identity, is seen in both dsg mutants DK429 and DK439.

Anti-Dsg antibody experiments on the Dsg protein of Myxococcus xanthus reveal the presence of constant levels of Dsg gene at all times of vegetative growth and development. Dsg complements Escherichia coli inf mutants which are defective in their autoregulatory ability of infC translation and its 66 amino-acid C-terminal extension is essential for function in M. xanthus.

The presence of the gene devR aids the completion of sporulation and aggregation activities. Negative auto regulation causes devR gene expression. The rescued dev/dev+ tandem duplication strain has a lower accumulation of mRNA than that of the dev strain's devRS region. Sporulation levels reveal a 1000-fold difference when devRS+ is moved from the mutant devRS locus by varying the distance from 20 kb to 3 Mb.

Roland Thaxter, in 1892, discovered that the imperfect fungus, Chondromyces crocatus, was actually a bacterium. It was considered an imperfect fungus as it had a complex fruiting body. Thaxter identified it as a myxobacteria and revealed its similarity to cellular slime molds. The cellular slime molds have been investigated by John Bonner and Kenneth B. Raper in 1942. They studied the morphogenesis of several mycobacteria.

Bacteria have developed an intricate system of intercellular communication. An answer was sought to the molecular basis of information flow between the cells and how it could be used in development. The problem was tackled by studying cell-to-cell interaction. It was found that in Myxococcus Xanthus, four sequential intercellular signals were responsible for the breaking of vegetative cells into spores. Bioassay was employed to isolate signal molecules in wild-type cells that allow A- mutants to portray a lacz transcriptional fusion based on A signal.

A-signalling and cell density sensing in Myxococcus xanthus were demonstrated. The addition of A-factor purified from buffer conditioned by the development of wild-type cells restored development of asg mutants, which are defective in fruiting body aggregation and sporulation. It has been shown that heat-stable A-factor is a set of amino acids and peptides while heat-labile A-factor consists of at least two proteases. Moreover, the experiments showed that the A-signal used by M. xanthus plays a role in specifying the minimum cell density required for development.

Two proteins showing A-factor activities and derived from conditioned mediums were purified and characterized. The first protein was identified as a heat-labile protein with a molecular mass of 27-kDa. This protein exhibited proteolytic activity aside from its A-factor function and its substrate specificity was observed to be similar to trypsin. The other protein was seen to be of low molecular weight and was heat stable. This polypeptide also demonstrated proteolytic activity and its substrate specificity differed from that of the first protein.

The heat labile, low molecular weight protein that exhibited A-factor activity was demonstrated to be composed of amino acids and peptides. Eleven of the 15 amino acids showing A-factor activity were derived from conditioned medium. The total activity of mixtures of amino acid were found to be equal to the sum of the activities of their constituents. Peptides from conditioned medium also exhibited A-factor activity that is equal to the sum of the activities of their amino acid compositions. Inducement of A-dependent gene expression by heat-stable A-factor was postulated to be caused by a chemosensory circuit.

Inactivation of the MXAN_0853 gene blocked the production in Myxococcus xanthus of straight-chain fatty acids which otherwise represent 30% of total fatty acids. Despite this drastic change in the fatty acid profile, no change in phenotype could be observed, which contrasts with previous interpretations of the role of straight-chain fatty acids in the organism's development.

A stimulation assay that allows contact stimulation of Tgl and type IV pili in Myxococcus xanthus is presented. In this process, Tgl stimulation is monitored by measuring the pili assembly as opposed to the swarming of S motile cells, thereby creating several advantages. First, swarming as well as nonswarming strains can be used. Second, the stimulation can be examined in a matter of hours instead of days.

DNA segments belonging to the highly conserved central sequence of sigma(super 54) activator proteins of Myxococcus xanthus have been studied. M. xanthus is a gram-negative soil bacterium which uses metabolically dormant myxospores that are resistant to heat and dessication when faced with starvation. A PCR technique developed by Kaufman and Nixon was used to amplify sequences of the central binding domain of sigma(super 54). They were able to detect 14 NtrC-like fragments of 475 bp from M. xanthus chromosomal DNA. These fragments were used to create internal disruptions in the genes.

Bacteria have an ability to survive in varied circumstances, making their study interesting. The bacterial cell environment is not static and the cells need to respond to changes it order to survive. Some cells activate adaptive genes and some move toward richer environments away from toxic ones. The mechanisms that activate communication between the bacterial cell and ligands, between two bacterial cells or between a bacterial cell and a plant cell provide rich examples of signal production and signal transduction.

Research was conducted to examine the hypothesis that the Myxococcus xanthus pilQ (sglA) gene encodes a secretin homolog required for type IV pilus biogenesis, social motility, and development. The mapping, cloning and the sequence of the sglA locus were also analyzed. Results reveal that pilQ (sglA) encodes a secretin homolog. The characterization of pilQ extends the striking similarities between proteins involved in S-motility and those required for twitching motility in N. gonorrhoeae and P. aeruginosa.